Escherichia coli is the common engineered bacterium used to produce PCB. However, it still suffers from low production level, precursor deficiency, and low catalytic efficiency. In this study, a highly efficient PCB-producing strain was created. First, chassis strains and enzyme sources were screened, and copy numbers were optimized, affording a PCB titer of 9.1 mg/L. Most importantly, the rate-limiting steps of the PCB biosynthetic pathway were determined, and the supply of precursors necessary for PCB synthesis was increased from endogenous sources, affording a titer of 21.4 mg/L. Then, the key enzymes for PCB synthesis, HO1 and PcyA, were assembled into a multi-enzyme complex using the short peptide tag RIAD-RIDD, and 23.5 mg/L of PCB was obtained. Finally, the basic conditions for PCB fermentation were initially determined in 250 mL shake flasks and a 5-L bioreactor to obtain higher titers of PCB. The final titer of PCB reached 147.0 mg/L, which is the highest reported titer of PCB so far. This research provided the foundation for the industrial production of PCB and its derivatives.Phycocyanobilin (PCB) is widely used in healthcare, food processing, and cosmetics.  PCB has a wide range of medical applications due to its antioxidant, anti-inflammatory, anti-cancer, and other properties . It can l-alanine, phenylpropanoid acids, and 5-aminolevulinic acid (ALA) [E. coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae by screening multiple sources of hemA and hemL as well as constructing biosensors [3+ into protoporphyrin IX to form heme [. Therefore, based on the current study, it is possible to de novo synthesize PCB [In recent years, biosynthesis has become an effective way to increase the titer of some natural compounds that are widely used but limited in natural synthesis, such as 3,4-dihydroxyphenyl-id (ALA) . ALA synid (ALA) ,11. Reseosensors . ALA is orm heme . After hsize PCB .Fig. 1EnE. coli has been widely utilized to synthesize a variety of substances [ho1 and pcyA genes to enhance PCB titer to 6.64 mg/L in E. coli [ho1 and pcyA, improving the cofactor cycle, adding the precursor ALA directly, and overexpressing hemB and hemH [E. coli. However, the genes involved in the PCB synthesis pathway were different, and the titers of PCB were still low. In addition, the endogenous synthesis of heme, an important precursor for PCB synthesis, remains limited. Thus, the biosynthesis of PCB in E. coli has many issues that need to be solved urgently.As an engineered bacteria with well-studied genetic and metabolic information, bstances ,22. Prev E. coli . Immedia E. coli . More reand hemH , the higho1 and pcyA gene expression, the catalysis rate of heme to PCB was enhanced. In addition, several commonly used fermentation media in laboratories were compared, along with induction temperatures, and a fermentation temperature of 25 °C and GMD-Gly medium were selected. Finally, an expanded culture of PCB synthesis was performed in a 5-L bioreactor to verify the potential of the best-engineered strain for PCB production. We obtained 147.0 mg/L, the highest PCB titer among microbial synthesis reported to date.In this study, an efficient PCB-producing strain was constructed through metabolic engineering. First, to create optimal conditions for PCB synthesis, a better chassis strain was screened. Then, the PCB titer was further increased by integrated expression of key genes involved in precursor synthesis. Moreover, through the binding of the RIAD-RIDD short peptide tag to 22.1High fidelity DNA polymerase Phanta® Max Master Mix and a FastPure® Gel DNA Extraction Mini Kit were purchased from Vazyme Biotech Co., Ltd . All chemical reagents not described above were purchased from Sangon Biotech Co., Ltd . All genes had been cloned in previous work . Oligonu2PO4, 12.42 g/L K2HPO4.TB medium: 10 g/L tryptone, 24 g/L yeast extract, 5 g/L glycerol, 2.31 g/L KH4)2SO4, 3 g/L KH2PO4, 3 g/L MgSO4⸱7H2O, 1 g/L NaCl, 1.5 g/L Trisodium citrate dihydrate, 0.015 g/L CaCl2⸱6H2O, 0.1125 g/L FeSO4⸱7H2O, 0.075 g/L vitamin B1, 4 g/L tryptone, 2 g/L yeast extract.FLB medium: 35 g/L glucose, 5 g/L 2SO4, 1 g/L citric acid, 1 g/L MgSO4⸱7H2O, 10 g/L yeast extract, 30 g/L glycerol, 10 g/L maltodextrin, 0.1 g/L vitamin B1, and 1 mL/L trace elements.GMD-Gly fermentation medium: 6 g/L KH3, 14.64 g/L MnSO4·H2O, 0.75 g/L CuSO4·5H2O, 2 g/L Na2MoO4·2H2O, 2 g/L CaCl2.2H2O, 3.0 g/L H3BO3, 2.5 g/L CoCl2.6H2O, 2.5 g/L NiSO4.6H2O, and 100 mL HCl.The trace elements: 100 g/L Fe (III) citrate, 18 g/L ZnCl2.2E. coli JM109 was used for plasmid construction. E. coli strain St06, possessing heterologous genes for de novo production of PCB, was constructed in previous work [E. coli were activated in Luria-Bertani (LB) medium at 37 °C. Ampicillin (50 mg/L), kanamycin (50 mg/L), chloramphenicol (25 mg/L), or spectinomycin (50 mg/L) was used to culture the E. coli strains as required.ous work . All str2.3Molecular cloning and enzyme digestion techniques were used for plasmid construction. The plasmids and primers are listed in 2.4600 was 0.6–0.8, the appropriate concentrations of isopropyl-β-d-thiogalactopyranoside (IPTG) were added to induce the synthesis of PCB at 25 °C. The concentration of the IPTG was 0.5 mM. According to plasmid requirements, appropriate concentrations of antibiotics were added, including ampicillin (50 mg/L), kanamycin (50 mg/L), chloramphenicol (25 mg/L), or streptomycin (50 mg/L).Shake flash conditions: The seed solution was incubated at 37 °C and 220 rpm for 10–12 h. The seed solution was then transferred to the fermentation medium at an inoculum level of 2%. When the ODFed-batch fermentation conditions: The secondary seed solution was added to the 5-L bioreactor with 2.5-L of the fermentation medium. During the early stage, the temperature was controlled at 37 °C. The IPTG was added when there was a rebound in dissolved oxygen (DO), at which time the temperature was cooled to 25 °C. In the fermentation process, the pH was controlled at 7.0 by the automatic addition of 50% ammonia. The dissolved oxygen and speed were adjusted appropriately according to the situation.2.5The fermentation broth was prepared with an equal volume of methanol. The mixture was heated in a water bath at 50 °C for 1 h and then shaken for 5 min. After centrifugation, the supernatant was filtered through a 0.22 μm membrane. The concentration of PCB was analyzed by High Performance Liquid Chromatography (HPLC) with a C18 column (30 cm × 0.25 cm) at 25 °C. A wavelength of 380 nm and a flow rate of 0.8 mL/min were used .33.1ho1 and pcyA was better for the production of PCB compared with both genes controlled by a T7 promoter [E. coli strains for the synthesis of PCB, the strains JM109(DE3), B834(DE3), W3110(DE3), BL21 Star(DE3), Origami B(DE3), and BL21(DE3) were compared. The plasmid pRSFDuet-PT7lac-ho1T-PT7lac-pcyAS was introduced into these strains, and the strain St06 was the best . To compare the ability of the two key enzymes to synthesize PCB, they were individually expressed or co-expressed in E. coli BL21(DE3) to obtain strains St07, St08, St09, St10, St11, St12, and St13. The strains St12 and St13 yielded 4.2 mg/L and 8.4 mg/L of PCB, respectively. The strains St10 and St11 produced 3.5 mg/L and 4.3 mg/L PCB, respectively. These titers were much lower than that of the strain St07 (E. coli (Ho1S and PcyAS derived from PCC6803 are abbreviated as Ho1 and PcyA).Previous studies suggested that using two independent T7 promoters to express promoter . To discthe best A. Ho1S aain St07 C. The tiho1 and pcyA from PCC 6803 were selected for further study. On this basis, ho1 and pcyA, which were derived from Synechocystis sp. PCC6803, were simultaneously cloned into other plasmids with different copy numbers to obtain pETDuet-ho1-pcyA (medium copy number), pCDFDuet-ho1-pcyA (medium copy number), and pACYCDuet-ho1-pcyA (low copy number) (E. coli BL21(DE3) to generate the strains St14, St15, and St16. However, the strain St07  was still the highest PCB-producing strain. The titers of the strains St14, St15, and St16 were 6.0 mg/L, 5.9 mg/L, and 5.2 mg/L, respectively  B. Then, ectively D. The re3.2HemB, hemC, hemD, hemE, hemF, hemG, and hemH. Heme was a precursor to PCB biosynthesis (E. coli [T7-hemBCD was integrated into the arsB locus of the E. coli BL21(DE3) genome using the CRISPR/Cas9 gene editing system to increase the endogenous synthesis of heme. Then, the correctly sequenced strain St17 was obtained. The plasmid PW07 (pRSFDuet-ho1-pcyA) was introduced into the strain St17 to obtain the strain St18. It was found that the strain St18 increased the PCB of the initial strain St07 by 64% to 15.0 mg/L. Next, PT7-hemEFGH was integrated into the yfeX locus of the strain St17 to obtain the strain St19. The gene yfeX from E. coli had been reported to inhibit heme synthesis [Heme synthesis was encoded by seven genes: ynthesis . However(E. coli . Therefoynthesis . Similar3.3CysG and CyoE on the genome of the strain St19 (E. coli BL21(DE3) ΔarsB:hemBCD ΔyfeX:hemEFGH) were knocked-out. The gene CysG can convert uroporphyrinogen III to the precursor to cobalamin and siroheme, while CyoE degrades heme into Heme O  of the strain St19 was mutated to improve the glycerol utilization [glpK gene lead to a decrease in PCB production  were compared for the synthesis of PCB A. Moreovlization . Then, toduction B. This m4·7H2O, FeCl3, (NH4)2SO4, and glycerol was added, and the IPTG was added to induce PCB synthesis when the DO was increased to 40. The titer of PCB increased to 147.0 mg/L  at two locus, forming the final strain E. coli BL21(DE3) ΔarsB:hemBCD ΔyfeX:hemEFGH (St19). Finally, heme was supplied in significant levels, and this promoted the synthesis of PCB. This is a relatively common mode of conversion, and it is also a very efficient one.In the process of biosynthesis, precursor supply was the key factor to improve the titer of follow-up target products. During metabolic engineering to modify target strains for the synthesis of useful biobased chemicals, the synthesis of the target products requires a large supply of precursors ,32. For n of OAH . Heme is studies , but the studies ,19,34. THigh substrate concentrations have a substantial effect on the catalytic efficiency of enzymes. Some organelles in eukaryotes can accumulate large amounts of substrates, promoting the catalytic efficiency of specific enzymes. However, there are no organelles in prokaryotes, resulting in substrates being dispersed in the cytoplasm. Therefore, many researchers have tried to enhance the substrate concentration near the enzyme to promote the conversion rate of the target reaction. In a multi-enzyme reaction, the product synthesized by the previous enzyme is the substrate of the latter enzyme. By bringing the two enzymes closer to each other, the product released by the previous one can be directly captured by the latter enzyme. If two enzymes are connected by a linker, the target product can be efficiently synthesized , 36. FurhemAL of the C5 pathway was also integrated, but unexpectedly, PCB was not even detected. We speculate that the use of the T7 promoter placed a significant burden on the chassis strain, resulting in a growth inhibition affecting subsequent synthesis, which is still being explored. In addition, we have not fully optimized Ho1 and PcyA, the two key enzymes for PCB synthesis. We plan to use enzyme engineering to systematically and rationally modify these enzymes to further improve the synthesis of PCB.In this study, the initial strain for PCB synthesis was constructed by screening the chassis strains, the source of the key enzymes, and the copy numbers. Most importantly, precursor supply was increased by genomic integration of key genes, which increased PCB titer by 10.5 mg/L compared with the initial strain. Furthermore, the key genes were self-assembled through the short peptide tags RIAD-RIDD to further increase PCB synthesis. The best fermentation medium was determined in a 250-ml shake flask. We optimized the feed composition, dissolved oxygen, and induction time in a 5-L bioreactor, which afforded a yield of 147.0 mg/L, the highest titer