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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.

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.

The objective of this study was to describe prolonged surgical anaesthesia and recovery in fire salamanders ( Salamandra salamandra) using tricaine methanesulfonate (MS-222). A total of 14 salamanders were anaesthetised for electromyography wire implantation. Sodium bicarbonate buffered solutions (0.5–4 g l–1) of MS-222 were prepared (adjusted to pH 7.0). Anaesthesia was induced by partial immersion in pre-oxygenated 3 g l–1 solution for 20 min. Buprenorphine (0.5 mg kg–1) was administered subcutaneously. During microsurgery, heart rate (HR), solution pH and temperature were recorded. Reflectance pulse oximeter (SpO2) (Masimo Rad-57) was recorded in two salamanders. Anaesthetic plane and MS-222 pH stability (pH 7.6) were maintained by renewing administration of oxygenated MS-222 solution (0.5–3 g l–1) onto swabs that partially covered the body. Recovery started at the end of surgery (MS-222 0 g l–1). Postoperatively, salamanders were given oral meloxicam (0.2 mg kg–1). Mean time for loss of righting reflex during induction was 13.7 ± 2.2 min. Duration of anaesthesia and time to recovery were 111 ± 24.2 and 31 ± 10.3 min, respectively. Due to complications, two salamanders did not recover. Baseline HR was 67.4 ± 34.5 beats/min, and it decreased significantly until recovery ( p ≤ 0.0001). In two salamanders, baseline SpO2 was 85.5% ± 14.5, SpO2 during surgery was 61% ± 6.4, improving to 80.5% ± 2.1 on recovery. In conclusion, prolonged recovery anaesthesia is achievable with MS-222 dilutions in salamander. Reflectance SpO2 could prove valuable during immersion anaesthesia.

Crayfish (Decapoda: Astacoidea and Parastacoidea) are among the few animals that have stem cells in hemolymph, with the capacity to continuously produce differentiated neuronal structures throughout life. As the use of crayfish and other invertebrates increases in biomedical research, we must develop laboratory standards and guidelines for performing clinical procedures. This manuscript presents introductory protocols for anesthesia in crayfish during diagnostic imaging. Five anesthetic protocols were evaluated: immersion in buffered tricaine methanesulfonate (MS222; 50 mg/L); immersion in buffered MS222 (150 mg/L); immersion in propofol (65 mg/L); injection of propofol (50 mg/kg); and injection of propofol (100 mg/kg) into the ventral surface of an abdominal somite. MS222 immersion (50 and 150 mg/L) had no observable effect on crayfish. After an extended period of time, immersion in propofol (65 mg/L) created a sedative effect suitable for short-term handling. Propofol injection (50 mg/kg) into the ventral surface of an abdominal somite created an effective plane of anesthesia without adverse effects during or after recovery. Propofol injection at 100 mg/kg had adverse effects and is not recommended for use in crayfish. CT imaging was performed successfully as proof of concept for handling anesthetized crayfish. These findings provide initial data for the anesthetization of crayfish used in research settings.

OBJECTIVE Doses of buffered tricaine methanesulfonate (MS-222) up to 1000 mg/L for 15 minutes are reported inefficient to produce euthanasia in goldfish. The goal of this study was to determine if goldfish can be euthanized by more prolonged immersion in MS-222. ANIMALS 24 healthy goldfish (weight range: 1 to 10 g) were randomly assigned to 4 groups of 6 fish. PROCEDURES The first group (G1) was exposed to 500 mg/L buffered MS-222 for 15 minutes then placed in freshwater for 3 hours. The second (G2) and third groups (G3) were exposed to 1000 mg/L of buffered MS-222 for 15 minutes then placed in freshwater for 3 hours and 18 hours respectively. The fourth group (G4) was exposed to 1000 mg/L of buffered MS-222 for 60 minutes then placed in freshwater for 3 hours. Time to cessation and return of operculation were recorded. If the goldfish did not resume operculation, heart rate was evaluated by Doppler ultrasonic flow detector. RESULTS Median times to apnea were 35 seconds at 1000 mg/L and 65 seconds at 500 mg/L. Re-operculation occurred only in G1 in 5 out of 6 individuals. All fish from G1, 3 fish from G2, 0 fish from G3, 1 fish from G4 had remaining heartbeats at the end of the observation period. CLINICAL RELEVANCE Overall, a dose of 1000 mg/L of buffered MS-222 for 15 minutes was efficient to euthanize juvenile goldfish at 20 °C. Different fish body mass and water quality parameters might explain different results compared to previous studies.

