Does the anesthetic tricaine methanesulfonate (MS-222) distort oxidative status parameters in tadpoles?

The effect of anesthesia/euthanasia with ethyl 3-aminobenzoate methanesulfonate (MS-222) on the oxidative status of Hyla arborea tadpoles was examined to determine whether the use of the anesthetic can confound the experimental results of the oxidative stress-based investigation. The experiment was conducted on two groups of tadpoles reared at different temperatures to produce differences in antioxidant capacity between the groups. After development at different temperatures (20 °C and 25 °C), the animals were exposed to different concentrations of MS-222 (0, 0.1, 1, and 5 g/L) for 15 min. The higher temperature decreased catalase activity, glutathione and protein carbonyl levels and increased glutathione reductase activity. The glutathione level and glutathione/thiol-related parameters were significantly changed after MS-222 exposure. However, individuals from the different temperature groups responded differently to the tested anesthetic, pointing to the possible influence of the initial levels of antioxidant capacity. The analysis of the interaction between the factors (temperature and MS-222) confirmed that the anesthetic can confound the results regarding the effects of temperature on the oxidative status parameters. The concentration of 0.1 g/L MS-222 had the lowest influence on the alterations in oxidative status and the results of the effect of temperature. A brief review of the current literature on the use of MS-222 in tadpoles made clear the absence of precise information on anesthetic concentration and exposure time. Similar studies should be repeated and extended to other amphibian species and other factors of interest to provide better guidance on tadpole anesthesia/euthanasia for future experiments that consider oxidative status parameters.

Development of platensimycin, platencin, and platensilin overproducers by biosynthetic pathway engineering and fermentation medium optimization.

The platensimycin (PTM), platencin (PTN), and platensilin (PTL) family of natural products continues to inspire the discovery of new chemistry, enzymology, and medicine. Engineered production of this emerging family of natural products, however, remains laborious due to the lack of practical systems to manipulate their biosynthesis in the native-producing Streptomyces platensis species. Here we report solving this technology gap by implementing a CRISPR-Cas9 system in S. platensis CB00739 to develop an expedient method to manipulate the PTM, PTN, and PTL biosynthetic machinery in vivo. We showcase the utility of this technology by constructing designer recombinant strains S. platensis SB12051, SB12052, and SB12053, which, upon fermentation in the optimized PTM-MS medium, produced PTM, PTN, and PTL with the highest titers at 836 mg L-1, 791 mg L-1, and 40 mg L-1, respectively. Comparative analysis of these resultant recombinant strains also revealed distinct chemistries, catalyzed by PtmT1 and PtmT3, two diterpene synthases that nature has evolved for PTM, PTN, and PTL biosynthesis. The ΔptmR1/ΔptmT1/ΔptmT3 triple mutant strain S. platensis SB12054 could be envisaged as a platform strain to engineer diterpenoid biosynthesis by introducing varying ent-copalyl diphosphate-acting diterpene synthases, taking advantage of its clean metabolite background, ability to support diterpene biosynthesis in high titers, and the promiscuous tailoring biosynthetic machinery. ONE-SENTENCE SUMMARY: Implementation of a CRISPR-Cas9 system in Streptomyces platensis CB00739 enabled the construction of a suite of designer recombinant strains for the overproduction of platensimycin, platencin, and platensilin, discovery of new diterpene synthase chemistries, and development of platform strains for future diterpenoid biosynthesis engineering.

EVALUATION OF PLASMA CORTISOL AND LACTATE IN KOI (CYPRINUS CARPIO) EUTHANIZED BY IMMERSION IN TRICAINE METHANESULFONATE, CLOVE OIL, OR CARBON DIOXIDE GAS DISSOLVED IN WATER.