reported so far. However, there are still some unsolved problems in our research. The gene 10.13039/501100012166National Key Research and Development Program of China (2019YFA0904800) and the Foundation for Innovative Research Groups of the 10.13039/501100001809National Natural Science Foundation of China (32021005).This work was supported by the Yuqi Wang: Methodology, Investigation, Formal analysis, Writing – original draft, Writing – review & editing. Ning Li: Formal analysis, Writing – review & editing. Xiaoyu Shan: Methodology, Supervision. Xinrui Zhao: Investigation, Formal analysis. Yang Sun: Methodology, Supervision. Jingwen Zhou: Methodology, Supervision, Funding acquisition, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The [89Zr]Zr-desferrioxamine (DFO)-TST001 showed high radiochemical purity  and specific activity (24.15 ± 1.34 GBq/μmol), and was stable in 5% human serum albumin, and phosphate buffer saline (>85% RCP at 96 h). The EC50 values of TST001 and DFO-TST001 were as high as 0.413 ± 0.055 and 0.361 ± 0.058 nM (P > 0.05), respectively. The radiotracer had a significantly higher average standard uptake values in CLDN18.2-positive tumors than in CLDN18.2-negative tumors  2 days post injection (p.i.). BGC823CLDN18.2 mice models showed high tumor/muscle ratios 96 h p.i. with [89Zr]Zr-DFO-TST001 was much higher than those of the other imaging groups. Immunohistochemistry results showed that BGC823CLDN18.2 tumors were highly positive (+++) for CLDN18.2, while those in the BGC823 group did not express CLDN18.2 (−). The results of ex vivo biodistribution studies showed that there was a higher distribution in the BGC823CLDN18.2 tumor bearing mice (2.05 ± 0.16 %ID/g) than BGC823 mice (0.69 ± 0.02 %ID/g) and blocking group (0.72 ± 0.02 %ID/g). A dosimetry estimation study showed that the effective dose of [89Zr]Zr-DFO-TST001 was 0.0705 mSv/MBq, which is within the range of acceptable doses for nuclear medicine research. Taken together, these results suggest that Good Manufacturing Practices produced by this immuno-positron emission tomography probe can detect CLDN18.2-overexpressing tumors.Claudin18.2 (CLDN18.2) is a tight junction protein that is overexpressed in a variety of solid tumors such as gastrointestinal cancer and oesophageal cancer. It has been identified as a promising target and a potential biomarker to diagnose tumor, evaluate efficacy, and determine patient prognosis. TST001 is a recombinant humanized CLDN18.2 antibody that selectively binds to the extracellular loop of human Claudin18.2. In this study, we constructed a solid target radionuclide zirconium-89 (  •anti-CLDN18.2 antibody PET probe.Development of radiolabeled a GMP grade •High affinity to CLDN18.2 in vitro and in vivo.•This tracer can noninvasively report CLDN18.2 expression in different tumors. In Chin18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) parameters including SUVmax, metabolic tumor volume, and total lesion glycolysis did not predict CLDN 18.2 expression status in diffuse-type gastric cancer [89Zr]-anti-CLDN18.2 VHH-ABD as the most appropriate imaging agent (high tumor uptake and low uptake in the liver) in preclinical studies. However, in a subsequent clinical study, [89Zr]-VHH-Fc was found to be more specific and persistent than [89Zr]-anti-CLDN18.2 VHH-ABD, and was also considered to be a molecular imaging tracer with potential value for cancer diagnosis, as it contains CLDN18.2 Zr-desferrioxamine (DFO)-TST001. The goal of this study was to assess the ability of [89Zr]Zr-DFO-TST001 to characterize CLDN18.2 expression.Due to the superior targeting specificity and high sensitivity of molecular imaging technology, we used the TST001 antibody produced under Good Manufacturing Practices (GMP) conditions to construct the immuno-PET molecular probe ethanesulfonic acid and 1 M Na2CO3 buffer, and then mixed with DFO-TST001 for 60 min at 37 °C. The reaction mixture was purified by a PD-10 column with 0.01 M phosphate buffer saline .For 2.3CLDN18.2/BGC823 model nude mice were injected with 7.4 MBq of [89Zr]Zr-DFO-TST001 via the tail vein (n = 3). Then 10 min static PET scans were acquired at each time point ). As a non-specific control group, BGC823CLDN18.2 mice (n = 3) were fasted 6 h in advance, then injected with 7.4 MBq of 18F- FDG via the tail vein. The mice were anesthetized with 2% isoflurane before and during the 18F-FDG PET imaging. With a small-animal PET/CT scanner , the PET images were reconstructed by Avatar 3 , and the regions of interest (ROIs)-derived SUV was calculated by drawing ROIs over these organs. All animal experiments were performed according to the National Institutes of Health guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care and Ethics Committee of Peking University Cancer Hospital .Normal Kunming (KM) mice and BGC8232.489Zr]Zr-DFO-TST001 via the tail vein and were then sacrificed at 2, 24, 48, 72 and 144 h p.i. (n = 4). The tissues including the blood, heart, liver, spleen, lung, kidneys, stomach, intestines, muscle, bone, and brain were dissected. The radioactivity of the tissues was measured using a γ-counter . The radioactivity of each organ was calculated as percentage of injected dose per gram (%ID/g). For the tumor model's ex vivo biodistribution, female nude mice bearing BGC823CLDN18.2 and BGC823 tumor xenografts were injected by tail vein with 0.74 MBq of [89Zr]Zr-DFO-TST001 to evaluate the distribution of [89Zr]Zr-DFO-TST001 in major organs and tumors (n = 4 per group). The mice were sacrificed and dissected at 48 h p.i. (n = 4), and the tumor, kidney, blood, and other major organs were collected and weighed. The blocking study was also performed in BGC823CLDN18.2 mice by a co-injection of 0.74 MBq of [89Zr]Zr-DFO-TST001 with an excess dose of cold TST001 (1 mg). At 48 h p.i., the blocked mice were sacrificed and dissected. Then, the organ biodistribution of [89Zr]Zr-DFO-TST001 was determined.The KM mice were intravenously injected with 0.74 MBq of [2.589Zr]Zr-DFO-TST001 in KM mice by OLINDA/EXM 2.0 software .For human radiation dosimetry, animal biodistribution data were obtained by the standard method of organ dissection. The human organ radiation dosimetry data were extrapolated from the biodistribution data of Zr-DFO-IgG, quality control of [89Zr]Zr-DFO-TST001, cell lines and tumor-bearing model, assessment of CLDN18.2 expression, cellular experiments, and immunohistochemistry studies are all described in the Supplementary data.Quantitative data are expressed as the mean ± standard deviation (SD), with all error bars denoting the SD. The means were compared using Student's 33.150 value of DFO-TST001 binding to CLDN18.2 was not significantly different from that of TST001 . The radiochemical purity (RCP) of [89Zr]Zr-DFO-TST001 was more than 99% in 0.01 M PBS (pH 7.4)  B. The inperature C. The ex3.3CLDN18.2 cells was significantly different from that in BGC823 cells , while no significant changes were observed in the BGC823 group . The uptake by BGC823CLDN18.2 cells (CLDN18.2 positive) was significantly higher than that by BGC823 cells (CLDN18.2 negative) at each selected time point (P < 0.0004). Meanwhile, an excess of unlabeled TST001 significantly blocked the uptake of [89Zr]Zr-DFO-TST001  (89Zr]Zr-DFO-TST001 by BGC823CLDN18.2 cells at 60 min was 2.51-fold higher than that of BGC823 cells and 3.54-fold higher than that of the blocking group. The specificity of [89Zr]Zr-DFO-TST001 for CLDN18.2 was thus demonstrated at the cellular level.Western blotting results confirmed that the expression of CLDN18.2 in BGC82323 cells A. The re.2 group C. The di120 min) D. In the3.489Zr]Zr-DFO-TST001 demonstrated favorable pharmacokinetics with a relatively long half-life in vivo  after injection of [d spleen . The SUV89Zr]Zr-DFO-TST001 were evaluated in real time and noninvasively via small-animal PET/CT imaging at 2, 24, 48, 72, and 96 h p.i. of the radiotracer. Meanwhile, we set up the following three control groups, which were blocked by excess TST001, negative CLDN18.2 expression in BGC823 cells, and nonspecific targeting of [89Zr]Zr-DFO-IgG (7.4 MBq), respectively  , the [89Zr]Zr-DFO-IgG tumor uptake value at all other time points  was significantly lower than that of [89Zr]Zr-DFO-TST001   . Using Zr-DFO-TST001 were significantly higher than those of the other control groups (For comparison with the gold-standard probe 1 h p.i. A. The rectively) B. The tul groups C and D, 124I imaging, which greatly reduces patient compliance Zr-DFO-TST001, which might be a result of nonspecific binding and hepatobiliary clearance. This is very similar to previous studies on the 89Zr-labeled antibody Zr-DFO-TST001 was higher than that of 18F-FDG at the tumor sites in the BGC823CLDN18.2 model, and the T/M value of [89Zr]Zr-DFO-TST001 was also much higher than that of 18F-FDG (10.23 ± 1.30 vs. 1.80 ± 0.22)  A and D.CLDN18.2 tumors, and the BGC823 xenograft tumors were negative for CLDN18.2 , while the BGC823 tumors were negative (−), which was consistent with the imaging and Western blotting results  was also observed in the gastric mucosa of all mice, but neither PET/CT imaging nor biodistribution showed any obvious uptake and retention of the probe in the stomach, likely because the expression of CLDN18.2 in vivo was limited to the gastric mucosa, and monoclonal antibodies had difficulty accessing the hidden CLDN18.2 binding epitope in the gastric mucosa [The results of IHC revealed high and homogenous CLDN18.2 expression in BGC823CLDN18.2 E. The st results . These rc mucosa .3.689Zr]Zr-DFO-TST001 in BGC823CLDN18.2 and BGC823 tumor-bearing mice is presented in CLDN18.2 group, 9.28 ± 0.19 %ID/g in BGC823 group, and 20.96 ± 0.88 %ID/g in blocking group, respectively). The uptake value of the spleen was second to that of the liver . Tumor uptake in BGC823CLDN18.2 tumor bearing mice was higher (2.05 ± 0.16 %ID/g) than that in the BGC823 mice (0.69 ± 0.02 %ID/g) and blocking group (0.72 ± 0.02 %ID/g)  (CLDN18.2 vs. 1.47 ± 0.50 in blocking group)  A. The tug group) B and D. g group) C. Consis489Zr labeling of a GMP grade anti-CLDN18.2 recombinant humanized antibody TST001. [89Zr]Zr-DFO-TST001 exhibited good specificity at the cellular level and rapid tumor accumulation which remained positive from 24 to 96 h. It provides a promising molecular probe for detecting the treatment effects of therapeutic antibodies in humans in real time. It also provides a possibility for the screening and efficacy evaluation of patients targeted for CLDN18.2 therapy in the future.We successfully prepared Yan Chen and Xingguo Hou: Investigation, Methodology, Software, Formal analysis, Data curation, Writing - Original draft preparation; Dapeng Li: Investigation, Methodology, Software, Writing - Original draft preparation; Jin Ding: Conceptualization, Investigation, Resources, Validation; Jiayue Liu: Methodology, Software, Formal analysis; Zilei Wang: Methodology, Software, Formal analysis; Fei Teng, Hongjun Li, Fan Zhang, Yi Gu, and Steven Yu: Resources, Validation, Supervision; Xueming Qian: Investigation, Resources, Validation, Supervision; Zhi Yang: Conceptualization, Methodology, Investigation, Resources, Validation, Supervision; Hua Zhu: Conceptualization, Methodology, Investigation, Resources, Validation, Writing - Reviewing and Editing, Supervision.Intellectual properties protection have been filed by Suzhou Transcenta Therapeutics Co., Ltd., inventor of Xueming Qian; Fei Teng; Hongjun Li; Yi Gu, and Beijing Cancer Hospital, inventor of Hua Zhu; Yang Zhi; Jin Ding; Feng Wang. All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Yersinia pestis bacteria from a ruptured bubo to the lungs or by directly inhaling aerosolized bacteria. The fatality rate associated with pneumonic plague is significant unless effective antibiotic therapy is initiated soon after an early and accurate diagnosis is made. As with all bacterial pathogens, drug resistance is a primary concern when developing strategies to combat these Yersinia pestis infections in the future. While there has been significant progress in vaccine development, no FDA-approved vaccine strategy exists; thus, other medical