Amphibians commonly are managed under human care for research, education, conservation, and companionship and frequently are in need of sedation, anesthesia, or end-of-life care involving euthanasia. Objective investigation of sedative and anesthetic protocols in these taxa still is in its infancy, but knowledge of current best practices is paramount to appropriate care. Tricaine methanesulfonate delivered via immersion (bath) is the most common anesthetic agent in amphibians, but several other effective techniques have been identified. This summary provides a comprehensive review of the current evidence-based literature regarding amphibian sedative, anesthetic, and euthanasia techniques.

PtmU3 is a newly identified nonheme diiron monooxygenase, which installs a C-5 β-hydroxyl group into the C-19 CoA-ester intermediate involved in the biosynthesis of unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 possesses a noncanonical diiron active site architecture of a saturated six-coordinate iron center and lacks the μ-oxo bridge. Although the hydroxylation process is a simple reaction for nonheme mononuclear iron-dependent enzymes, how PtmU3 employs the diiron center to catalyze the H-abstraction and OH-rebound is still unknown. In particular, the electronic characteristic of diiron is also unclear. To understand the catalytic mechanism of PtmU3, we constructed two reactant models in which both the Fe1II-Fe2III-superoxo and Fe1II-Fe2IV═O are considered to trigger the H-abstraction and performed a series of quantum mechanics/molecular mechanics calculations. Our calculation results reveal that PtmU3 is a special monooxygenase, that is, both atoms of the dioxygen molecule can be incorporated into two molecules of the substrate by the successive reactions. In the first-round reaction, PtmU3 uses the Fe1II-Fe2III-superoxo to install a hydroxyl group into the substrate, generating the high-reactive Fe1II-Fe2IV═O complex. In the second-round reaction, the Fe1II-Fe2IV═O species is responsible for the hydroxylation of another molecule of the substrate. In the diiron center, Fe2 adopts the high spin state (S = 5/2) during the catalysis, whereas for Fe1, in addition to its structural role, it may also play an assistant role for Fe1 catalysis. In the two successive OH-installing steps, the H-abstraction is always the rate-liming step. E241 and D308 not only act as bridging ligands to connect two Fe ions but also take part in the electron reorganization. Owing to the high reactivity of Fe1II-Fe2IV═O compared to Fe1II-Fe2III-superoxo, besides the C5-hydroxylation, the C3- or C18-hydroxylation was also calculated to be feasible.

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 (

To investigate the potential for secondary metabolite biosynthesis by

Halogenation can be utilized for the purposes of labeling and molecular imaging, providing a means to, e.g., follow drug distribution in an organism through positron emission tomography (PET) or study the molecular recognition events unfolding by nuclear magnetic resonance (NMR) spectroscopy. For cancer therapeutics, where often highly toxic substances are employed, it is of importance to be able to track the distribution of the drugs and their metabolites in order to ensure minimal side effects. Labeling should ideally have a negligible disruptive effect on the efficacy of a given drug. Using a combination of NMR spectroscopy and cytotoxicity assays, we identify a site susceptible to halogenation in monomethyl auristatin F (MMAF), a widely used cytotoxic agent in the antibody-drug conjugate (ADC) family of cancer drugs, and study the effects of fluorination and chlorination on the physiological solution structure of the auristatins and their cytotoxicity. We find that the cytotoxicity of the parent drug is retained, while the conformational equilibrium is shifted significantly toward the biologically active trans isomer, simultaneously decreasing the concentration of the inactive and potentially disruptive cis isomer by up to 50%. Our results may serve as a base for the future assembly of a multifunctional toolkit for the assessment of linker technologies and exploring bystander effects from the warhead perspective in auristatin-derived ADCs.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia (CLL), typically to an aggressive lymphoma. Treatment options for RS are limited and the disease is often fatal. Receptor tyrosine kinase–like orphan receptor 1 (ROR1) is expressed on CLL cells and other cancers but not on healthy adult tissues, making it an attractive, tumor-specific therapeutic target. VLS-101 is being developed as an antibody-drug conjugate (ADC) for therapy of ROR1-expressing (ROR1+) cancers. VLS-101 comprises UC-961 (a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE). VLS-101 binding to ROR1 results in rapid cellular internalization and delivery of MMAE to induce tumor cell death. We studied 4 RS patient-derived xenografts (RS-PDXs) with varying levels of ROR1 expression (11%, 32%, 85%, and 99% of cells). VLS-101 showed no efficacy in the lowest-expressing RS-PDX but induced complete remissions in those with higher levels of ROR1 expression. Responses were maintained during the posttherapy period, particularly after higher VLS-101 doses. In systemic ROR1+ RS-PDXs, VLS-101 dramatically decreased tumor burden in all RS-colonized tissues and significantly prolonged survival. Animals showed no adverse effects or weight loss. Our results confirm ROR1 as a target in RS and demonstrate the therapeutic potential of using an ADC directed toward ROR1 for the treatment of hematological cancers. A phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with RS and other hematological malignancies.