Little research has taken place on the effect of euthanasia methods on biophysical and biochemical changes at the time of euthanasia in fish. These changes are used in multiple species to determine stress levels before death. Koi (Cyprinus carpio) are an important fish species often used in laboratory research, kept in backyard ponds, and managed in zoological and aquarium collections. The current study evaluated euthanasia of koi by immersion in 0.5 g/L tricaine methanesulfonate (MS-222) (n = 10), 0.5 g/L clove oil (n = 8), 1 g/L clove oil (n = 10), and CO2 (n = 7) on time to cessation of opercular movement, plasma lactate levels, and plasma cortisol levels. CO2 had the longest mean time to cessation of opercular movement, and MS-222 had the shortest (mean CO2: 24.9 min, range 13.18-31.35 min; MS-222: 2.68 min, range 1.33-4.5 min). The difference was not significant between any of the groups for plasma cortisol or lactate levels. MS-222 demonstrated the highest cortisol levels, and CO2 had the lowest (mean CO2: 108.7 ng/ml, range 33.9-195.8 ng/ml; MS-222: 650.6 ng/ml, range 77.3-2374.9 ng/ml). Average lactate levels were highest for 1 g/L clove oil and lowest for 0.5 g/L clove oil (mean 0.5 g/L clove oil: 5.1 mmol/L, range 1.8-8.1 mmol/L; 1 g/L clove oil: 7.4 mmol/L, range 5.6-10.5 mmol/L).

Role of p53 transcription factor in determining the efficacy of telomerase inhibitors in cancer treatment.

AIM: Telomerase expression is unique to cancer cells, making it a promising target for therapy. However, a major drawback of telomerase inhibition is that it affects cancer cell proliferation only when telomeres shorten, creating a lag phase post-continuous drug treatment. Acute cytotoxicity of telomerase inhibitors is dependent on their ability to induce DNA damage. p53 senses DNA damage and is the primary effector required for sensitizing cells towards apoptosis. MAIN METHODS: Isogenic p53+/+ and p53-/- ovarian cancer cell lines were generated using the CRISPR/Cas9 system and the anti-cancer effect of telomerase inhibitors MST-312 and BIBR1532 were determined. Flow cytometry, real-time PCR, and western blot were performed to study cell cycle, apoptosis, and gene expression. KEY FINDINGS: We report that MST-312 exhibits p53-dependent cytotoxicity, while BIBR1532 exhibits p53-independent cytotoxicity. Colony-forming ability also confirms the p53-dependent effect of MST-312. Re-expression of p53 in p53-/- cells could rescue MST-312 sensitivity. In p53+/+ cells, MST-312 causes S phase arrest and activation of p53-dependent target genes like anti-apoptosis markers (Fas and Puma) and cell cycle markers (p21 and cyclinB). In p53-/- cells, MST-312 causes S/G2/M arrest. BIBR1532 induces S/G2/M phase cell cycle arrest irrespective of p53 status. This correlates with the expression of the DNA damage marker (γ-H2AX). Long-term continuous treatment with MST-312 or BIBR1532 results in p53-independent telomere shortening. SIGNIFICANCE: In summary, we demonstrate that acute anti-cancer effects of MST-312 are dependent on p53 expression. Hence, it is important to consider the p53 expression status in cancer cells when selecting and administering telomerase inhibitors.

Branched pegylated linker-auristatin to control hydrophobicity for the production of homogeneous minibody-drug conjugate against HER2-positive breast cancer.

Trastuzumab emtansine (Kadcyla®) was the first antibody-drug conjugate (ADC) approved by the Food and Drug Administration in 2013 against a solid tumor, and the first ADC to treat human epidermal growth factor receptor 2 positive (HER2+) breast cancer. However, this second generation ADC is burden by several limitations included heterogeneity, limited activity against heterogeneous tumor (regarding antigen expression) and suboptimal tumor penetration. To address this, different development strategies are oriented towards homogeneous conjugation, new drugs, optimized linkers and/or smaller antibody formats. To reach better developed next generation ADCs, a key parameter to consider is the management of the hydrophobicity associated with the linker-drug, increasing with and limiting the drug-to-antibody ratio (DAR) of the ADC. Here, an innovative branched pegylated linker was developed, to control the hydrophobicity of the monomethyl auristatin E (MMAE) and its cathepsin B-sensitive trigger. This branched pegylated linker-MMAE was then used for the efficient generation of internalizing homogeneous ADC of DAR 8 and minibody-drug conjugate of DAR 4, targeting HER2. Both immunoconjugates were then evaluated in vitro and in vivo on breast cancer models. Interestingly, this study highlighted that the minibody-MMAE conjugate of DAR 4 was the best immunoconjugate regarding in vitro cellular internalization and cytotoxicity, gamma imaging, ex vivo biodistribution profile in mice and efficient reduction of tumor size in vivo. These results are very promising and encourage us to explore further fragment-drug conjugate development.