countermeasures are needed. Antibody treatment has been shown to be effective in animal models of plague. We produced fully human polyclonal antibodies in transchromosomic bovines vaccinated with the recombinant F1-V plague vaccine. The resulting human antibodies opsonized Y. pestis bacteria in the presence of RAW264.7 cells and afforded significant protection to BALB/c mice after exposure to aerosolized Y. pestis. These data demonstrate the utility of this technology to produce large quantities of non-immunogenic anti-plague human antibodies to prevent or possibly treat pneumonic plague in human.Plague is an ancient disease that continues to be of concern to both the public health and biodefense research communities. Pneumonic plague is caused by hematogenous spread of  Yersinia pestis is a gram-negative Tier 1 select bacterial biothreat agent that can cause rapidly fatal infections [Y. pestis is a major biothreat due to its capacity for aerosol dissemination and its contagious nature in the pneumonic form. The illness can be treated with several different classes of antibiotics, including aminoglycosides  and quinolones  [fections ,3. Whilefections ,5. Y. peloxacin) ,7. Howevloxacin) ,9. Recenloxacin) ,11,12,13caf1 gene located on a large plasmid (pMT) and is robustly expressed at 37 °C [Y. pestis that are F1 negative  have been identified and retain their virulence in mice [Yersinia-induced immune suppression [Y. pestis. The dual antigens were theorized to be able to protect against emerging/engineered threats that may be F1 negative in spite of known heterogeneity amongst the LcrV proteins from different isolates [Two protective antigens have been used to make subunit vaccines, including the F1 capsular antigen and the LcrV antigen ,15. The at 37 °C ,17. F1 iat 37 °C ,19,20. Iat 37 °C . However in mice ,24,25,26 in mice ,28. The  in mice ,30. Thispression ,32,33,34pression ,35,36. Tpression ,37. Actiisolates ,39,40. Risolates ,44,45,46Y. pestis LcrV (mAb 7.3) and F1 (mAb F1-04-A-G1) antigen is protective in mouse models of bubonic and pneumonic plague [Y. pestis [The vaccine studies using the F1 and LcrV antigens suggest that antibodies play a role in protection. When administered prophylactically or 48 h post-infection, either alone or in combination, passive immunization with two monoclonal antibodies (mAbs) generated against c plague . The in . pestis .Y. pestis infection [Y. pestis by macrophages and protected macrophages from Y. pestis-induced cell death. The observed in vitro phenotype, however, is dependent upon the culture conditions used to grow the bacteria in the laboratory, as these conditions affect the expression of these protective antigens by the bacteria.Passive protection has been accomplished using antibodies directed against either the F1 or the LcrV antigen ,52,53,54nfection ,49,55. Tnfection ,47,56. BIn this report, we characterize and evaluate anti-plague polyclonal antibodies generated in transchromosomic (Tc) bovines using ELISA, functional macrophage assays, and in vivo mouse models of pneumonic plague. Tc bovines endogenously produce fully human IgG polyclonal antibodies in response to environmental and vaccine-delivered antigens ,58,59. WY. pestis pgm- pPst- strain was generated at USAMRIID and was kindly provided by Susan Welkos  [Y. pestis pgm- pPst- is an attenuated strain derived from the fully virulent Y. pestis CO92 strain, which is cured of the pPst plasmid containing the plasminogen activator (Pla) virulence locus (pla) and is pigmentation locus (pgm)-deficient [Y. pestis was grown on Remel® Sheep Blood Agar (SBA) plates  and incubated at 28 °C or 37 °C for 24 h. Bacterial colonies were harvested and used to inoculate 10 mL of brain heart infusion (BHI) broth  and incubated in the BHI medium for 2 h at 37 °C with shaking at 200 rpm prior to infecting the macrophages. To ensure the bacteria are harvested in the log phase of growth, the OD600 of the culture post-incubation was not allowed to exceed 1.0 prior to incubation with antibodies. For in vivo challenge studies the fully virulent Y. pestis CO92 was used. Broth cultures were inoculated using growth from freshly inoculated tryptose blood agar base  slants (grown at 28–30 °C for approximately 48 h) which were suspended in Heart Infusion broth  + 0.2% Xylose  (HIBX) and incubated approximately 24 h at 28–30 °C and shaking at 150 rpm.The MD, USA) . Y. pesteficient ,64. Y. pThe anti-F1 mouse IgG1 monoclonal antibody (mAb) F1-04-A-G1 was provided by James Burans and Jennifer Aldrich  . The antbIGHM − / −, bIGHML1 − / −, bIGL − / −) and carry a human artificial chromosome (HAC) vector labeled as isKcHACD with an IgG1 production bias. This HAC vector consists of human chromosome 14 fragment and 2 fragment. The 14 fragment contains the entire human immunoglobulin heavy chain locus except that the IGHM constant region remains bovine, and the key regulatory sequences were bovinized. The 2 fragment contains the entire human immunoglobulin k light chain locus [Tc bovines were produced as previously described ,66. The in locus ,66.E. coli and purified, was provided by DynPort Vaccine Company  through the Joint Program Executive Office (JPEO) for Chemical and Biological Defense. Up to 2.1% of body weight of hyperimmune plasma per animal was collected from immunized Tc bovines on days 8, 11, and 14 after vaccination V3 throughV7. Plasma was collected using an Autopheresis C, Model 200, automated plasmapheresis system . Plasma samples were stored frozen at −20 °C until purifications were performed.Two Tc bovines were immunized with 2 mg recombinant F1-V (rF1-V), a fusion protein of the F1 capsular antigen and the virulence-associated LcrV gene product, formulated with SAB’s proprietary adjuvant formulation (SAB-adj-1) for the first vaccination (V1) and the second vaccination (V2) at a 3-week interval. The bovines were then boosted with 5 mg rF1-V formulated with SAB-adj-1 for third vaccination (V3) to seventh vaccination (V7) at 4-week intervals. Recombinant F1-V, produced with fermentation and expressed in SAB-183 (lot PD2001332PG) was purified from pooled Tc bovine plasma from V3 to V7 as previously described . Negativ2 in low glucose Dulbecco’s Modified Eagle Medium (DMEM) containing  1% L-glutamine, 1% non-essential amino acids, 1% HEPES buffer , and 10% fetal bovine serum . Cells were used before passage 15 and seeded in 96-well plates using an automated Multidrop Combi Reagent Dispenser .RAW264.7 murine macrophage-like cells derived from an Abelson murine leukemia virus tumor (ATCC TIB-71) were grown at 37 °C in 5% CO600 of 1.0 (~5.34 × 108 colony forming units (CFU) per milliliter). Depending upon the targeted protein, the in vitro assays were performed using bacteria grown under different temperatures to adequately characterize the antibody–bacteria interactions. Y. pestis requires at least 4 h of growth at 37 °C prior to infection to produce enough capsule to prevent phagocytosis [4 cell/well) were seeded into 96-well plates one day prior to infection. Y. pestis, at 8 × 106 CFU/mL was pre-incubated with 10 µg/mL or 100 µg/mL antibodies in DMEM for 1 h at 37 °C prior to infection. Macrophages were then infected at an MOI of approximately 10, in triplicate wells. The plates were centrifuged at 200× g for 5 min to initiate infection and then incubated at 37 °C with 5% CO2. After 1 h of infection, gentamicin (8 μg/mL)  was added to the wells to kill extracellular bacteria, and the plates were incubated for an additional hour at 37 °C with 5% CO2. After incubation macrophages were washed two times in PBS and lysed using 0.1% Triton X-100 in PBS. Serial dilutions of lysates were plated in duplicate on SBA plates and incubated for 2 days at 28 °C for CFU enumeration. Additional control wells were also infected with Y. pestis that was not pre-incubated with any antibody (Yp only).A schematic representation of this assay is provided in ocytosis , while aocytosis ,69. For Y. pestis CO92 (pneumonic plague model) were prepared as previously described [50 equivalents. Exposure of mice to aerosolized bacteria was accomplished as previously described [Y. pestis strain CO92 (encapsulated) created with a three-jet collision nebulizer. Samples were collected from the all-glass impinger (AGI) vessel and analyzed with CFU calculations to determine the inhaled dose of Y. pestis. The median lethal dose for Y. pestis CO92 in female BALB/c mice is approximately 6.8 × 104 inhaled CFUs [Aerosolized challenge doses of virulent escribed ,71. The escribed ,71. Brieled CFUs ,72.Y. pestis CO92 (TS CO92) and its non-encapsulated derivative strain Y. pestis C12 (TS C12) were diluted, as described above, but at a concentration of 10 µg/mL [Immunoglobulin (Ig) class IgG titers  from vaccinated bovines were determined with an ELISA performed in 96-well, Immulon 2 HB, round-bottom plates . Recombinant F1-V , F1 , and LcrV  were individually used as antigens diluted in 0.1 M carbonate buffer, pH 9.5, to a concentration of 2 µg/mL. Irradiated temperature-shifted 10 µg/mL ,64. Plat5 Y. pestis CO92 pgm-/pPst- (Yp pgm-/pPst-) cells were treated with anti-F1 or anti-LcrV antibodies as described above. The inoculum samples were centrifuged at 2800× g for 10 min and resuspended in 350 µL dPBS . The samples were then read on a FACSCanto II flow cytometer , gating on Y. pestis pgm-/pPst- untreated cells in the FSC vs. SSC dot plot and noting the aggregation and shift of subsequent antibody-treated Y. pestis pgm-/pPst- bacterial cells outside of that gate.Approximately 4 × 10pgm-/pPst- cells were resuspended in 1X dPBS, then diluted to 8 × 106 CFU/mL in DMEM and pre-incubated with 10 µg/mL primary antibody (F1-04-A-G1 or 7.3) for 1 h at 37 °C. After washing in FACS buffer , cells were resuspended in FACS buffer with secondary goat anti-mouse antibody  at 10 µg/mL. After 30 min at room temperature, cells were washed, resuspended in FACS buffer, and read on a FACSCanto II.For assessment of F1 and LcrV levels on bacterial surface, Yp 50 is estimated at selected time points with logistic regression. In addition, a predicted value at each day postexposure is given with accelerated failure time model. Any p values of ≤ 05 were considered significant. Analyses were performed in SAS version 9.4 .For ELISA, an exact, one-sided, two-sample Wilcoxon test was performed between treatments. No adjustment was applied for multiple comparisons. The comparison was made between the three technical replicates taken on each animal. For in vitro macrophage assays and gentamicin protection assay, results were compared using a Wilcoxon rank sum test, stratified by date of experiments. The survival rates at selected time points were compared using Fisher exact test. The log-rank test was used to compare mouse survival curves post challenge. The EDY. pestis challenge. Furthermore, in an effort to discern how the human polyclonal antibodies affect phagocytosis and/or growth of Y. pestis pgm-/pPst- within host macrophages, we first optimized the Y. pestis growth conditions to induce expression of selective temperature-dependent virulence factors. For instance, at low or ambient temperature (26–28 °C), similar to that in the flea vector, there is little to no expression of the F1 capsule protein [Y. pestis needs to be grown at 37 °C for >2 h before the anti-phagocytic activity of the capsule is appreciable [Y. pestis was initially grown at 28 °C or 37 °C for 24 h on SBA plates and then was sub-cultured in BHI medium and grown for an additional 2 h at 37 °C. Y. pestis grown at 28 °C for 24 h followed by 37 °C for 2 h (28–37 °C) expresses a very limited F1 capsule that would not likely obscure the LcrV antigen, a component of the T3SS, on the surface of the bacterial cell. In contrast, Y. pestis grown at 37 °C for 24 h followed by 37 °C for 2 h (37–37 °C) expresses a much more robust F1 capsule and also appreciable levels of the LcrV antigen. These bacterial cell descriptions based upon the previous literature were confirmed in our laboratory with Western blot and flow cytometric analyses , produced in Tc bovines immunized with the recombinant F1-V fusion protein vaccine, to