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 ([N

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.

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.

Alfaxalone anesthesia and residue clearance profiles in rainbow trout Alfaxalone, a synthetic neuroactive steroid, has been tested as an immersion anesthetic in ornamental fish, but its safety and efficacy in sport fish have not been investigated. In the current study, we compared the physiologic and behavioral effects of alfaxalone with those of tricaine methanesulfonate (MS222) for anesthesia of rainbow trout (Oncorhynchus mykiss) via water immersion. We also analyzed alfaxalone-exposed tissues to determine residue clearance times. Fish were anesthetized for 10 min by immersion in low-dose alfaxalone (A(low); 5 mg/L induction, 1 mg/L maintenance), high-dose alfaxalone (A(high); 5 mg/L induction, 2 mg/L maintenance), or MS222 (MS; 150 mg/L induction, 100 mg/L maintenance). Fish received all 3 treatments, separated by a washout period of at least 18 d in a blinded, complete crossover design. We hypothesized that immersion in A(low) or A(high) would provide a stable plane of anesthesia in rainbow trout, with dose-dependent time to recovery, and that opercular rates and depths of anesthesia would be equivalent to that of MS222. The time to anesthesia induction was longer for alfaxalone than MS222 but averaged less than 100 s. The time to recovery from anesthesia was also longer for alfaxalone than MS222, with significantly shorter recovery time for A(low) than for A(high). All treatments decreased opercular rate and response to noxious stimuli. Alfaxalone residue clearance was greater than 80% from all tissues within 1 h, greater than 99% from muscle within 4 h, and 100% from all tissues within 36 h after exposure. We conclude that alfaxalone immersion at 5 mg/L for induction and 2 mg/L for maintenance provides a safe, viable alternative to MS222 for the anesthesia of rainbow trout.

In this research, we designed a rapid tricolour immunochromatographic test strip with double test lines (TS-DTL) and two-colour AuNP probes, which realised the simultaneous detection of tricaine mesylate (TMS) and malachite green (MG). Through a distinct tricolour system (red T1 line, blue T2 line and purple C line), a visual identification of TMS (0.2 μg/mL) and MG (0.5 μg/mL) was quickly achieved on site, which improved the accuracy of naked eye observations. The LODs of TMS in aquaculture water, fish and shrimp were 11.0, 29.6 and 61.4 ng/mL, respectively. MG LODs were 47.0 ng/mL (aquaculture water), 82.8 ng/mL (fish) and 152.4 ng/mL (shrimp). The LOD of MG was close to the similar TS methods. However, visual detection of TMS could meet the requirements of the residue limit (1 μg/mL) of TMS in the USA, and the quantitative detection of TMS was over 16 times lower than the USA standard. The developed platform was rapid (~20 min, HPLC~3 h) and accurate, which was verified using a traditional HPLC method. The recovery rates ranged from 82.2% to 108.6% in three types of real samples, indicating a potential application in on-site fast screening or multiple detection for TMS and MG residues in aquatic products.