Optical Elastography for Micropressure Characterization of Zebrafish Embryonic Cardiac Development.

The proper formation of the vertebrate embryonic heart relies on various mechanical forces which determine its form and function. Measuring these forces at the microscale of the embryo is a challenge. We propose a new tool utilizing high-resolution optical elastography and stiffness measurements of surrounding tissues to non-invasively track the changes in the pressure exerted by the heart on the neighboring yolk, as well as changes in contractile patterns during early cardiac growth in-vivo, using the zebrafish embryo as a model system. Cardiac development was characterized every three hours from 24 hours post-fertilization (hpf) to 30 hpf and compared between wildtype fish and those treated with MS-222, a commonly used fish anesthetic that decreases cardiac contractility. Wildtype embryos from 24 to 30 hpf showed an average yolk indentation pressure of 0.32 mmHg to 0.41 mmHg, respectively. MS-222 treated embryos showed an average yolk indentation pressure of 0.22 mmHg to 0.29 mmHg. Yolk indentation pressure between control and treated embryos at 24 hpf and 30 hpf showed a significant difference (p < 0.05). Our method allowed for contractility and pressure evaluation at these early developmental stages, which have not been previously reported in published literature, regardless of sample or imaging modality. This research could lead to a better understanding of heart development and improved diagnostic tools for congenital heart disease.

Comparison of Tricaine Methanesulfonate (MS-222) and Alfaxalone Anesthesia in Zebrafish (Danio rerio).

The research use of zebrafish has risen exponentially over the past decade while anesthetic options have remained largely unchanged.6 ricaine methanesulfonate (MS-222) is widely accepted as an anesthetic for routine husbandry procedures, however it has limitations and safety concerns. 11 A greater variety of effective anesthetic options for surgical procedures would be advantageous for the research community. Adult zebrafish were randomly assigned to one of the following groups (n = 10, 5 males and 5 females): 200 mg/L MS-222; 6-, 10-, 13-, and 16-mg/L alfaxalone, and control. All zebrafish in the MS-222 group reached a surgical plane of anesthesia within 95 ± 32 s. By contrast, only 2 of 10, 1 of 10, 0 of 10, and 0 of 4 of the 6, 10, 13, and 16 mg/L alfaxalone groups, respectively, reached a surgical plane of anesthesia within the allotted 10-min period. Recovery time was also significantly slower in the alfaxalone groups as compared with MS-222, with some fish taking greater than 10 min to recover. In addition, 33 of 34 zebrafish (the 16 mg/L group was not completed due to safety concerns) in the alfaxalone groups lost opercular movements for greater than one minute during their anesthetic event and had to be removed to the recovery tank. The results demonstrated that alfaxalone was unable to provide a reliable and safe surgical plane of anesthesia at any of the drug doses tested. Therefore, we recommend alfaxalone not be used as an anesthetic for painful procedures on zebrafish and conclude that MS-222 remains a more viable anesthetic for immersion anesthesia in zebrafish.

Effects of SETD2 on telomere length and malignant transformation property of Met-5A after one-month crocidolite exposure.