the well-characterized mouse monoclonal (mAbs) anti-LcrV (7.3) and anti-F1 (F1-04-A-G1), which have previously been shown to be highly protective in mice against 3, produc protein ,20. Subsreciable . In ordeanalyses .Y. pestis grown at 28–37 °C or 37–37 °C with mouse monoclonal anti-F1 (F1-04-A-G1), anti-LcrV (7.3) mAbs, or with human polyclonal anti-rF1-V antibodies (SAB-183) derived from Tc bovines. Changes in the bacterial population, such as alterations in size or granularity, were assessed with flow cytometry. Incubation of Y. pestis grown at 28–37 °C with antibodies resulted in no overt change in bacteria relative to Y. pestis cells in the absence of antibodies (Yp only) or human polyclonal antibody from non-immunized Tc bovines (SAB Neg Ctrl) , the bacteria grown at 37 °C −37 °C and incubated with 100 μg/mL of anti-LcrV antibody 7.3 exhibited an appreciable alteration as detected using flow cytometry, but the degree of aggregation was clearly less than that observed when the bacteria were incubated with the anti-F1 mAb, and this observation is not consistent between iterations. Although no change in bacteria was observed in the presence of 10 µg/mL of polyclonal SAB-183 antibodies, there was a marked shift in the bacterial population in the presence of 100 µg/mL of polyclonal SAB-183 material  -Top. Aftmaterial -Bottom.Y. pestis 28–37 °C at 2 h post-infection. The level of bacterial internalization was more pronounced with Y. pestis pre-incubated with 100 μg/mL of mAb compared to Y. pestis preincubated with 10 μg/mL. The levels of phagocytosis enhancement were also similarly observed with SAB-183 polyclonal antibodies relative to the anti-LcrV mAb (7.3) (Mouse anti-LcrV mAb (7.3) enhanced initial opsonization and phagocytosis of Ab (7.3) .Y. pestis 37–37 °C at 2 h post-infection. Both capsule production and T3SS upregulation are induced at 37 °C, therefore enhancement of bacterial uptake is observed with both anti-F1 (F1-04-A-G1) and anti-LcrV (7.3). However, the increase in phagocytosis is substantially greater in the presence of the anti-F1 mAb relative to the anti-LcrV mAb, likely due to increased F1 production and overall epitope availability. The levels of phagocytosis enhancement also reached statistical significance post-incubation with SAB-183 relative to the Y. pestis only or the SAB Negative Control. (Mouse anti-F1 mAb (F1-04-A-G1) opsonized and enhanced phagocytosis of Control. .Y. pestis temperature-shifted CO92 (TS CO92) and irradiated whole-cell Y. pestis temperature-shifted non-encapsulated C12 (TS C12) strains.Previous studies identified that alterations in vaccine formulations, such as inclusion of various adjuvants, aside from impacting antibody titers can also change the epitope binding profile of the polyclonal antibody responses, with some epitopes conferring superior protection versus others on the same antigen ,74. In aY. pestis were grown at 28 °C and then temperature switched (TS) to 37 °C for approximately 3.5 h in order to upregulate the expression of the bacterial capsule and T3SS components. The anti-F1 and anti-LcrV antibody responses were significantly increased in the SAB-183 material relative to the SAB Negative Control (p < 0.05 vs SAB Negative Control). The antibody response was substantially greater against the LcrV antigen (8.3 × 104–2.4 × 105) relative to the F1 antigen (1.6 × 104–2.4 × 104) (p = 0.05 vs SAB Negative Control) was also elicited against Y. pestis TS CO92 and Y. pestis TS C12. The antibody response was higher against Y. pestis TS CO92 (4.2 × 103–8.8 × 103) relative to Y. pestis TS C12 (1.3 × 103–1.4 × 103) (During antigen preparation both strains of 4 × 104) . In addi4 × 103) , likely Y. pestis CO92. Mice received 0.5 mg, 1.0 mg, or 2.0 mg of IgG purified antibodies derived from Tc bovines vaccinated with the rF1-V vaccine. Mice were then estimated to have inhaled approximately 8.6 × 105 CFU of Y. pestis CO92 . While all mice that received 0.5 mg of this polyclonal antibody succumbed to infection, there was a statistically significant delay in time-to-death or euthanasia compared to mice receiving PBS alone (p = 0.0003) or mice receiving purified IgG derived from Tc bovines not vaccinated with rF1-V  (p = 0.033) or with the 2.0 mg treatment group (p < 0.001). Likewise, there was a significant increase in survival rate when comparing the 1.0 mg treatment group with the 2.0 mg treatment group (p = 0.033).Mice were treated with antibodies approximately 12 h pre-exposure to aerosolized  0.0001) . There w 0.0001) . Day 21 p < 0.001 in both comparisons and p = 0.012 when comparing 1.0 mg and 2.0 mg treatment groups) (n = 3) received either 0.1 mg of anti-LcrV mAb 7.3 or 0.2 mg of anti-F1 mAb F1-04-A-G1 and all positive control mice survived the infection.For the animals that did succumb to infection or that were euthanized in accordance with early endpoint euthanasia criteria the time-to-death was similarly significant in a dose-dependent manner. When comparing the 0.5 mg treatment group with the 1.0 mg and 2.0 mg treatment groups, these differences in time-to-death were statistically significant ( groups) . Mouse-d50) of the polyclonal material was calculated for days 5, 10, 15, and 21 postexposure to aerosolized Y. pestis  and anti-LcrV (7.3) mAbs that have previously been shown to be highly protective in vivo, and to use the antibodies as benchmarks that may later be used to rapidly screen potential novel antibody therapeutics directed against F1 or LcrV antigens. Second, to assess in vitro function and in vivo protective efficacy of a novel anti-plague human polyclonal antibody therapeutic (SAB-183) from genetically engineered cattle, which were vaccinated with the recombinant F1-V subunit vaccine, relative to the anti-F1 (F1-04-A-G1) and anti-LcrV (7.3) mAbs.Y. pestis was grown at 28 °C for 24 h followed by 37 °C for 2 h (28–37 °C). These growth conditions promote the induction of the T3SS and hence the LcrV antigen production but limit the formation of a robust F1 capsule [Y. pestis grown at 37 °C for 24 h followed by 37 °C for 2 h (37–37 °C), growth conditions that promote a more robust F1 and LcrV production (Y. pestis (37–37 °C) relative to Y. pestis (28–37 °C). This could be due to overall lower titers against the F1 protein compared to titers directed against LcrV (Mouse anti-LcrV (7.3) antibody enhanced bacterial uptake by RAW264.7 macrophages. This enhancement was more pronounced when  capsule ,68. The oduction . Since tnst LcrV . AdditioY. pestis (28–37 °C), due to a lack of a robust capsule along with other temperature-induced factors, is potentially more susceptible to nonspecific antibody binding as seen with much higher invasion in the presence of SAB Negative Control antibodies 2 h post-invasion. However, this enhancement attributed to the SAB negative control is less pronounced with Y. pestis (37–37 °C), suggesting the robust F1 capsule formed is obscuring possible cross-reactive antigens on the surface of the bacteria (Y. pestis (28–37 °C) that is likely attributable to cross-reactive antibodies or possibly endogenous antibodies against other species of Yersinia.It appears that bacteria . FurtherY. pestis. The studies reported here examined a single administration of antibodies approximately 12 h pre-exposure to aerosolized Y. pestis. In future studies it would be important to examine the effect of multiple doses of the antibodies, to determine if these antibodies could be used as postexposure prophylaxis or as a therapeutic, or if the efficacy can be improved with direct delivery to the lungs.Importantly, the human polyclonal antibodies derived from Tc bovines can protect mice from a substantial challenge with aerosolized Y. pestis have been isolated in several regions of Madagascar during relatively recent plague outbreaks and multi-drug resistant isolates continue to be of the utmost concern in both public health and biodefense arenas [Y. pestis infection and subsequent plague disease are urgently needed.Although a larger amount of this polyclonal antibody material is required to achieve complete protection in the BALB/c mouse model of pneumonic plague (relative to mouse-derived mAbs), even the lower dose (0.5 mg) did significantly increase time-to-death or euthanasia. Whereas mAbs required less material in this mouse model, neither the immunogenicity nor the half-life of mouse-derived mAbs versus fully human antibodies was addressed in this study. Furthermore, the concept of Tc bovines producing antibodies against emerging pathogens is important due to the rapidity of the production and the large amount of material that can be generated. Due to the heterogeneity amongst the LcrV proteins from different isolates ,75, the e arenas ,80. Thus


We have prepared novel potent breast cancer drug molecules from non-toxic and inexpensive method. Column chromatography is not necessary for purification of target molecules. The value of overall atom economy, environmental factor, environmental catalyst and product mass intensity gives additional merits for this synthetic method. Synthesized flexible dimeric imidazolium bromides showed less toxicity and gives excellent anticancer response against normal mammary epithelial cells. Novel dimeric pyridinium bromides showed excellent anticancer response against tested cancer cell lines. In cell cycle, novel flexible dimeric pyridinium bromides showed significant arrest in the G2/M phase by nearly three folds, when compared with control drug. We have studied the targeting epidermal growth factor receptor for all the synthesized flexible amino substituted and methyl substituted dimeric pyridinium bromides. Organic cation like imidazolium, pyridinium and pyrazolium ion with simple inorganic anions are mostly used in pharma industries due to its unique physiochemical behaviors3. The quarternization of sp2 nitrogen of pyridine and imidazole nucleus is the important method in the synthesis of ionic liquids10. Pyridine derivatives are very important and useful moieties to reach wide popularity in the Bronsted acidic ionic liquids to prepare the drug molecules18. Binary mixture are prepared from 1-butuyl-3-methyl imidazolium cation with acetate anion and Doxorubicin or Mitoxantrone and its cytotoxicity is studied against human colorectal adenocarcinoma CaCo-2 cells and showed higher cytotoxicity than the Colo 320 HSRcells20. Monomeric imidazolium cation based ionic liquids showed excellent anti-tumor activities21. Alkyl chain length from C2–C8 substituted imidazolium bromide/hydroxide type of ionic liquids showed moderate to significant vermicidal response22. Water soluble betulinic acid counter anion with trihexyl tetradecyl phosphonium cation type of ionic liquids are acted as selective antitumor agent against Hep G2 (liver), MG63 (Osteosarcoma), T47 D (breast), A459(lung) and RKO (colon) cancer cells23. Electron donating substituted benzofused phosphorlactones showed potential anti-pancreatic cancer cell which was identified by cell cycle experiment24. Ammonium and phosphonium cation with simple and alkyl substituted anion showed better cytotoxicity response against leukemia cell lines. Higher alkyl chain containing phosphonium type of ionic liquids showed excellent anti-cancer activity against breast cancer and ovarian cancer cells with GI50 values25. Saeid et al. reported that, the anti-cancer properties of lipid nano encapsulated and starch nano-encapsulated form of the Samarium complex was studied against cell lines of A-549 and MCF-7 by MTT method. Lipid and starch nano-encapsulated molecules showed higher antitumor and antimicrobial response than the Samarium complex alone26. Recent researchers are focusing their finding towards the preparation and identification the non-toxic anti-cancer drug molecule which is not yet available32. We wish to prepare cheaper and water soluble anticancer drug molecules with lipophilic and hydrophilic moieties from substituted pyridinium salts under shortest synthetic methodology; we hope that, will get good response in the study of anti-cancer properties of substituted pyridinium salts against breast cancer cell line under MTT assay.Organic cation binds with inorganic anion (or) Inorganic cation binds with organic anion (or) both cation and anion from organic moieties which are in liquid nature at room temperature are called room temperature ionic liquids (RTIL). In the structure description of ionic liquids, it is an electrically neutral molten salt consisting of equal number of cation and anion in the liquid form. RTIL shows, unique thermo physical characters such as low melting salt, highly viscous in nature, thermally more stable and have greater electrochemical properties1–4 from easily available and cheapest starting materials under simple synthetic procedure. 