Recent advancement in small molecules as HCV inhibitors.

Hepatitis C virus (HCV) has caused a considerable threat to human health. To date, no treatments are without side effects. The proteins and RNA associated with HCV have specific functions during the viral life cycle. The vulnerabilities to virus are associated with those proteins or RNA. Thus, targeting these proteins and RNA is an efficient strategy to develop anti-HCV therapeutics. The treatment for HCV-infected patients has been greatly improved after the approval of direct-acting antivirals (DAAs). However, the cost of DAAs is unusually high, which adds to the economic burden on patients with chronic liver diseases. So far, many efforts have been devoted to the development of small molecules as novel HCV inhibitors. Investigations on the inhibitory activities of these small molecules have involved the target identification and the mechanism of action. In this mini-review, these small molecules divided into four kinds were elaborated, which focused on their targets and structural features. Furthermore, we raised the current challenges and promising prospects. This mini-review may facilitate the development of small molecules with improved activities targeting HCV based on the chemical scaffolds of HCV inhibitors.

A highly selective switch-on fluorescence sensor targeting telomeric dimeric G-quadruplex.

The fluorescence probes with high selectivity and sensitivity for telomeric multimeric G-quadruplexes have attracted much attention. Nevertheless, few small molecules have exhibited telomeric multimeric G-quadruplexes recognition specificity. Thus, there is an urgent demand to develop specific fluorescence probes for telomeric multimeric G-quadruplexes. We reported herein the specific sensing of telomeric dimeric G-quadruplex TTA45 via a fluorescence light-up response using a commercially available triazine derivative HPTA-1 as a probe. HPTA-1 could discriminate the telomeric dimeric G-quadruplex TTA45 against other types of DNA structures accompanied by a drastic enhancement of the emission intensity without compromising the conformation and stability. Compared with most multimeric G-quadruplex recognition ligands, HPTA-1 had much simpler structure and lower molecular weight. The binding mechanism studies suggested that the distinct fluorescence response was caused by electrostatic and π-π stacking interactions of HPTA-1 with the pocket between two G-quadruplex units of telomeric dimeric G-quadruplex TTA45..

Recent advances in the development of small molecules targeting RNA G-quadruplexes for drug discovery.

Extensive evidence indicates that RNA G-quadruplexes have associated with some important cellular events. Investigation of RNA G-quadruplexes is thus vital to revealing their biofunctions. Several small molecules have been developed to target RNA G-quadruplexes to date. Some of the small molecules showed significantly light-up fluorescence signals upon binding to RNA G-quadruplexes, while some of them regulated the biofunctions of RNA G-quadruplexes. In this mini-review, the small molecules divided into four kinds are expounded which focused mainly on their structural features and biological activities. Moreover, we raised the current challenges and promising prospects. This mini-review might contribute to exploiting more sophisticated small molecules targeting RNA G-quadruplexes with high specificity based on the reported chemical structural features.

Selective recognition of c-MYC Pu22 G-quadruplex by a fluorescent probe.

Nucleic acid mimics of fluorescent proteins can be valuable tools to locate and image functional biomolecules in cells. Stacking between the internal G-quartet, formed in the mimics, and the exogenous fluorophore probes constitutes the basis for fluorescence emission. The precision of recognition depends upon probes selectively targeting the specific G-quadruplex in the mimics. However, the design of probes recognizing a G-quadruplex with high selectivity in vitro and in vivo remains a challenge. Through structure-based screening and optimization, we identified a light-up fluorescent probe, 9CI that selectively recognizes c-MYC Pu22 G-quadruplex both in vitro and ex vivo. Upon binding, the biocompatible probe emits both blue and green fluorescence with the excitation at 405 nm. With 9CI and c-MYC Pu22 G-quadruplex complex as the fluorescent response core, a DNA mimic of fluorescent proteins was constructed, which succeeded in locating a functional aptamer on the cellular periphery. The recognition mechanism analysis suggested the high selectivity and strong fluorescence response was attributed to the entire recognition process consisting of the kinetic match, dynamic interaction, and the final stacking. This study implies both the single stacking state and the dynamic recognition process are crucial for designing fluorescent probes or ligands with high selectivity for a specific G-quadruplex structure.