Crocidolite is a carcinogen contributing to the pathogenesis of malignant mesothelioma. This study aimed to characterize the possible telomere-related events mediating the malignant transformation of mesothelial cells with and without SETD2 under crocidolite exposure. The crocidolite concentration resulting in 90% viable SETD2 knockout Met-5A (Met-5ASETD2-KO) and Met-5A were estimated to be 0.71 µg/cm2 and 1.8 µg/cm2, respectively, during 72 h of exposure, which was further employed in chronical crocidolite exposure during a 72 h exposure interval per time up to 1 month. Chronical crocidolite-exposed Met-5ASETD2-KO (chronical Cro-Met-5ASETD2-KO) had higher colony formation and increased telomerase reverse transcriptase (TERT) protein levels than chronical crocidolite-exposed Met-5A (chronical Cro-Met-5A) and Met-5ASETD2-KO. Chronical Cro-Met-5ASETD2-KO had longer telomere length (TL) than chronical Cro-Met-5A, although there were no changes in TL for either chronical Cro-Met-5A or chronical Cro-Met-5ASETD2-KO compared with their corresponding cells without crocidolite exposure. BIBR 1532, an inhibitor targeting TERT, partially reduced colony formation and TL for chronical Cro-Met-5ASETD2-KO, while BIBR 1532 reduced TL but had no effect on colony formation for chronical Cro-Met-5A. Therefore, SETD2 deficient mesothelial cells are susceptible to malignant transformation during chronical crocidolite exposure, and TERT-dependent TL modification likely partially drives SETD2 loss-mediated early onset of mesothelial malignant transformation.

BIBR1532 Affects Endometrial Cell Proliferation, Migration, and Invasion in Endometriosis via Telomerase Inhibition and MAPK Signaling.

OBJECTIVES: The effect of telomerase inhibitor BIBR1532 on endometriotic cells was investigated to explore the inhibitory effect of targeting telomerase on endometriosis. DESIGN: In vitro primary cell culture study. Participants/Materials: Primary endometrial cells derived from eutopic and ectopic endometrium in patients with endometriosis. SETTING: The study was conducted in the university hospital. METHODS: Paired eutopic and ectopic endometrial cells were collected from 6 patients from January 2018 to July 2021. A TRAP assay was performed to detect the telomerase activity of the cells. MTT, cell cycle, apoptosis, migration, and invasion assays were performed to study the inhibitory effect of BIBR1532. Enrichment analysis was performed to identify the key pathways involved in endometriosis progression and telomerase action. Then, Western blotting was used to investigate the expression of related proteins. RESULTS: BIBR1532 treatment significantly inhibited the growth of eutopic and ectopic endometrial cells, with apoptosis and cell cycle signaling involved. Migration and invasion, important characteristics for the establishment of ectopic lesions, were also inhibited by BIBR1532. The MAPK signaling cascade, related to telomerase and endometriosis, was decreased in eutopic and ectopic endometrial stromal cells with the treatment of BIBR1532. LIMITATIONS: The severe side effects of telomerase inhibitors might be the main obstacle to clinical application, so it is necessary to find better drug delivery methods in vivo. CONCLUSIONS: The telomerase inhibitor BIBR1532 affects endometrial cell proliferation, migration, and invasion in endometriosis.

Tandem C-H Annulation Reaction of Benzaldehydes and Aminobenzoic Acids with Two Equivalents of Alkyne toward Isocoumarin-Conjugated Isoquinolinium Salts: A Family of Organic Luminophores.

A novel rhodium-catalyzed tandem C-H annulation of commercially available benzaldehydes and aminobenzoic acids with 2 equiv of alkyne is reported for the construction of isocoumarin-conjugated isoquinolinium salts that demonstrate diverse outstanding photoactivity. Depending on the substituents in the isoquinolinium moiety, they display either highly efficient fluorescence (up to 99% of quantum yield) or strong fluorescence quenching, which is provided by the transfer of the HOMO from the isoquinolinium to the isocoumarin moiety. Importantly, the functional groups in the benzaldehyde coupling partner also strongly affect the reaction selectivity, shifting the pathway to the formation of the photoinactive isocoumarin-substituted indenone imines and indenyl amines. Selective formation of the latter can be achieved by using a reduced amount of the oxidizing additive.

Bioinspired Synthesis of Platensimycin from Natural ent-Kaurenoic Acids.

The biomimetic formal synthesis of the antibiotic platensimycin for the treatment of infection by multidrug-resistant bacteria was accomplished starting from either ent-kaurenoic acid or grandiflorenic acid, each of which is a natural compound available in multigram scale from its natural source. Apart from the natural origin of the selected precursors, the keys of the described approach are the long-distance functionalization of ent-kaurenoic acid at C11 and the efficient protocol for the A-ring degradation of the diterpene framework.