1,5-Dibromopentane/m-xylene dibromide and 2.02 equivalents of 4-methlpyridine/2-amino-3-methylpyridine are dissolved in CH3CN followed by refluxtion for about2-3 h afforded the quarternized flexible dimeric pyridinium bromide 1–4 in 92–95% yield  Atom economy of 1, 5-dibromo pentanereacts with 4-methyl pyridine is defined as how much reactant are converted into dimeric pyridinium bromide 1.The atom economy is calculated for the following reaction Fig. .\documenEnvironmental factor (EF) The environmental factor is calculated for the following reaction by ratio of mass value of unwanted (or) wasted materials/Mass value of product molecule.Mass of waste is calculated as follow Fig. :\documenSo,Product mass intensity (PMI) The ratio between total mass of (reactant + solvent) and mass of expected product.We have calculated product mass intensity of the above reaction by using following formula Fig. \documentSimilarly, we have calculated the atom economy, environmental factors and product mass intensity of remaining reactions and presented in the Table 33. Therefore, it is very important to evaluate the anticancer activity of newly discovered compounds against various cancer cells.Ionic liquids have rarely or never been tested for anticancer studies and our previous ionic liquid compounds showed potent anticancer activity50 values are greater than 20 for MCF-7 and MDA-MB-231 breast cancer cell lines, IC50 values greater than 30 for MCF-10A breast cancer cell lines in 24 h incubation period with reference to doxorubicin which is used as a reference drug molecules34. IC50 values for Phyllanthus niuri DCM extract against MCF-7 and MCF-7 ADR breast cancer cell lines showed 100 in 24 h incubation period35. Sumit et al. prepared from triazole tethered Ospemifene-isatin conjugated drug molecules using expensive catalyst for McMurry coupling followed by additional four steps to get their triazole tethered Ospemifene-isatin conjugated drug molecules. IC50 values of different alkyl chain length containing triazole tethered ospemifene-isatin derivatives are 53.10 and 70.71 for two and three methylene linkers respectively36. Dibiotin ester linked Ruthenium complex and cis-platin showed IC50 values of 31.5 ± 4.7, 38 ± 1.41 against MCF 7 breast cancer cell line in 24 h incubation37. Mina and coworker reported the breast cancer effect of Metformin drug molecules on T47D breast cancer cell lines. IC50 value for Metformin and Silibinin drug molecules are 21.20, 106.50 respectively in 24 h incubation period against T47D breast cancer cell lines38.IC1–4) was tested against three cancer cell lines namely human breast adenocarcinoma (MCF-7), (MDA-MB-231), and ductal carcinoma (T47D), and one human mammary epithelial (MCF-10A) cell line by MTT assay. The investigated anticancer activity of the compounds was time-dependent, i.e., cell viability decreased with increasing incubation, and their IC50 values are listed in Table 50 values of the compounds showed less toxicity against normal human mammary epithelial cells and more toxicity against cancer cells. All the compounds have efficiently killed the MDA-MB-231 cancer cell line compared to other tested cells  and ethidium bromide (EB) after 24 h. Morphological assessment demonstrates viable cells (control) with normal morphology and pale green nuclei. However, the MDA-MB-231 cells treated with compounds for 24 h confirmed morphological changes as a greater part of cells become shrinkage, and green apoptotic cells containing apoptotic bodies and red necrotic cells were observed , on MDA-MB-231 cell apoptosis and necrosis. MDA-MB-231 cells were exposed to the compounds for 48 h. Following treatment, cell nuclei were stained with annexin V-FITC and propidium iodide (PI) and analyzed through flow cytometry. The findings revealed significant alterations in the apoptotic and necrotic cell populations. In the absence of treatment, only 0.09% of the cells exhibited late apoptotic characteristics, as indicated by their placement in quadrant Q2. However, when exposed to 25 μM of compounds 1 and 2, the late apoptotic cell population increased to 29.3% and 21.8%, respectively  in inducing early apoptosis. Interestingly, compound 2 also induced necrotic cell death relative to the control , 0.18% of the control cells displayed such features. This proportion significantly increased to 19.1% and 21.8% for compounds 1 and 2, respectively, post-treatment 33. Applications of the Swiss ADME web tool has recently contributed greatly to the design and development of anticancer, tubercular, and antimicrobial agents41. The bioavailability radar demonstrated six physicochemical indices such as lipophilicity (XLOGP3), size, polarity, solubility, saturation, and flexibility  and this confirms that these compounds easily cross the BBB. Furthermore, the skin permeability coefficient (log Kp) was calculated, which appeared to be related to lipophilicity and molecular size. A very negative value of the log Kp estimated for all compounds (− 5.26 to − 6.12 cm/s) indicates low skin penetration.Swiss drug design tools were used to predict the bioavailability of compounds (ty Table . The bio1–4) follow Lipinski's five rules, as a result of which these compounds are expected to have excellent drug-like properties. The fact that these new compounds have molecular weights below 500 indicates that these compounds are easier to transport, diffuse and absorb than larger molecules. Compounds 1 and 2 show no hydrogen bond acceptors (nHA) whereas 3 and 4 show two hydrogen bond acceptors and none of the donor (nHD) atoms in these compounds, which are less than 10 and 5, respectively  molecular weight ≤ 500 Da, (ii) LogP < 5, (iii) nHBD ≤ 5, (iv) nHBA ≤ 10, (v) TPSA < 140 Å2, which leads to adherence to criteria for oral drugs.The number of rotatable bonds in these compounds ranges from 4 to 6 which is within the range. The TPSA values of compounds 42. It plays an important role in cell proliferation and is often targeted by anticancer drugs43. EGFR has been especially studied in breast cancer, as it is an important target for the development of new treatment44. Research has shown that blocking EGFR can lead to decreased tumor growth and improved survival rates45. Many current therapies are designed to specifically target EGFR, making it a key focus for cancer research. Targeting EGFR is important for this study as dimeric pyridinium bromides show potent activity against breast cancer cells.Epidermal growth factor receptor (EGFR) is a protein found on the surface of cells1 showed a carbon–hydrogen bond (C–H) interaction formed between the carbon atom of the CH2 moiety and ASP831 with a distance of 3.05 Å  interaction formed between the carbon atom of the CH2 moiety and THR766 with a distance of 2.90 Å )bis(4-methylpyridinum)bromide 0 Å Fig. . Also, s0 Å Fig. . 1,1′bis(2-amino-3-methylpyridin-1-ium)bromide 4 Å Fig. . 1,1′)bis(-2-amino-3-methylpyridin)bromide 8 Å Fig. . Also, s8 Å Fig. . 1,1' and 4-methyl pyridine/2-amino-3-methylpyridine(2.02 equi.) were dissolved in 80 mL of dry acetonitrile under refluxing condition for 2 h afforded the desired compound1H NMR  δ(ppm): 1.44–1.51; 2.10–2.18 ; 2.70 ; 4.62–4.65 ; 7.95–7.96 ; 8.75–8.76 ; 13CNMR δ (ppm): 20.1, 20.6, 28.4, 59.0, 127.3, 141.7, 158.5, HRMS: m/z 128.1000; Anal. Calculated for C17H24N2Br2: C, 49.06; H, 5.76; N, 6.73; Found: C, 49.02; H, 5.72; N, 6.69  δ (ppm): 1.37–1.43 ; 1.77–1.84 ; 2.0 ; 4.12–4.15 ; 6.76–6.80 ; 7.63–7.65; ; 7.71–7.72 ; 13C NMR  δ (ppm):16.9, 22.2, 26.4, 54.1, 113.6, 123.7, 137. 4, 141.7, 152.5, HRMS: m/z143.1150; Anal. Calculated for C17H26N4Br2: C, 45.73; H, 5.82; N, 12.55; Found: C, 45.69; H, 5.78; N, 12.51  and 4-methyl pyridine/2-amino-3-methylpyridine(2.02 equi.) were dissolved in 80 mL of dry acetonitrile at room temperature for 2 h afforded the desired compound 2, 4.1H NMR  δ (ppm): 2.55 ; 5.68 ; 7.78–7.80 ; 7.45–7.44 ; 7.25–7.30 ; 7.15–7.18 ; 8.61–8.62 ; 7.80–7.77 ; 13C NMR  δ (ppm): 29.4, 63.0, 143.2, 134.3, 130.6, 130.0, 129.6, 129.1, 128.7, HRMS: m/z145.1100; Anal. Calculated for C20H22N2Br2: C, 53.33; H, 4.88; N, 6.22; Found: C, 53.29; H, 4.84; N, 6.18  δ (ppm): 4.70 ; 5.42 ; 6.17 ; 6.84–6.87 ; 7.27–7.29 ; 7.44–7.48 ; 7.74–7.76 ; 7.70–7.72 ; 13C NMR  δ (ppm): 16.6, 56.4, 113.8, 122.4, 124.1, 127.3, 130.1, 133.2, 137.6, 142.3, 153.1, HRMS: m/z 160.1200; Anal. Calculated for C20H24N4Br2: C, 50.02; H, 5.04; N, 11.66; Found: C, 49.98; H, 4.99; N, 11.63 , MDA-MB-231 (ATCC: CRM-HTB-26™), T-47D (ATCC: HTB-133™), and MCF 10A (ATCC: CRL-10317™) were purchased from the American type culture collection .3 cells are seeded in a 96-well plate and incubated for growth under the conditions mentioned above. Once confluency is reached, the cells are treated with different concentrations of simple/amino substituted dimeric pyridinium bromides 1–4 and incubated for 24 h. After incubation, the media is removed, and 20 μL of MTT is added and incubated for further 3 h. After 3 h, MTT is removed, and DMSO is added to dissolve the formazan crystals. The absorbance is measured at 570 nm, and the percentage of cell death is calculated using the formula5 × 10This article does not contain any studies with human participants or animals performed by the authors.We comply with the ethical standards. We provide our consent to take part.Supplementary Figures.


Stringent cleaning procedures during spacecraft assembly are critical to maintaining the integrity of life-detection missions. To ensure cleanliness, NASA spacecraft are assembled in cleanroom facilities, where floors are routinely cleansed with Kleenol 30 (K30), an alkaline detergent.Through metabolomic and cultivation approaches, we show that cultures of spacecraft-associated Acinetobacter tolerate up to 1% v/v K30 and are fully inhibited at ≥2%; in comparison, NASA cleanrooms are cleansed with ~0.8-1.6% K30.For A. johnsonii 2P08AA (isolated from a cleanroom floor), cultivations with 0.1% v/v K30 yield (1) no changes in cell density at late-log phase, (2) modest decreases in growth rate (~17%), (3) negligible lag phase times, (4) limited changes in the intracellular metabolome, and (5) increases in extracellular sugar acids, monosaccharides, organic acids, and fatty acids. For A. radioresistens 50v1 (isolated from a spacecraft surface), cultivations yield (1) ~50% survivals, (2) no changes in growth rate, (3) ~70% decreases in the lag phase time, (4) differential changes in intracellular amino acids, compatible solutes, nucleotide-related metabolites, dicarboxylic acids, and saturated fatty acids, and (5) substantial yet differential impacts to extracellular sugar acids, monosaccharides, and organic acids.These combined results suggest that (1) K30 manifests strain-dependent impacts on the intracellular metabolomes, cultivation kinetics, and survivals, (2) K30 influences extracellular trace element acquisition in both strains, and (3) K30 is better tolerated by the floor-associated strain. Hence, this work lends support towards the hypothesis that repeated cleansing during spacecraft assembly serve as selective pressures that promote tolerances towards the cleaning conditions. While dFor NASA cleanrooms (ISO class 8) where Mars and Europa spacecraft are assembled, the floors are routinely cleansed with ~0.8-1.6% v/v Kleenol 30 , which i3–104 16S rRNA m−2 (intact cells), ~104–105 16S rRNA m−2 , 1–40 OTU m−2, ~102–104 colony forming units m−2, and ~ 101 spores m−2  via scission of the ether linkage  which were subjected to differing cleaning regimes . Hence, comparisons