Recent advances in the development of ligands specifically targeting telomeric multimeric G-quadruplexes.

Long human telomeric DNA sequence could form higher-order G-quadruplex structures, namely telomeric multimeric G-quadruplexes. The formation of telomeric multimeric G-quadruplexes has been demonstrated. Several efforts have been devoted to the development of ligands targeting telomeric multimeric G-quadruplexes in recent years. The reported ligands specifically targeting telomeric multimeric G-quadruplexes exhibited either high anticancer activity with effective stabilization ability or distinct fluorescence responses to telomeric multimeric G-quadruplexes. In this review, the ligands including three types of small molecules are discussed which focus on their structural features and binding modes. Furthermore, we put forward the promising prospects and current challenges. This presented review might provide new strategies to exploit more sophisticated ligands targeting telomeric multimeric G-quadruplexes.

Effectiveness of traditional Chinese herbal medicine, San-Huang-San, in combination with enrofloxacin to treat AHPND-causing strain of Vibrio parahaemolyticus infection in Litopenaeus vannamei.

The effects of oral administration of enrofloxacin (ENR) and San-Huang-San (SHS), singly or in combination, on the survival performance, disease resistance, and immunity of Litopenaeus vannamei were investigated. After challenge with an AHPND-causing strain of Vibrio parahaemolyticus (VPAHPND), shrimp were immediately fed a drug-free diet, diets containing only ENR (20 mg·kg-1) or SHS (500 mg·kg-1) or diets containing low-dose (10 mg·kg-1 ENR + 250 mg ·kg-1 SHS), medium-dose (20 mg·kg-1 ENR + 500 mg ·kg-1 SHS), and high-dose (40 mg·kg-1 ENR + 1000 mg ·kg-1 SHS) drug combinations for 5 days. The cumulative shrimp mortality over 5 days after injection of VPAHPND in the ENR + SHS combination groups was significantly lower than that in the ENR or SHS alone groups (p < 0.05). Immune parameters, including the vibrio density, total hemocyte counts (THCs), hemocyanin (HEM) concentration, antibacterial activity, activity levels of lysozyme (LZM), acid phosphatase (ACP), alkaline phosphatase (AKP), and phenoloxidase (PO) in cell-free hemolymph, and the expression levels of the immune-related genes anti-lipopolysaccharide factor (ALF), cathepsin B (catB), crustin, lectin (Lec), lysozyme (LZM), and Toll-like receptor (TLR) in hemocytes were determined in the shrimp. The results showed that the shrimp in drug combination groups cleared more VPAHPND than that in the ENR or SHS group in the same time. The values for other immune parameters in the drug combination groups were higher than those in the ENR or SHS group (p < 0.05). Finally, in the histological examinations, the histological structural alignment and integrity of the hepatopancreatic tubules in the drug combination groups were better than that in the ENR and SHS groups. Under the experimental conditions, compared with ENR or SHS used alone, the combination use of ENR and SHS could improve immunity and disease resistance in shrimp after VPAHPND infection, and could reduce the use of ENR when the better therapeutic effect was achieved.

Evaluation of combination effects of Astragalus polysaccharides and florfenicol against acute hepatopancreatic necrosis disease-causing strain of Vibrio parahaemolyticus in Litopenaeus vannamei.