Identification of aminobenzoic acids as selective inhibitors of the N-terminal phosphatase of soluble epoxide hydrolase.

Soluble epoxide hydrolase (EC 3.3.2.10) is a key enzyme in the regulation of inflammation and metabolism, whereas, the role of its N-terminal phosphatase activity (N-phos) has been poorly understood because of a lack of selective inhibitors. Here we report 4-aminobenzoic (Ki 15.3 µm) and 3-amino-4-hydroxy benzoic acid (Ki 11.7 µm) as selective competitive inhibitors of N-phos.

Comparative Metabolomics Reveals a Bifunctional Antibacterial Conjugate from Combined-Culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596.

To investigate the potential for secondary metabolite biosynthesis by Streptomyces species, we employed a coculture method to discover natural bioactive products and identified specific antibacterial activity from a combined-culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596. Molecular networking using ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) data revealed a specific clade of metabolites in this combined-culture that were not detected in both monocultures. Using the chemical profiles, a previously unidentified conjugate between FabF inhibitor and catechol-type siderophore was successfully identified and named harundomycin A. Harundomycin A was a conjugate between the 2,4-dihydroxy-3-aminobenzoate moiety of platensimycin and N,N'-bis(2,3-dihydroxybenzoyl)-O-seryl-cysteine (bisDHBA-Ser-Cys) with a thioester linkage. Along with the production of harundomycin A, platensimycin, its thiocarboxylic acid form thioplatensimycin, enterobactin, and its degradation product N,N'-bis(2,3-dihydroxybenzoyl)-O-l-seryl-dehydroalanine (bisDHBA-Ser-Dha) were also induced in the combined-culture. Genomic data of S. hygroscopicus HOK021 and T. pulmonis TP-B0596 indicated that strain HOK021 possessed biosynthetic gene clusters for both platensimycin and enterobactin, and thereby revealed that T. pulmonis stimulates HOK021 and acts as an inducer of both of these metabolites. Although the harundomycin A was modified by bulky bisDHBA-Ser-Cys, responsible for the binding to the target molecule FabF, it showed a similar antibacterial spectrum to platensimycin, including against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, suggesting that the pharmacophore is platensimycin. Additionally, Chrome Azurol S assay showed that harundomycin A possesses ferric iron-chelating activity comparable to that of enterobactin. Our study demonstrated the transformation of existing natural products to bifunctional molecules driven by bacterial interaction.

The Combined Escherichia coli Nissle 1917 and Tryptophan Treatment Modulates Immune and Metabolome Responses to Human Rotavirus Infection in a Human Infant Fecal Microbiota-Transplanted Malnourished Gnotobiotic Pig Model.

Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.

Platensimycin-berberine chloride co-amorphous drug system: Sustained release and prolonged half-life.

Co-amorphous technology is an emerging approach for pharmaceutical engineering of drugs and drug leads with improved physicochemical properties and bioavailability. Platensimycin (PTM) is a promising natural antibiotic lead that acts on bacterial fatty acid synthase and exhibits excellent antibacterial activity. Despite great strides to improve its poor pharmacokinetics by medicinal chemistry and nanotechnology, there are no convenient oral delivery systems developed. Here, a co-amorphous system of PTM and berberine chloride (BCL) was developed for oral delivery of PTM. Co-amorphous PTM-BCL was prepared by rotary vacuum evaporation method, and systematically characterized by powder X-ray diffraction, temperature modulated differential scanning calorimetry, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). Compared with PTM or BCL alone, the equilibrium solubility and dissolution rate of both of them in the co-amorphous systems decreased significantly, showing the characteristics of sustained release. The molecular interactions between PTM and BCL were mediated by strong charged-mediated hydrogen bonds, based on FTIR, XPS, and NMR-based techniques. The co-amorphous PTM-BCL system showed excellent physiochemical stability at room and elevated (40 °C) temperature under dry conditions. The combination of PTM and BCL showed increased killing of a clinical isolated methicillin-resistant Staphylococcus aureus strain in killing checkerboard assays. Finally, co-amorphous PTM-BCL exhibited 2- or 3-fold longer half-life in rats than that of crystalline and amorphous PTM upon oral administration, respectively. Our study suggests a rational approach to realize the full potential of potent antibiotic PTM, which may be conveniently adapted for engineering of other important pharmaceutics.