across these sub-environments lend support toward the hypothesis that repeated cleansing under the conditions of spacecraft assembly serve as selective pressures that favor or promote biochemical tolerances toward the local cleaning conditions.Therefore, to obtain molecular and quantitative insights into the tolerances of the cleanroom-associated 2.2.1.A. johnsonii 2P08AA and A. radioresistens 50v1 were obtained through the Planetary Protection Culture Collection at the Jet Propulsion Laboratory. Stocks of ethanol (ENG Scientific) and Kleenol 30  were sterile filtered and stored as 200 μL aliquots at 4°C. Stock solutions of 25 mM Fe2+ were prepared using ferrous ammonium sulfate (Fe(NH4)2(SO4)2·6H2O; EM Science) in ultrapure water (18 MΩ−1), followed by sterile filtration , and storage as 200 μL aliquots at 4°C (with no visible precipitation over long term storage).Bacterial strains of 2HPO4·7H2O (Sigma), 15.0 g KH2PO4 (EM Science), 2.50 g NaCl (Fisher Scientific), 5.00 g NH4Cl (EM Science) per liter of ultrapure water. Low-osmolarity media (0.2x M9) were prepared by adding 0.4929 g MgSO4·7H2O (EM Science) and 0.0147 g CaCl2·2H2O (EM Science) to 200.0 mL 5x MM and dilution to 1.00 L using ultrapure water (to yield 1x M9), followed by an additional 5-fold dilution (200 mL 1x M9 in 1.00 L) to yield 0.2x M9. Final concentrations for ions in 0.2x M9 were 20 μM Ca2+, 400 μM Mg2+, 4.4 mM K+, 10.7 mM Na+, and 3.7 mM NH4+, along with 5.4 mM Cl−, 4.5 mM H2PO4−, 4.4 mM HPO42−, and 0.4 mM SO42−.Concentrated minimal media (5x MM) were prepared using 30.0 g NaLysogeny broth media (LB) were prepared using 10.0 g tryptone (VWR), 5.0 g yeast extract (Amresco), 5.0 g NaCl (Fisher Scientific) and 1 mL of 1 M NaOH (Sigma-Aldrich) per liter of ultrapure water. Agar plates were prepared using 1.0 L LB and 15 g agar (Amresco).Cultivations were performed in 15 mL screw cap test tubes , which were washed with tap water, rinsed with distilled water, and autoclaved between experiments. All screw cap test tubes were capped tightly and wrapped with parafilm during cultivation to prevent loss of ethanol, which served as a sole supplied carbon source. Cultivations were performed with mild agitation (200 rpm) using a New Brunswick Scientific Innova 4,200 incubator. Cell densities were monitored during cultivation by following agar plate counts and changes in optical density (OD) at 600 nm (Spectronic 20 Genesys). All media were autoclaved at 121°C and 15 psi for 45 min.2.2.A. radioresistens 50v1 and A. johnsonii 2P08AA were separately streaked onto LB agar plates and incubated at 32°C for ~24 h. Isolated colonies were inoculated into 2.0 mL 0.2x M9 containing 25 μM Fe2+ and 0–2.0% v/v Kleenol 30 (or 0–40.0 μL). Pre-cultures were initiated by addition of 20.0 μL 90% v/v ethanol to yield final concentrations of 150 mM or 1.0% v/v ethanol. Pre-cultures were grown to late log phase (OD ~0.6 at ~12 h). Target cultures were prepared using fresh media (2.00 mL) which were inoculated with 20.0 μL (1:100 dilution) of the respective pre-cultures and initiated by addition of 1.0% v/v ethanol.Glycerol stocks of k), lag time (L), and an estimate of the maximum change in relative biomass  using a modified version of the Gompertz equation , which d6-fold using 0.2x M9, and spread (20 μL) onto LB agar plates using sterile plastic cell spreaders. All plates were sealed with parafilm and incubated at 32°C for 24 h. Plates with ≤300 colonies were enumerated and expressed as colony forming using per mL of the parent culture (cfu mL−1).Survivals of late-log phase cultures (OD ~0.4) were assessed by plating onto LB agar plates. In control experiments, plate counts using 0.2x M9 agar plates supplemented with ethanol (just prior to use) yielded irreproducible results, when compared to LB agar plate, likely due to variances in adsorption of ethanol (under our conditions). For the plate count assays, therefore, aliquots (100 μL) of the cultures (in 0.2x M9/Fe) were transferred to 2.5 mL microcentrifuge tubes, decimally diluted by 102.3.A. radioresistens 50v1 and A. johnsonii 2P08AA were prepared as described and harvested during early stationary phase  by centrifugation  at 4°C for 15 min (Beckman Coulter Allegra 21R). After centrifugation, the cell pellets and supernatant fractions were separated and, respectively, treated. The supernatants were saved as 500 μL aliquots, dried using a DNA 110 Savant DNA SpeedVac, and stored at −80°C. The pelleted cells were washed twice with 0.2x M9 and stored at −80°C.Cultures of Samples were characterized by the West Coast Metabolomics Center using gas chromatography and time-of-flight mass spectrometry (GC-TOF/MS). In brief, dried cells and culture broth were separately resuspended in 2 mL of pre-chilled (−20°C) and degassed extraction solvent , vortexed for 30 s, shaken for 5 min at 4°C, clarified by centrifugation , and the resulting supernatant evaporated to dryness. Samples were derivatized by resuspension in 10 μL of 40 mg/mL methoxyamine hydrochloride in pyridine  followed by addition of 41 μL N-methyl-N-(trimethylsilyl) trifluoroacetamide ; fatty acid methyl esters  were additionally added to serve as retention index markers .Samples were transferred to crimp top vials and separated on an Agilent 6,890 Gas Chromatograph equipped with a Gerstel automatic liner exchange system (ALEX), multipurpose sample (MPS2) dual rail, Gerstel CIS cold injection system , and built-in gas purifier . Chromatographic separation was afforded using a 30 m (0.25 mm i.d.) Rtx-5Sil MS column (0.25 μm 95% dimethyl 5% diphenyl polysiloxane film) with an additional 10 m integrated guard column . Carrier gas was 99.9999% pure Helium with a constant flow of 1 mL/min.−1 injection speed on a spitless injector with a purge time of 25 s. Temperature profile included 1 min at 50°C for the oven temperature, an increase of 20°C min−1 to 330°C across 14 min, with a final hold for 5 min at 330°C. A temperature of 280°C was used for the transfer line between the gas chromatograph and Leco Pegasus IV time of flight mass spectrometer . The measured mass range was 85–500 Da, scan rate was at 17 spectra s−1, electron energy was 70 eV, ion source temperature was 250°C, and detector voltage was 1850 V. Spectra were acquired using the Leco ChromaTOF software vs. 2.32  and processed using the BinBase database system , where statistical relevance was accepted at t-tests (Microsoft Excel) with corrections for multiple testing using a false discovery rate (FDR) of FDR ≤ 0.20 (n = 3), metabolites exhibiting thresholds for normality of p > 0.03 (Shapiro–Wilk Tests) and significance of p < 0.05 (Student’s t-tests) were carried forward in the univariate and visual assessments of the data.Discrete changes in the metabolomes  were identified using unpaired Student’s R ≤ 0.20 . For thep < 0.05) using MetaMappBroad structural and metabolic trends were obtained by visualizing the changes in the metabolomes  under low-osmolarity aqueous conditions with ethanol serving as the sole supplied carbon source. Cultivations on 2% v/v K30 did not yield measurable cell densities.In A. johnsonii 2P08AA show no apparent loss in survival, as plate counts (~late log phase) effectively show no difference (p > 0.05) in the presence (2.1 ± 0.5 × 107 cfu mL−1) and absence (2.6 ± 0.1 × 107 cfu mL−1) of the detergent. These trends indicate quantitative survival of the 2P08AA strain in the presence of 0.1% v/v K30.In 0.1% v/v K30, cultures of the floor-associated A. radioresistens 50v1, in contrast, survivability in 0.1% v/v K30 readily decreases (p = 0.006) to 46 ± 6% (propagated error) – as calculated by comparison of cultures cultivated in the presence (4.0 ± 0.4 × 107 cfu mL−1) and absence (8.8 ± 0.7 × 107 cfu mL−1) of 0.1% v/v K30. These trends are indicative of ~50% survival in the presence of 0.1% v/v K30.For the spacecraft surface-associated 7 cfu mL−1; 50v1, 1.9 ± 1.1 × 107 cfu mL−1) and absence  of 1.0% v/v K30. These trends are indicative of ~70–80% inhibition in the presence of 1.0% v/v K30 across both strains.In 1.0% v/v K30, survivabilities for the 2P08AA strain (26 ± 12%) and 50v1 strain (22 ± 8%) decrease to similar values – as indicated by comparison of cultures cultivated in the presence , estimated time in lag phase , and log of the maximum increase in relative biomass .To account for optical scattering, the maximum increases in relative biomass (k) show ~18% decreases for cultivations of the 2P08AA strain in 0.1% v/v K30 (p = 0.029) – as calculated by comparison of the difference in growth rates (−1) and presence (0.41 ± 0.02 h−1) of 0.1% v/v K30. The 50v1 strain, in contrast, shows no change in growth rate (p = 0.05) in 0.1% v/v K30 – as calculated by comparison of rates (−1) and presence (0.40 ± 0.02 h−1) of 0.1% v/v K30. When cultivated in 1.0% v/v K30, growth rates for the 2P08AA strain (0.22 ± 0.03 h−1) and 50v1 strain (0.35 ± 0.01 h−1) decrease by ~56% (p = 0.001) and ~ 13% (p = 0.043), respectively, as calculated by comparison rates  of the detergent.Comparisons of the growth rates  and 50v1  strains. Additionally, two factor ANOVA analyses confirm that cultivations in the presence of K30 differentially impact the growth rates of the 2P08AA and 50v1 strains . These combined trends for exponential phase behavior are indicative of higher tolerances toward K30 by the 50v1 strain at 0.1 and 1.0% K30. These trends are opposite to those from plate counts at late-log phase, which indicate a higher tolerance for the 2P08AA strain.Comparisons by one factor ANOVA analyses confirm that cultivations in the presence of 0.1–1.0% v/v K30 significantly impact the respective growth rates for the 2P08AA (L) in 0.1 and 1.0% v/v K30 show decreases for both strains during cultivation (−1 (absence of K30) reduces to negligible values (~0 h) in 0.1 and 1.0% v/v K30. For the 50v1 strain, the native lag time of 2.61 ± 0.24 h−1 is ~3-fold longer than 2P08AA strain and decreases by ~70% in 0.1% v/v K30 (0.76 ± 0.15 h−1) and to negligible values in 1.0% v/v K30.Comparisons of the lag times  and 50v1  strains. Additionally, two factor ANOVA analyses confirm that cultivations in the presence of K30 differentially impact the lag times for the 2P08AA and 50v1 strains . These combined trends indicate that K30 induces accelerated entry into the exponential phase in a concentration dependent manner, where the lag phase for the 2P08AA strain is effectively eliminated at ~10-fold lower K30 abundances when compared to the 50v1 strain – under these conditions.Comparisons by one factor ANOVA analyses confirm that cultivations in the presence of 0.1–1.0% v/v K30 significantly impact the respective lag times for the 2P08AA (p = 0.026) in maximum apparent biomass when comparing values obtained in absence (2.30 ± 0.02) and presence of (2.44 ± 0.04) of 0.1% v/v K30 and accounting for the log transformation. Similarly, for the 50v1 strain, cultivations in 0.1% v/v K30 yield ~55% increases (p = 0.048) in maximum apparent biomass when comparing values obtained in absence (2.28 ± 0.01) and presence of (2.47 ± 0.04) of 0.1% v/v K30 and accounting for the log transformation. When cultivated in 1.0% v/v K30, the 2P08AA strain (1.53 ± 0.18) and 50v1 strain (2.07 ± 0.03) exhibit decreases of ~80% and ~ 40% in maximum apparent biomass, respectively, which is consistent with the decreased growth rates under these conditions .Comparisons of the maximum change in relative biomass  and 50v1 strains . Additionally, two factor ANOVA analyses confirm that cultivations in the presence of K30 differentially impact the maximum changes in relative biomass for the 2P08AA and 50v1 strains . Combined, these trends suggest that 0.1% v/v K30 induces changes in apparent biomass in stationary phase.Comparisons by one factor ANOVA analyses confirm that cultivations in the presence of 0.1–1.0% v/v K30 significantly impact the respective maximum changes in relative biomass for the 2P08AA (3.3.3.3.1.A. johnsonii 2P08AA and A. radioresistens 50v1 were compared after cultivation in the absence and presence of 0.1% v/v Kleenol 30 (early stationary phase). Per strain, whole cell extracts yielded a total of 119 intracellular