Prostate-specific membrane antigen (PSMA) is a promising target for the treatment of advanced prostate cancer (PC) and various solid tumors. Although PSMA-targeted radiopharmaceutical therapy (RPT) has enabled significant imaging and prostate-specific antigen (PSA) responses, accumulating clinical data are beginning to reveal certain limitations, including a subgroup of non-responders, relapse, radiation-induced toxicity, and the need for specialized facilities for its administration. To date non-radioactive attempts to leverage PSMA to treat PC with antibodies, nanomedicines or cell-based therapies have met with modest success. We developed a non-radioactive prodrug, SBPD-1, composed of a small-molecule PSMA-targeting moiety, a cancer-selective cleavable linker, and the microtubule inhibitor monomethyl auristatin E (MMAE). SBPD-1 demonstrated high binding affinity to PSMA (Ki = 8.84 nM) and selective cytotoxicity to PSMA-expressing PC cell lines (IC50 = 3.90 nM). SBPD-1 demonstrated a significant survival benefit in two murine models of human PC relative to controls. The highest dose tested did not induce toxicity in immunocompetent mice. The high specific targeting ability of SBPD-1 to PSMA-expressing tumors and its favorable toxicity profile warrant its further development.

Natural product congeners serve a useful role in the understanding of natural product biosynthesis and structure-activity relationships. A minor congener with superior activity, selectivity, and modifiable functional groups could serve as a more effective lead structure and replace even the original lead molecule that was used for medicinal chemistry modifications. Currently, no effective method exists to discover targeted congeners rapidly, specifically, and selectively from producing sources. Herein, a new method based on liquid-chromatography tandem-mass spectrometry combination is evaluated for targeted discovery of congeners of platensimycin and platencin from the extracts of Streptomyces platensis. By utilizing a precursor-ion searching protocol, tandem mass spectrometry not only confirmed the presence of known congeners but also provided unambiguous detection of many previously unknown congeners of platensimycin and platencin. This high-throughput and quantitative method can be rapidly and broadly applied for dereplication and congener discovery from a variety of producing sources, even when the targeted compounds are obscured by the presence of unrelated natural products.

Herein, we describe the novel reactivity of hexafluoroisopropyl 2-aminobenzoates. The metal-free synthesis of 1,4-benzodiazepines and quinazolinones from hexafluoroisopropyl 2-aminobenzoates has been developed at room temperature. These procedures feature good functional group tolerance, mild reaction conditions, and excellent yields. The newly formed products can readily be converted to other useful N-heterocycles. Moreover, the products and their derivatives showed potent anticancer activities in vitro by MTT assay.

Abstract: Chemical reactivity studies of the organotin(IV) carboxylates diphenyltin(IV) di-4-amino-benzoate (1) and triphenyltin(IV) 4-aminobenzoate (2) were conducted using cyclic voltammetry. Then, their antioxidant activities were tested by the 2,2-diphenyl-1-picrylshydrazyl (DPPH) method. Cyclic voltammetry was used to determine the kinetic constants of compounds 1 and 2 for the forward chemical reaction (kf). The constant values of the chemical reaction rate of 1 and 2 on cyclic voltammogram by experiment were obtained by comparing with the values from digital simulation methods obtained using Polar software 5.8.3. The results demonstrated that the constant value of the rate of the subsequent chemical reaction is a function of the rate of its potential (slope = kf/i); that is, 6.481 and 6.069 1/V for 1 and 2, respectively. The type of chemical reaction mechanism that occurs around the surface of the working electrode follows reaction mechanism of electrochemical reaction is quasi reversible and chemical reaction is irreversible (EqCi). The antioxidant activities of compounds 1 and 2 produced IC50 values of 5.91 and 12.57 ig/mL, respectively. These results indicate that both compounds are active as antioxidants. However, their antioxidant activities were lower than that of ascorbic acid, which has an IC50 value of 0.66 ig/mL.

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.

Methemoglobin is hemoglobin containing ferric iron rather than ferrous iron which renders it incapable of binding to oxygen. Blood sampling of fish is done under sedation or general anesthesia. Tricaine methanesulfonate (TMS) or benzocaine is commonly used but both can cause oxidation of hemoglobin to methemoglobin. Our objective was to determine if methemoglobin concentrations in healthy rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), or Atlantic salmon (Salmo salar) increase during sedation with 25 mg/L of a 10% benzocaine solution or with repeated short anesthetizations by 65 mg/L of 10% benzocaine solution or 65 mg/L of TMS. Sedation by benzocaine caused a significant increase in methemoglobin in all species over time (P