The effects of oral administration of Astragalus polysaccharides (APS) and florfenicol (FFC), singly or in combination, on the survival performance, disease resistance, and immunity of Litopenaeus vannamei were investigated. After challenge with an AHPND-causing strain of Vibrio parahaemolyticus (VPAHPND), shrimp were immediately fed a drug-free diet, diets containing only APS (200 mg·kg-1) or FFC (15 mg·kg-1), or diets containing low-dose (7.5 mg·kg-1 FFC + 100 mg·kg-1 APS), medium-dose (15 mg·kg-1 FFC + 200 mg·kg-1 APS), and high-dose (30 mg·kg-1 FFC+400 mg·kg-1 APS) drug combinations for 5 days. The cumulative shrimp mortality over 5 days after injection of VPAHPND in the APS + FFC combination groups was significantly lower than that in the APS or FFC alone groups (p < 0.05). Immune parameters, including the total hemocyte counts (THCs), hemocyanin (HEM) concentration, antibacterial activity, activity levels of lysozyme (LZM), and levels of acid phosphatase (ACP), alkaline phosphatase (AKP), and phenoloxidase (PO) in cell-free hemolymph, and the expression levels of the immune-related genes anti-lipopolysaccharide factor (ALF), cathepsin B (catB), crustin, lectin (Lec), lysozyme (LZM), and Toll-like receptor (TLR) in hemocytes and hepatopancreas were determined in the shrimp. The values for these immune parameters in the drug combination groups were higher than those in the APS or FFC group (p < 0.05). Finally, in the histological examinations, the histological structural alignment and integrity of the hepatopancreatic tubules in the drug combination groups was better than that in the APS and FFC groups. Under the experimental conditions, dietary APS and FFC had a synergistic effect on immunity and disease resistance among shrimp after VPAHPND infection.

Comparison of florfenicol pharmacokinetics in Exopalaemon carinicauda at different temperatures and administration routes.

Florfenicol is a broad-spectrum antibacterial drug. Exopalaemon carinicauda is important in the prawn aquaculture industry in China. Florfenicol pharmacokinetics in E. carinicauda were studied at different temperatures and via different routes of administration to provide a scientific basis for the rational use of drugs for E. carinicauda production. At water temperatures of 22 ± 0.4°C and 28 ± 0.3°C, after intramuscular (IM) injection and oral (per ora (PO)) administration of florfenicol at 10 mg/kg body weight (BW) and 30 mg/kg BW, respectively, the florfenicol concentration in the plasma, hepatopancreas, gills, muscles, and carapace of E. carinicauda was determined by high-performance liquid chromatography. After IM injection at different temperatures, the metabolism of florfenicol in E. carinicauda conformed to a two-compartment open model with zero-order absorption. After PO administration, the metabolism of florfenicol in E. carinicauda was consistent with a two-compartment open model with first-order absorption. Using an identical administration route but different water temperatures, the metabolism of florfenicol in E. carinicauda was quite different. Overall, florfenicol was absorbed rapidly and distributed widely in E. carinicauda, but elimination was slow and the bioavailability was not high. A low temperature and PO administration resulted in a low elimination rate.

Pharmacokinetic profiles of florfenicol in spotted halibut, Verasper variegatus, at two water temperatures.

The pharmacokinetic profiles of florfenicol in the spotted halibut (Verasper variegatus) were investigated at 15 and 20°C water temperatures, respectively. Florfenicol content in plasma samples was analyzed using an HPLC method. Drug concentration versus time data were best fitted to a three-compartment model after a single intravenous administration (15 mg/kg BW), and fitted to a two-compartment model after an oral administration (30 mg/kg BW) at 15 and 20°C. The florfenicol concentration in the blood increased slowly during the 12 hr following an oral administration at 15°C, with a peak concentration (Cmax ) of 9.1 mg/L, and then declined gradually. The half-lives of absorption, distribution, and elimination phase were 2.18, 5.66 and 14.25 hr, respectively. The bioavailability (F) was calculated to be 24.14%. After an oral administration at 20°C, shorter half-lives of absorption (1.33 hr), distribution (2.51 hr) and elimination (9.71 hr), a higher Cmax (12.2 mg/L), and a similar F (23.98%) were found. Based on the pharmacokinetics and pharmacodynamics, an oral dose of 30 mg/kg BW was suggested to be efficacious for bacterial disease control in spotted halibut farming.