metabolites and free metabolites from the membrane . Metabolites from both strains showing important changes (p < 0.05) due to cultivation with K30 are listed in p < 0.05, FDR ≤ 0.20) after correction for multiple testing are underlined.Metabolomes from whole cells of via networks of KEGG reactant pairs (black edges or arrows) and Tanimoto chemical similarity scores (gray edges or arrows). Detected metabolites are presented as nodes (circles). Important changes are highlighted as red nodes (increases in relative abundance), blue nodes (decreases in relative abundance), or yellow nodes (no change). Node sizes signify the relative degree of change (or fold-change).Displayed in n = 119). No changes are observed when parsing the data using ChemRich. While potential increases (p < 0.05) are observed in gluconate, analyses in ChemRich provide no support for broad changes in sugar acid content. Despite the potential (p < 0.05) decreases in arabinose and increases in sorbitol, analyses in ChemRich show no support for broad changes in monosaccharide composition. Similarly, ChemRich analyses provide no support for broad changes in lipid-related metabolites despite the potential (p < 0.05) decreases in phosphoethanolamine and increases in ethanolamine and behenic acid .For the 2P08AA strain , cultivamultiple changes in the intracellular metabolome due to cultivation in 0.1% v/v K30  are supported by decreases in the relative abundances of standard amino acids , non-standard amino acids , and a peptide . Dimerization of glycine under the analytical conditions is suggested by detection of 2,5-dihydroxypyrazine  are supported by decreases in mannitol  are supported by decreases in 5’-AMP, MTA, pyrophosphate, thymidine, and uracil, along with increases in cytidine-5′-monophosphate (5’-CMP). Impacts to dicarboxylic acids  are supported by increases in fumarate, malate, and tartrate. No impacts to saturated fatty acids are observed  despite the decreases in isopentadecanoic acid  and increases in stearic acid .Impacts to compatible solutes  is supported by decreases in intracellular glycerate, 3-phosphoglycerate, serine, glycine, and homoserine. Inhibition or reduction in Lysine Biosynthesis (map00300) and Valine, Leucine, and Isoleucine Biosynthesis (map00290) is supported by decreases in lysine and valine; however, no other changes are noted in the respective pathways.Inhibition or reduction in proline and arginine biosynthesis through Arginine and Proline Metabolism (map00330) and Arginine Biosynthesis (map00220) are supported by decreases in ornithine, citrulline, and proline. The lack of changes in abundances for urea indicate no measurable impact on the flux of the urea cycle – despite the decreases in ornithine and citrulline. The lack of observed arginine is suggestive of conversion of arginine to ornithine under the analytical conditions  or the lInhibition or reduction in Peptidoglycan Biosynthesis (map00550) and/or peptide (protein) synthesis is suggested by decreases in alanine–alanine. Inhibition or reduction in β-alanine metabolism (map00410) is supported by decreases in β-alanine, which in turn is suggestive of reductions in arginine and uracil degradation. Differential changes in fatty acid metabolism (map01212) are suggested by the decreases in free isopentadecanoic acid (Reaction R02663) and increases in free stearic acid.Inhibition or altered flux through glycolysis (map00010) is suggested through decreases in 3-phosphoglycerate. Activation or altered flux through components of the TCA cycle are suggested by increases in fumarate and malate. Inhibition or reduced flux of selected monosaccharides through Fructose and Mannose Metabolism (map00051) and Pentose and Glucuronate Interconversions (map00040) are, respectively, suggested by decreases in mannitol and arabinose.Differential changes in Nucleotide Metabolism (map01232) are suggested by increases in 5’-CMP and 5′-MTA, along with decreases in 5’-AMP, pyrophosphate, thymidine, and uracil. Additionally, inhibition or reduction in Pyrimidine Metabolism (map00240) is suggested decreases by uracil and thymidine.3.4.A. johnsonii 2P08AA and A. radioresistens 50v1 (early stationary phase). Per strain, dried cultivation broth (cell free) yielded a total of 56 metabolites, which were processed and visualized as described above. Listed in Displayed in p = 1.4×10−5; Q = 6.9×10−5; ChemRich) are supported by substantial increases in extracellular gluconolactone (22-fold) and glycerate (34-fold). Impacts to di- and monocarboxylic acids, which were grouped as organic acids , are supported by increases in oxalate and decreases in adipic acid (hexanedioic acid) and 3,4-dihydroxybenzoate (protocatechuate). Impacts to extracellular monosaccharides  are supported by substantial increases in extracellular fructose (4.5-fold), galactose (22-fold), and mannose (57-fold) and decrease (2.5-fold) in extracellular ribose (pentose).When grouped by biochemical classes, cultivations of the 2P08AA strain in K30 yield differential impacts to extracellular sugar acids, monosaccharides, and organic acids (di- and monocarboxylic acids). Yet, univariate tests show no changes after correction for multiple testing. Impacts to sugar acids (n = 56) – yield significant changes in the extracellular galactonate, gluconate, gluconolactone, glycerate, mannose, ribose, tartrate, 4-hydroxybutyrate, and α-ketoglutarate. Likewise, grouping by biochemical classes are reveals significant changes in the extracellular metabolome.In contrast, cultivations of the 50v1 strain in K30 – after correction for multiple testing  are supported by substantial increases in extracellular galactonate (36-fold), gluconate (46-fold), gluconolactone (8.8-fold), glycerate (9.3-fold), and 2-methylglycerate (1.9-fold). Impacts to extracellular organic acids  are suggested by the significant increases in tartrate and significant decreases in α-ketoglutatate, along with the potential decreases in citramalate and 4-hydroxybutyrate. Impacts to extracellular monosaccharides  are suggested by significant increases in extracellular mannose (20-fold) and significant decreases in ribose (2.5-fold).Impacts to sugar acids (3.5.3.5.1.A. johnsonii 2P08AA (n = 3) and A. radioresistens 50v1 (n = 3) to differing quantitative descriptors characterizing changes in the cultivation media and growth kinetics in the absence and presence of 0.1% v/v K30. Correlations for the intracellular (or whole cell) metabolomes from late log-phase cultures were assessed against growth rates at exponential phase, plate counts at late-log phase, maximum relative biomass at stationary phase, and total detergent concentrations in the media, which were assumed to be 0.025% w/w in cultures containing 0.1% v/v K30.In The CCA plots include the total array of detected metabolites (text), listed quantitative descriptors (vector lines), and transformed data after dimension reduction (glyphs). For the 2P08AA and 50v1 strains, stronger correlations along CCA Axis 1  are observed when compared to CCA Axis 2 , respectively. Dimension reduction for the metabolomes from the 2P08AA and 50v1 strains ,B respec3.5.2.p > 0.05) or metabolite abundances for the 2P08AA strain in 0.1% v/v K30 (FDR > 0.20).When considering changes in survival , the vecp > 0.05 or FDR > 0.20). These trends suggest that the quantitative survival for the 2P08AA strain (in part) relates to lack of changes in central metabolism and compatible solute formation.From the CCA plot for the 2P08AA strain , metabolp < 0.05) and substantial changes in the metabolome .For the 50v1 strain , the vecFrom the CCA plot for the 50v1 strain , metabolp > 0.05) for the apparent decreases in phenylacetic acid (~0.1-fold decreases) and 4-hydroxybenzoate (~0.9-fold decreases). Alternatively, described further in Section 3.5.3, phenylacetic acid and 4-hydroxybenzoate are potential biodegraded products of K30. Hence, trends for the 50v1 strain hint at a potential for biodegradation of K30 through benzene metabolism.Trends from the CCA plot for the 50v1 strain  are addi3.5.3.p < 0.05), yet counter to the associated univariate tests for the metabolomic data that indicate no change in the relative metabolite abundances after correction for multiple testing (FDR > 0.20).When considering biomass, the vector describing the maximum relative biomass at stationary phase for the 2P08AA strain  overlapsFrom the CCA plot for the 2P08AA strain , the metp < 0.05) and metabolomic data , which yield support for increases in the maximum relative biomass and differential changes in metabolite abundances. In contrast to the 2P08AA strain, these trends for 50v1 strain suggest that increases in the maximum relative biomass are associated with several intracellular metabolomic changes.For the 50v1 strain , the vecΔ9). These trends suggest that increases in the maximum relative biomass for the 50v1 strain are related (at the minimum) to changes in butanoate metabolism , amino acid metabolism  and phosphate metabolism (phosphate and O-phosphoserine).From the CCA plot for the 50v1 strain , metabolp > 0.05), stearic acid , 1-monopalmitin , behenic acid , and 2-monoolein . However, in comparison, ChemRich analyses yield no support for changes in the intracellular fatty acids.The trends for the 50v1 strain  also sugFurther, trends from the CCA plot for the 50v1 strain  support Together, these observations are relevant since the accumulation of PHAs can result in increases in relative optical densities , which i3.5.4.p < 0.05) and the associated univariate tests for the metabolomes that imply no change in relative abundances (FDR > 0.20).When considering growth rates , the vector for the 2P08AA strain  overlapsΔ9), 5′-MTA, ribose, several amino acids, oleic acid, N-acetylglucosamine, and 5’-AMP. These trends suggest that the decreases in growth rates are related to changes in amino acid metabolism, cell wall metabolism, and nucleotide metabolism. The trends also suggest relations to changes in free unsaturated fatty acid content through associations with palmitoleic acid  and oleic acid . However, ChemRich analyses yield no support for changes in unsaturated fatty acid metabolism.From the CCA plot of the 2P08AA strain, metabolites trending alongside the growth rates include palmitoleic acid , while univariate tests on the metabolomic data support changes in the abundances for multiple metabolites . These combined trends are suggestive of substantial post-exponential phase changes in the metabolomes, as growth rates for the 50v1 strain during exponential phase are unaltered by – and potentially not associated with – the multiple metabolomic changes observed during late-log phase.For the 50v1 strain , in cont3.5.5.When considering the K30 formulation ,B, the vp = 0.39) and 4-hydroxybenzoate .For the 2P08AA strain, metabolites trending alongside the detergent concentrations include myristic acid , pelargonic acid , 4-hydroxybenzoate, phenylacetic acid, and behenic acid . The trends for 4-hydroxybenzoate and phenylacetic acid lend support toward the potential for biodegradation of dodecyl benzene sulfonate and polyethylene glycol mono-nonylphenyl ether, the detergents from the K30 formulation. As described in p < 0.05), myristic acid , and pelargonic acid . We note that behenic acid levels in fluvial biofilms increase after exposure to desiccation  include behenic acid , and octadecanol . These trends are suggestive of increases in an intracellular dicarboxylic acid (tartrate) and free lipid-related molecules (1-monoolein and octadecanol), which may, respectively, relate to metal retention in the cell and the aggregation of detergents in the intracellular or membrane space.For the 50v1 strain , metabol3.6.3.6.1.A. johnsonii 2P08AA (n = 3) and A. radioresistens 50v1 (n = 3) from late log-phase cultures to the quantitative descriptors of growth rates at exponential phase, plate counts at late-log phase, maximum relative biomass at stationary phase, and total concentration of EDTA and sodium metasilicate in the media, which was assumed to be 0.02% w/w in cultures containing 0.1% v/v K30.In The CCA plots in 3.6.2.When considering survival and