Analysis of persistent contaminants and personal care products by dispersive liquid-liquid microextraction using hydrophobic magnetic deep eutectic solvents.

In this work, hydrophobic magnetic deep eutectic solvents (HMDESs) were used in the development of a simple and rapid dispersive liquid-liquid microextraction (DLLME) approach coupled to high performance liquid chromatography with UV detection (HPLC-UV) for the determination of ten organic contaminants including five polycyclic aromatic hydrocarbons, four UV filters, and a pesticide from water at trace levels. The HMDESs were prepared by mixing a hydrogen bond acceptor, metal halide salt, and hydrogen bond donor in suitable molar ratios. Two HMDESs, 2 tetraoctylammonium bromide ([N8888+][Br-]): cobalt chloride (CoCl2): 4 octanoic acid (OA) and 3 trioctylphosphine oxide (TOPO): neodymium chloride (NdCl3): 3 OA, offered the highest analyte extraction efficiency overall and were chosen as suitable solvents for validation of the microextraction method. Under optimized extraction conditions, the method required 30 µL of HMDES as extraction solvent, acetone (87.5 µL) as disperser solvent, a NaCl concentration of 30% (w/v), and an extraction time of 120 s at 20°C. Enrichment factors of the analytes ranged from 44.6 for 3-(4-methylbenzylindene) camphor to 66.0 for 2-ethylhexyl-4-(dimethyl)aminobenzoate. The method provided low limits of detection (LODs) ranging from 0.5 to 4.5 µg L-1, and acceptable precision, with RSD values lower than 9.6%. Furthermore, the validated method was successfully applied for tap and lake water analysis, resulting in relative recoveries of spiked samples ranging between 94.7 and 119.2%.

Effects of replacing fish meal with cottonseed protein concentrate on the growth, immune responses, digestive ability and intestinal microbial flora in Litopenaeus vannamei.

The effects of cottonseed protein concentrate (CPC) in place of fishmeal on the growth performance, immune response, digestive ability and intestinal microbiota of Litopenaeus vannamei were investigated in this study. L. vannamei (initial body weight: 0.42 ± 0.01g) was fed for 8 weeks by four isonitrogenous and isolipid feeds with CPC replacing fishmeal (FM) at 0% (control), 15% (CPC15), 30% (CPC30) and 45% (CPC45), respectively. At the end of the study, the final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR) and protein efficiency ratio (PER) of L. vannamei in CPC15 and CPC30 groups were significantly increased, while the feed conversion ratio (FCR) of L. vannamei in the CPC30 group was significantly reduced when compared with the FM group (P < 0.05). After Vibrio parahaemolyticus infection, the cumulative mortality of L. vannamei in CPC15 within 24 hpi was significantly lower than that of the control group (P < 0.05). When compared with the control group, the activities and expression of the immunity-related enzymes in the hepatopancreas had almost the same obvious change trend in the CPC-containing groups, which indicated that the replacement for fishmeal by CPC led to significant immune response in L. vannamei. Besides, significant up-regulation of the digestive enzyme activities were observed in the CPC-containing groups. Analysis of intestinal microbiota showed that significant difference in alpha diversity existed between the CPC-containing groups and the control group. The relative abundances of several top 10 dominated species at the phylum and genus levels were significantly changed in the CPC-containing groups compared with the control group (P < 0.05). Functional prediction of the microbiota indicated that the pathway of protein digestion and absorption was significantly more abundant while the pathways of nitrotoluene degradation, aminobenzoate degradation, atrazine degradation, dioxin degradation and xylene degradation were significantly less abundant in the CPC-containing groups than the FM group (P < 0.05). In summary, optimal dietary CPC replacement of FM could improve the growth, immunity, digestive capacity and the diversities of the intestinal microbial flora of L. vannamei. However, parts of the functions of the intestinal microbial flora were decline.

An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer.