Background Antibody-drug conjugate (ADC) construction poses numerous challenges that limit clinical progress. In particular, common bioconjugation methods afford minimal control over the site of drug coupling to antibodies. Here, such difficulties are overcome through re-bridging of the inter-chain disulfides of cetuximab (CTX) with auristatin-bearing pyridazinediones, to yield a highly refined anti-epidermal growth factor receptor (EGFR) ADC. Methods In vitro and in vivo assessment of ADC activity was performed in KRAS mutant pancreatic cancer (PaCa) models with known resistance to CTX therapy. Computational modelling was employed for quantitative prediction of tumour response to various ADC dosing regimens. Results Site-selective coupling of an auristatin to CTX yielded an ADC with an average drug:antibody ratio (DAR) of 3.9, which elicited concentration- and EGFR-dependent cytotoxicity at sub-nanomolar potency in vitro. In human xenografts, the ADC inhibited tumour growth and prolonged survival, with no overt signs of toxicity. Key insights into factors governing ADC efficacy were obtained through a robust mathematical framework, including target-mediated dispositional effects relating to antigen density on tumour cells. Conclusions Together, our findings offer renewed hope for CTX in PaCa therapy, demonstrating that it may be reformatted as a next-generation ADC and combined with a predictive modelling tool to guide successful translation.

Fish behavioral responses are sensitive to chemicals in the water. Herein, rare minnow were tested for their shoal preference, and the shoal (school) factors including nutritional status, body size, and shoal (school) size which can make their preference most stable were measured. Then, shoal preference was measured again while fish and shoal were subjected to a concentration gradient of chemicals (cadmium ion [Cd2+ ], tricaine methanesulfonate [MS222], and p-chloroaniline). Results showed that single rare minnow preferred shoals over blank control tanks. And this preference was most stable when the shoal was well-fed and contained 20 individuals of 2 cm long. Although there was no significant response under exposure to p-chloroaniline, time spent of test fish from introduced into tank to start moving decreased greatly at concentrations of Cd2+ >3 mg/L and MS222 >11 mg/L. The time the test fish spent close to the shoal significantly decreased at Cd2+ >3 mg/L, MS222 >11 mg/L, and p-chloroaniline >10 mg/L, and the frequency of boundary line crossing increased at the same concentrations. The behavioral parameters changed by 20, 5, and 8 min once the lowest observed effect concentrations of Cd2+ , MS222, and p-chloroaniline, respectively, were added. This study provides useful information on rare minnow shoal preference that may be used for a biological early warning system. This article is protected by copyright. All rights reserved.

In spite of significant progress in the field of targeted anticancer therapy, the FDA has approved only five ADC-based drugs. Hence the search for new targeted anticancer agents is an unfulfilled necessity. Here, we present novel types of protein–drug conjugates (PDCs) that exhibit superior anticancer activities. Instead of a monoclonal antibody, we used fibroblast growth factor 2 (FGF2) as a targeting molecule. FGF2 is a natural ligand of fibroblast growth factor receptor 1 (FGFR1), a transmembrane receptor overproduced in various types of cancers. We synthesized site-specific and stoichiometric-controlled conjugates of FGF2 with a highly potent, hydrophilic derivative of auristatin called auristatin Y. To increase the hydrophilicity and hydrodynamic radius of conjugates, we employed PEG4 and PEG27 molecules as a spacer between the targeting molecule and the cytotoxic payload. All conjugates were selective to FGFR1-positive cell lines, effectively internalized via the FGFR1-dependent pathway, and exhibited a highly cytotoxic effect only on FGFR1-positive cancer cell lines.

Auristatins are important payloads used in antibody drug conjugates (ADCs), and the most well-known compound family member, monomethyl auristatin (MMAE), is used in two Food and Drug Administration (FDA)-approved ADCs, Adcetris® and Polivy®. Multiple other auristatin-based ADCs are currently being evaluated in human clinical trials and further studies on this class of molecule are underway by several academic and industrial research groups. Our group's main focus is to investigate the structure-activity relationships (SAR) of novel auristatins with the goal of applying these to next generation ADCs. Modifications of the auristatin backbone scaffold have been widely reported in the chemical literature focusing on the terminal subunits: P1 (N-terminus) and P5 (C-terminus). Our approach was to modulate the activity and hydrophilic character through modifications of the central subunits P2-P3-P4 and thorough SAR study on the P5 subunit. Novel hydrophilic auristatins were observed to have greater potency in vitro and displayed enhanced in vivo antitumor activity when conjugated via protease-cleavable linkers and delivered intracellularly. Analysis of ADC aggregation also indicated that novel hydrophilic payloads enabled the synthesis of high-drug-to-antibody ratio (DAR) ADCs that were resistant to aggregation. Modification of the central peptide subunits also resulted in auristatins with potent cytotoxic activity in vitro and these azide-modified auristatins contain a handle for linker attachment from the central portion of the auristatin backbone.