Transcriptome analysis of the hepatopancreas in Exopalaemon carinicauda infected with an AHPND-causing strain of Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus carrying toxin-producing plasmid, has led to severe mortalities in multiple shrimp species throughout Asia. In order to understand the immunological response of shrimp to infection by AHPND-causing strain of V. parahaemolyticus (VPAHPND), the transcriptomic profiles of the hepatopancreas from severe AHPND-infected (BS_G), AHPND-survived (KB_G) and non-infected (PBS_G) Exopalaemon carinicauda groups were obtained using HiSeq™ 2500 (Illumina). In total, 525 million high quality clean reads were obtained in nine libraries and de novo assembled into 130,082 unigenes with an average length of 724 bp. Based on sequence similarity, 22.75% unigenes were annotated in the public databases. Comparative analysis revealed that 3733 genes differentially expressed in VPAHPND infection compared with the controls, including 1114 and 3461 unigenes in BS_G vs PBS_G and KB_G vs PBS_G, respectively. A total of 229 differential expressed genes that have high homologies with the known proteins in crustacean species were identified, among which 127 genes are reported potentially related to immune function. We identified relative genes and pathways associated with AHPND pathogenesis and defenses. Our results provide valuable information for further analysis of the mechanisms of shrimp defense against AHPND infection.

Effects of ammonia-N stress on metabolic and immune function via the neuroendocrine system in Litopenaeus vannamei.

The purpose of this study was to evaluate the immunological responses, such as phenoloxidase (PO), antibacterial, and bacteriolytic activities, and metabolic variables, such as oxyhemocyanin, lactate, and glucose levels, of Litopenaeus vannamei exposed to ambient ammonia-N at 0, 2.5, 5, 7.5, and 10 mg/L for 0, 3, 6, 12, 24, and 48 h, and determine the effects of the eyestalk hormone on the metabolic and immune functions of unilateral eyestalk-ablated L. vannamei exposed to ambient ammonia-N at 10 mg/L. The actual concentrations of the control and test solutions were 0.04, 2.77, 6.01, 8.30, and 11.36 mg/L for ammonia-N and 0.01, 0.15, 0.32, 0.44, and 0.60 mg/L for NH3-N (unionized ammonia). The results showed a significant decrease in the PO, antibacterial, and bacteriolytic activities in the plasma as well as a significant increase in the glucose and lactate levels and decreased oxyhemocyanin levels in the hemolymph of L. vannamei exposed to elevated ammonia-N levels. These findings indicated that L. vannamei exposed to ammonia-N might demonstrate weakened metabolic and immunological responses. Moreover, eyestalk removal caused a dramatic decrease in PO, antibacterial, and bacteriolytic activities, which indicated that the eyestalk hormone in L. vannamei exhibited a higher immune response due to the induction of protective mechanisms against ammonia-N stress. Eyestalk removal also caused a dramatic decrease in glucose and lactate levels, suggesting that the eyestalk hormone is involved in glucose metabolism to meet the energy requirements under ammonia-N stress conditions.

Cytotoxic and mutagenic properties of O4-alkylthymidine lesions in Escherichia coli cells.