cultivations, the vectors for plate counts and growth rates for the 2P08AA strain  overlap and growth rates include 2-isopropylmalic acid, pyrophosphate, phosphate, arachidic acid , lauric acid , capric acid , 4-hydroxyphenyllactic acid, sorbitol, and isopentadecanoic acid (13-methyl-14:0). These trends for the 2P08AA strain suggest that survival (which was not impacted) and growth rates  relate to a lack of change in the abundances of extracellular saturated fatty acids, phosphate, pyrophosphate, and α-hydroxy acids . However, the potential increases in extracellular sorbitol  and a dicarboxylic acid (succinate); while univariate tests support ~9-fold decreases in extracellular α-ketoglutarate. Metabolites trending alongside the growth rates for the 50v1 strain include tagatose and N-acetylglucosamine, which suggests that changes in extracellular monosaccharides relate to maintaining stable growth rates.3.6.3.When considering changes in the maximum relative biomass (stationary phase) and chelator concentrations (EDTA and sodium metasilicate from the K30 formulation), similar trends are observed for the 2P08AA and 50v1 strains ,D. The vp < 0.05), no support for changes in the abundances of the associated metabolites  and never exposed to Kleenol 30.The strain Hence, comparisons across these differing sub-environments provide initial insights into the impacts of repeated cleansing, under the conditions of spacecraft assembly, toward biochemical and cultivation selection.4.2.Acinetobacter, which suggests a biocide-type of activity for K30.Cultivations containing 0–2.0% v/v Kleenol 30 (K30) were conducted in a low-osmolarity minimal media  containing 150 mM ethanol, which served as the sole supplied carbon source. Cultivation media containing 2.0% v/v K30 were bactericidal for the tested Acinetobacter harbor a tolerance toward the current floor cleansing practices for NASA cleanrooms.In contrast, in 1.0% v/v K30, appreciable cultivation tolerances were observed for the 2P08AA and 50v1 strains. Cultures containing 1.0% v/v K30 exhibited (1) ~20–30% survivals, (2) ~66 and 12% decreases in growth rates, and (3) eliminated lag phase times for the 2P08AA and 50v1 strains, respectively. These cultivation tolerances of <2% v/v K30 are significant since current NASA planetary protection practices utilize ~0.8-1.6% v/v K30 for floor cleansing. Our results, therefore, suggest that the spacecraft-associated In 0.1% v/v K30, differential impacts were observed across the cultivation parameters. For the 2P08AA strain, cultures exhibited (1) no measurable changes in cell density at late-log phase (plate counts), (2) modest decreases in the growth rate (~18%), and (3) negligible times in the lag phase. These results indicate that K30 impacts the 2P08AA strain by accelerating growth out of the lag phase, slightly inhibiting growth during the exponential phase, and by yielding no changes cell density after cultivation to late-log phase.For the 50v1 strain, cultures exhibited (1) ~50% survivals, (2) no changes in growth rate, and (3) ~70% decreases in the lag phase time. This is indicative of K30 impacting the 50v1 strain by mildly accelerating growth out of the lag phase, yielding no change in the growth rates during the exponential phase, and inhibiting cell density by ~50% when cultivated to late-log phase.The differential cultivation trends observed in 0.1% v/v K30 are indicative of strain and growth-phase dependent responses to K30. In turn, these trends suggest that K30 reduces nutrient bioavailability in culture  and/or elicits specific biochemical responses  – which together manifest as acceleration out of the lag phase for both strains, slight inhibition in rates of cell division during the exponential phase for the 2P08AA strain, and moderate reductions in total cell density at late-log phase for the 50v1 strain.4.3.A. johnsonii 2P08AA, cultivations with 0.1% v/v K30 exhibited (1) no significant and discreet changes in the abundances of intracellular metabolites, (2) several significant extracellular changes when considering broader classifications such as sugar acids, monosaccharides, organic acids (di- and monocarboxylic acids), and fatty acids, and (3) potential support for biodegradation and metabolism of both detergents in the K30 formulation.To obtain metabolic insights into the tolerances toward Kleenol 30 (K30), we used cultures grown in 0.1% v/v K30 to obtain reliable biomasses at late-log phase for both strains  differential changes in the abundances of intracellular amino acids, compatible solutes, nucleotide-related metabolites, dicarboxylic acids, and saturated fatty acids, (2) substantial and differential changes in the abundances of extracellular sugar acids, monosaccharides, and organic acids, and (3) no clear statistical support for biodegradation and metabolism of the detergents from the K30 formulation .For These results for the 50v1 strain indicate that the intracellular and extracellular metabolomes of the 50v1 strain significantly adjust to accommodate cultivation in the presence of 0.1% v/v K30. These trends also suggest that survival is associated with intracellular changes to amino acid and peptide metabolism, nucleotide metabolism, central metabolism, fatty acid metabolism, and pyrimidine metabolism.A. baumanni 1,656–2 (A. baumanni AB5075 with mixed antibiotics (polymyxin B and rifampicin) , which could include changes in early biofilm development.For the 50v1 strain, the changes in amino acid metabolism include decreases in lysine, proline, glycine, ornithine, homoserine, valine and others listed in  1,656–2 , while dampicin) . In E.coormation . In Bifoormation . These oTrends across nucleotide abundances and the cultivation terms are similarly suggestive of early biofilm-type development  and/or qE. coli UTI189 show decreases in 5′-MTA and 5’-CMP, as well increases in metal-binding siderophores, when Fe3+ concentrations in culture decrease from 10 to 1 μM 2(SO4)2), which slowly oxidized to Fe3+ under the aerobic cultivations in the capped glass tubes (per absorbance spectroscopy). However, in control experiments, cultures supplemented with [Fe(EDTA)]1− grew slower, when compared to Fe(NH4)2(SO4)2, while cultivations in acid-washed glassware yielded no growth. Therefore, under the cultivation conditions, trace elements and species such as Zn2+, Co2+, Cu2+, Mn2+, BO33− , and MoO42−  were likely obtained from trace contaminants in the media and/or the glassware.To obtain biochemical insights into the mechanisms of tolerance, the metabolomic trends were interpreted in context of the cultivation conditions. In this study, cultivations were conducted in low-osmolarity media (0.2x M9) supplemented with 25 μM Fe2+ and Mg2+ are also expected since sodium metasilicate (Na2SiO3) is a water softening agent that functions through ion exchange via chelation of Ca2+ or Mg2+ (and release of Na+).These details are relevant since K30 contains EDTA and sodium metasilicate, which are chelators that sequester transition and alkaline earth metals. Hence, in culture, chelation by EDTA would likely restrict the bioavailability of transition metals and inhibit growth – as observed in control studies. Limited bioavailabilities for Ca3+, Zn2+, Co2+, Cu2+, and Mn2+  in extracellular gluconate, galactonate, glycerate, gluconolactone, and tartrate. In fact, increases in glycerate export for the 50v1 strain are supported by the increases in extracellular glycerate and decreases in intracellular glycerate. Control of intracellular Ca2+ retention is also suggested by increases in extra- and intracellular tartrate. Multivariate tests support these total trends and suggest that chelator concentrations (from the K30 formulation) influence the abundances of extracellular metal-binding biochemicals.Biochemically, therefore, these observations suggest that the 2P08AA strain targets trace Caper se) in extracellular trace element acquisition strategies are common survival features to the spacecraft-associated Acinetobacter. Future studies using concentrated and clarified cultivation media may yield spectral support for the trace metal chelation. Additional common survival features may include regulation of the bulk osmolarity, as suggested by decreases in intracellular compatible solutes for the 50v1 strain , statistical associations (CCA plot) between survival and changes in intracellular compatible solutes  for the 2P08AA strain, and increase in an extracellular compatible solute for the 2P08AA strain .Thus, our total results suggest that up-regulations , while multivariate tests show correlations between intracellular PHA synthesis  and increases in the For the 2P08AA strain, multivariate analyses highlight a potential for partial metabolism of the detergents (from the Kleenol 30 formulation) – which would serve as an additional biochemical strategy toward survival. While these exact trends were not observed for the 50v1 strain, multivariate tests do support correlations between survival and changes in the degradation of aromatic compounds (benzene metabolism). In support, GC–MS studies in via ω-oxidation of the terminal carbon and subsequent cycles of β-oxidation) may serve as activators for acceleration out of the lag phase, and that degradation of the aromatic detergent cores (via benzene metabolism) may sustain the 50v1 strain during exponential phase, where growth rates show no inhibition in the presence of 0.1% v/v K30. If so, carbon and energy acquisition from the detergents could serve as an added biochemical strategy toward survival. Alternatively, the K30 detergents could potentially disperse any low abundance cellular aggregates during the lag phase, thereby yielding higher OD values and the apparent increases in biomass. Under this assumption, the observed concentration dependence across the strains would be suggestive of the cellular aggregates from both strains having differing stabilities.When considering impacts of K30 on the lag phase, we observe a differential concentration dependence, where the time in lag phase is eliminated in the presence of 0.1 v/v K30 for the 2P08AA strain, and in 1.0% v/v K30 for the 50v1 strain. We venture that metabolism of the alkyl sides chains on the detergents  used in NASA planetary protection practices. These results indicate that the spacecraft-associated Acinetobacter may harbor tolerances toward the floor cleaning conditions.In conclusion, our combined cultivation and metabolomic results lend supports toward the hypothesis that repetitive cleansing with Kleenol 30 presents as selective pressures toward members of the spacecraft microbiome – where the phenotypic outcomes likely include cultivation tolerances and measurable biochemical responses. Fittingly, our described results indicate that Kleenol 30 is more readily tolerated by the floor-associated Such tolerances are likely important considerations for lower bioburden planetary missions, such as orbiter missions to Europa (Planetary Protection Category III), life detection missions to Mars (Planetary Protection Category IVb) or Europa (Planetary Protection Category IV), and investigations of Mars Special Regions (Planetary Protection Category IVc). In this context, our results suggest that higher floor cleanliness levels may be achieved with higher concentrations of Kleenol 30  or rotations with differing concentrations of Kleenol 30 . Alternatively, rotation of differing floor cleansers could assist in achieving lower floor bioburdens , with the caveat that the detergents and/or biocides retain compatibility with the spacecraft assembly procedures and spacecraft materials.https://www.metabolomicsworkbench.org/) at the NIH Common Fund’s National Metabolomics Data Repository (NMDR) and can be accessed directly via the project DOI: http://dx.doi.org/10.21228/M8XH73.Metabolomic data  are available through the Metabolomics Workbench  from the Planetary Protection Research component of the National Aeronautics and Space Administration (NASA) Research Opportunities in Space and Earth Sciences program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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