MS-222 (tricaine methanesulfonate) and eugenol are the two frequently used fish anesthetics. This study intends to analyze the regulation of these anesthetics in Chinese sea bass, Lateolabrax maculatus, through transcriptomic analysis. L. maculatus were exposed to MS-222 or eugenol, and those without any treatments were regarded as controls. Gills and livers were extracted for transcriptomic analysis after recovery in fresh water for 6 h. Identified genes were assigned to NR, COG, SWISS, GO, and KEGG database for predicting gene functions. A FDR ≤ 0.05 and |log2(FC)| ≥ 1 were applied to determined differentially expressed gene (DEG). A total of 45,626 unigenes were annotated using at least one database. The eugenol-treated liver group presented less DEGs compared with that treated by MS-222. Both the MS-222- and eugenol-treated liver groups presented notable DEGs that participated in human disease and metabolism pathways. The eugenol group showed more pathways related to detoxification activity and xenobiotics biodegradation, and those from the MS-222 group were related to organismal system such as reproduction. By comparing gill and liver samples using the same drug, the enriched pathways were generally consistent among the three comparisons. In conclusion, eugenol and MS-222 could change the pathways related to metabolism and immunity in L. maculatus. MS-222 may trigger more damages on the fish liver and reproduction.

The most successful therapeutic strategies for locally advanced cancers continue to combine decades-old classical radiosensitizing chemotherapies with radiotherapy. Molecular targeted radiosensitizers offer the potential to improve the therapeutic ratio by increasing tumor-specific kill while minimizing drug delivery and toxicity to surrounding normal tissue. Auristatins are a potent class of anti-tubulins that sensitize cells to ionizing radiation damage and are chemically amenable to antibody conjugation. To achieve tumor-selective radiosensitization, we synthesized and tested anti-HER2 antibody–drug conjugates of two auristatin derivatives with ionizing radiation. Monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) were attached to the anti-HER2 antibodies trastuzumab and pertuzumab through a cleavable linker. While MMAE is cell permeable, MMAF has limited cell permeability as free drug resulting in diminished cytotoxicity and radiosensitization. However, when attached to trastuzumab or pertuzumab, MMAF was as efficacious as MMAE in blocking HER2-expressing tumor cells in G2–M. Moreover, MMAF anti-HER2 conjugates selectively killed and radiosensitized HER2-rich tumor cells. Importantly, when conjugated to targeting antibody, MMAF had the advantage of decreased bystander and off-target effects compared with MMAE. In murine xenograft models, MMAF anti-HER2 antibody conjugates had less drug accumulated in the normal tissue surrounding tumors compared with MMAE. Therapeutically, systemically injected MMAF anti-HER2 conjugates combined with focal ionizing radiation increased tumor control and improved survival of mice with HER2-rich tumor xenografts. In summary, our results demonstrate the potential of cell-impermeable radiosensitizing warheads to improve the therapeutic ratio of radiotherapy by leveraging antibody–drug conjugate technology.