Due to the abundant presence of alkylating agents in living cells and the environment, DNA alkylation is generally unavoidable. Among the alkylated DNA lesions, O(4)-alkylthymidine (O(4)-alkyldT) are known to be highly mutagenic and persistent in mammalian tissues. Not much is known about how the structures of the alkyl group affect the repair and replicative bypass of the O(4)-alkyldT lesions, or how the latter process is modulated by translesion synthesis polymerases. Herein, we synthesized oligodeoxyribonucleotides harboring eight site-specifically inserted O(4)-alkyldT lesions and examined their impact on DNA replication in Escherichia coli cells. We showed that the replication past all the O(4)-alkyldT lesions except (S)- and (R)-sBudT was highly efficient, and these lesions directed very high frequencies of dGMP misincorporation in E. coli cells. While SOS-induced DNA polymerases play redundant roles in bypassing most of the O(4)-alkyldT lesions, the bypass of (S)- and (R)-sBudT necessitated Pol V. Moreover, Ada was not involved in the repair of any O(4)-alkyldT lesions, Ogt was able to repair O(4)-MedT and, to a lesser extent, O(4)-EtdT and O(4)-nPrdT, but not other O(4)-alkyldT lesions. Together, our study provided important new knowledge about the repair of the O(4)-alkyldT lesions and their recognition by the E. coli replication machinery.

In vivo detection and replication studies of α-anomeric lesions of 2'-deoxyribonucleosides.

DNA damage, arising from endogenous metabolism or exposure to environmental agents, may perturb the transmission of genetic information by blocking DNA replication and/or inducing mutations, which contribute to the development of cancer and likely other human diseases. Hydroxyl radical attack on the C1', C3' and C4' of 2-deoxyribose can give rise to epimeric 2-deoxyribose lesions, for which the in vivo occurrence and biological consequences remain largely unexplored. Through independent chemical syntheses of all three epimeric lesions of 2'-deoxyguanosine (dG) and liquid chromatography-tandem mass spectrometry analysis, we demonstrated unambiguously the presence of substantial levels of the α-anomer of dG (α-dG) in calf thymus DNA and in DNA isolated from mouse pancreatic tissues. We further assessed quantitatively the impact of all four α-dN lesions on DNA replication in Escherichia coli by employing a shuttle-vector method. We found that, without SOS induction, all α-dN lesions except α-dA strongly blocked DNA replication and, while replication across α-dA was error-free, replicative bypass of α-dC and α-dG yielded mainly C→A and G→A mutations. In addition, SOS induction could lead to markedly elevated bypass efficiencies for the four α-dN lesions, abolished the G→A mutation for α-dG, pronouncedly reduced the C→A mutation for α-dC and triggered T→A mutation for α-dT. The preferential misincorporation of dTMP opposite the α-dNs could be attributed to the unique base-pairing properties of the nucleobases elicited by the inversion of the configuration of the N-glycosidic linkage. Our results also revealed that Pol V played a major role in bypassing α-dC, α-dG and α-dT in vivo. The abundance of α-dG in mammalian tissue and the impact of the α-dNs on DNA replication demonstrate for the first time the biological significance of this family of DNA lesions.

PRECLINICAL PHARMACOKINETIC ANALYSIS OF (E)-METHYL-4-ARYL-4-OXABUT-2-ENOATE, A NOVEL SER/THR PROTEIN KINASE B INIBITOR, IN RATS.

(E)-Methyl-4-aryl-4-oxabut-2-enoate, designated YH-8, is a novel Serflhr protein kinase B (PknB) inhibitor, which is designed for the treatment of tuberculosis. The aim of this study was to investigate the pharmacokinetics, bioavailability, tissue distribution and excretion characteristics of YH-8 in rats and study its plasma protein binding in vitro. The pharmacokinetic properties were examined after intravenously injected YH-8 at 10 and 20 mg/kg and oral administrated YH-8 at 50, 100 and 200 mg/kg to rats. The concentrations of YH-8 in plasma were determined with LC-MS/MS, with a liquid-liquid extraction. The tissue distribution and urinary, fecal and -biliary excretion patterns of YH-8 were investigated following a single oral dosing of 100 mg/kg. The plasma protein binding rates of YH-8 were determined using ultra-filtration method. After intra- venous and oral administration, YH-8 showed dose-independent pharmacokinetic characteristics, with T(1/2) of approximately 5.5 h and 7.1 h, respectively. The oral absolute bioavailability of YH-8 was relatively low (about 12%). YH-8 was widely distributed in various tissues and showed substantial deposition in intestine, stomach, liver, lung and kidney. The drug was mainly eliminated via fecal excretion and its binding rate with plasma protein was concentration-dependent. In conclusion, this study as first provided the full pharmacokinetic characteristics of YH-8, which would be helpful for its further development and clinical application.