Gastric cancer is a worldwide health problem. Chemotherapy and radiotherapy are of great importance in the management of advanced gastric cancer. However, their therapeutic efficacy is limited by off-target side effects. Peptide-drug conjugates (PDCs) are a novel strategy for tumor-targeted drug delivery to overcome the existing drug resistance mechanisms and improve antitumor effects. Kita-Kyushu lung cancer antigen 1 (KK-LC-1) is exclusively expressed in several types of cancer including gastric cancer, representing a promising target for drug delivery. Here, we suggested KK-LC-1 as a potential target for PDC design for the first time and reported the first KK-LC-1-targeting PDC product 1131-MMAE, which is composed of a KK-LC-1-targeting peptide and an antimitotic drug conjugated by an enzymatically cleavable linker. We observed that 1131-MMAE could be efficiently endocytosed by KK-LC-1 positive gastric cancer cells for subsequent drug release and arrest the cell cycle at the most radiosensitive G2/M phase. We demonstrated that 1131-MMAE could significantly delay tumor growth with reduced toxicity than free drugs as a monotherapy. We further confirmed that 1131-MMAE was also a potent radiosensitizer. 1131-MMAE could selectively enhance the radiation response of KK-LC-1 positive tumor cells and achieve improved tumor control when combined with low-dose radiation. Overall, our study proposed an optimized therapeutic regimen for precision chemoradiotherapy, which has translational potential in multiple types of cancer.

Lipopeptide surfactin ameliorates the cell uptake of platensimycin and enhances its therapeutic effect on treatment of MRSA skin infection.

OBJECTIVES: The rapid development of drug-resistant bacteria, especially MRSA, poses severe threats to global public health. Adoption of antibiotic adjuvants has proved to be one of the efficient ways to solve such a crisis. Platensimycin and surfactin were comprehensively studied to combat prevalent MRSA skin infection. METHODS: MICs of platensimycin, surfactin or their combinations were determined by resazurin assay, while the corresponding MBCs were determined by chequerboard assay. Growth inhibition curves and biofilm inhibition were determined by OD measurements. Membrane permeability analysis was conducted by propidium iodide staining, and morphological characterizations were performed by scanning electron microscopy. Finally, the therapeutic effects on MRSA skin infections were evaluated in scald-model mice. RESULTS: The in vitro assays indicated that surfactin could significantly improve the antibacterial performance of platensimycin against MRSA, especially the bactericidal activity. Subsequent mechanistic studies revealed that surfactin not only interfered with the biofilm formation of MRSA, but also disturbed their cell membranes to enhance membrane permeability, and therefore synergistically ameliorated MRSA cellular uptake of platensimycin. Further in vivo assessment validated the synergistic effect of surfactin on platensimycin and the resultant enhancement of therapeutical efficacy in MRSA skin-infected mice. CONCLUSIONS: The combination of effective and biosafe surfactin and platensimycin could be a promising and efficient treatment for MRSA skin infection, which could provide a feasible solution to combat the major global health threats caused by MRSA.

Monomethyl auristatin antibody and peptide drug conjugates for trimodal cancer chemo-radio-immunotherapy.

Locally advanced cancers remain therapeutically challenging to eradicate. The most successful treatments continue to combine decades old non-targeted chemotherapies with radiotherapy that unfortunately increase normal tissue damage in the irradiated field and have systemic toxicities precluding further treatment intensification. Therefore, alternative molecularly guided systemic therapies are needed to improve patient outcomes when applied with radiotherapy. In this work, we report a trimodal precision cytotoxic chemo-radio-immunotherapy paradigm using spatially targeted auristatin warheads. Tumor-directed antibodies and peptides conjugated to radiosensitizing monomethyl auristatin E (MMAE) specifically produce CD8 T cell dependent durable tumor control of irradiated tumors and immunologic memory. In combination with ionizing radiation, MMAE sculpts the tumor immune infiltrate to potentiate immune checkpoint inhibition. Here, we report therapeutic synergies of targeted cytotoxic auristatin radiosensitization to stimulate anti-tumor immune responses providing a rationale for clinical translational of auristatin antibody drug conjugates with radio-immunotherapy combinations to improve tumor control.