Intratumoral heterogeneity is a leading cause of treatment failure resulting in tumor recurrence. For the antibody-drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1), two major types of resistance include changes in human epidermal growth factor receptor 2 (HER2) expression and reduced payload sensitivity, which is often exacerbated by heterogenous HER2 expression and ADC distribution during treatment. ADCs with bystander payloads, such as trastuzumab-monomethyl auristatin E (T-MMAE), can reach and kill adjacent cells with lower receptor expression that cannot be targeted directly with the ADC. Additionally, coadministration of T-DM1 with its unconjugated antibody, trastuzumab, can improve distribution and minimize heterogeneous delivery. However, the effectiveness of trastuzumab coadministration and ADC bystander killing in heterogenous tumors in reducing the selection of resistant cells is not well understood. Here, we use an agent-based model to predict outcomes with these different regimens. The simulations demonstrate that both T-DM1 and T-MMAE benefit from trastuzumab coadministration for tumors with high average receptor expression (up to 70% and 40% decrease in average tumor volume, respectively), with greater benefit for nonbystander payloads. However, the benefit decreases as receptor expression is reduced, reversing at low concentrations (up to 360% and 430% increase in average tumor volume for T-DM1 and T-MMAE, respectively) for this mechanism that impacts both ADC distribution and efficacy. For tumors with intrinsic payload resistance, coadministration uniformly exhibits better efficacy than ADC monotherapy (50%-70% and 19%-36% decrease in average tumor volume for T-DM1 and T-MMAE, respectively). Finally, we demonstrate that several regimens select for resistant cells at clinical tolerable doses, which highlights the need to pursue other mechanisms of action for durable treatment responses. SIGNIFICANCE STATEMENT: Experimental evidence demonstrates heterogeneity in the distribution of both the antibody-drug conjugate and the target receptor in the tumor microenvironment, which can promote the selection of resistant cells and lead to recurrence. This study quantifies the impact of increasing the antibody dose and utilizing bystander payloads in heterogeneous tumors. Alternative cell-killing mechanisms are needed to avoid enriching resistant cell populations.

Purpose Telomerase is reactivated in non-small cell lung cancer (NSCLC), and it increases cell resistance to irradiation through protecting damaged telomeres and enhancing DNA damage repair. We investigated the radiosensitizing effect of BIBR1532, a highly selective telomerase inhibitor, and its corresponding mechanism in NSCLC. Methods and Materials Cell proliferation, telomerase activity, and telomere dysfunction-induced foci were measured with CCK-8 assay, real-time fluorescent quantitative polymerase chain reaction, and immunofluorescence. The effect of BIBR1532 on the response of NSCLC cells to radiation was analyzed using clonogenic survival and xenograft tumor assays. Cell death and cell senescence induced by BIBR1532 or ionizing radiation (IR), or both, were detected with western blotting, flow cytometry, and senescence-association β-galactosidase staining assay. Results We observed dose-dependent direct cytotoxicity of BIBR1532 at relatively high concentrations in NSCLC cells. Low concentrations of BIBR1532 did not appear toxic to NSCLC cells; however, they substantially increased the therapeutic efficacy of IR in vitro by enhancing IR-induced apoptosis, senescence, and mitotic catastrophe. Moreover, in a mouse xenograft model, BIBR1532 treatment synergized with IR at nontoxic dose levels promoted the antitumor efficacy of IR without toxicity to hematologic and internal organs. Mechanistically, lower concentrations of BIBR1532 effectively inhibited telomerase activity and increased IR-induced telomere dysfunction, resulting in disruption of chromosomal stability and inhibition of the ATM/CHK1 (ataxia-telangiectasia-mutated/Checkpoint kinase 1) pathway, which impaired DNA damage repair. Conclusions Our findings demonstrate that disturbances in telomerase function by nontoxic dose levels of BIBR1532 effectively enhance the radiosensitivity of NSCLC cells. This finding provides a rationale for the clinical assessment of BIBR1532 as a radiosensitizer.

Throughout history, natural products have significantly contributed to the discovery of novel chemistry, drug leads, and tool molecules to probe and address complex challenges in biology and medicine. Recent microbial genome sequencing efforts have uncovered many microbial biosynthetic gene clusters without an associated natural product. This means that the natural products isolated to date do not fully reflect the biosynthetic potential of microbial strains. This observation has rejuvenated the natural product community and inspired a return to microbial strain collections. Mining large microbial strain collections with the most current technologies in genome sequencing, bioinformatics, and high-throughput screening techniques presents new opportunities in natural product discovery. In this review, we report on the newly expanded microbial strain collection at The Scripps Research Institute, which represents one of the largest and most diverse strain collections in the world. Two complementary approaches, i.e. structure-centric and function-centric, are presented here to showcase how to leverage a large microbial strain collection for natural product discovery and to address challenges and harness opportunities for future efforts. Highlighted examples include the discovery of alternative producers of known natural products with superior growth characteristics and high titers, novel analogs of privileged scaffolds, novel natural products, and new activities of known and new natural products. We anticipate that this large microbial strain collection will facilitate the discovery of new natural products for many applications.