Syntheses and characterizations of the in vivo replicative bypass and mutagenic properties of the minor-groove O2-alkylthymidine lesions.

Endogenous metabolism, environmental exposure, and treatment with some chemotherapeutic agents can all give rise to DNA alkylation, which can occur on the phosphate backbone as well as the ring nitrogen or exocyclic nitrogen and oxygen atoms of nucleobases. Previous studies showed that the minor-groove O(2)-alkylated thymidine (O(2)-alkyldT) lesions are poorly repaired and persist in mammalian tissues. In the present study, we synthesized oligodeoxyribonucleotides harboring seven O(2)-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu or sBu, at a defined site and examined the impact of these lesions on DNA replication in Escherichia coli cells. Our results demonstrated that the replication bypass efficiencies of the O(2)-alkyldT lesions decreased with the chain length of the alkyl group, and these lesions directed promiscuous nucleotide misincorporation in E. coli cells. We also found that deficiency in Pol V, but not Pol II or Pol IV, led to a marked drop in bypass efficiencies for most O(2)-alkyldT lesions. We further showed that both Pol IV and Pol V were essential for the misincorporation of dCMP opposite these minor-groove DNA lesions, whereas only Pol V was indispensable for the T→A transversion introduced by these lesions. Depletion of Pol II, however, did not lead to any detectable alterations in mutation frequencies for any of the O(2)-alkyldT lesions. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O(2)-alkyldT lesions and revealed the roles of the SOS-induced DNA polymerases in bypassing these lesions in E. coli cells.

Cytotoxic and mutagenic properties of regioisomeric O²-, N3- and O4-ethylthymidines in bacterial cells.

Exposure to environmental agents and endogenous metabolism can both give rise to DNA alkylation. Thymine is known to be alkylated at O(2), N3 and O(4) positions; however, it remains poorly explored how the regioisomeric alkylated thymidine lesions compromise the flow of genetic information by perturbing DNA replication in cells. Herein, we assessed the differential recognition of the regioisomeric O(2)-, N3- and O(4)-ethylthymidine (O(2)-, N3- and O(4)-EtdT) by the DNA replication machinery of Escherichia coli cells. We found that O(4)-EtdT did not inhibit appreciably DNA replication, whereas O(2)- and N3-EtdT were strongly blocking to DNA replication. In addition, O(4)-EtdT induced a very high frequency of T→C mutation, whereas nucleotide incorporation opposite O(2)- and N3-EtdT was promiscuous. Replication experiments with the use of polymerase-deficient cells revealed that Pol V constituted the major polymerase for the mutagenic bypass of all three EtdT lesions, though Pol IV also contributed to the T→G mutation induced by O(2)- and N3-EtdT. The distinct cytotoxic and mutagenic properties of the three regioisomeric lesions could be attributed to their unique chemical properties.

In vivo antibacterial activity of MRX-I, a new oxazolidinone.

MRX-I is a potent oxazolidinone antibiotic against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), penicillin-intermediate S. pneumoniae (PISP), and vancomycin-resistant enterococci (VRE). In this study, the in vivo efficacy of orally administered MRX-I was evaluated using linezolid as a comparator. MRX-I showed the same or better efficacy than linezolid in both systemic and local infection models against the tested strains.

Design, Synthesis, and Antifungal Activities of Novel Potent Fluoroalkenyl Succinate Dehydrogenase Inhibitors.

The development of new fungicide molecules is a crucial task for agricultural chemists to enhance the effectiveness of fungicides in agricultural production. In this study, a series of novel fluoroalkenyl modified succinate dehydrogenase inhibitors were synthesized and evaluated for their antifungal activities against eight fungi. The results from the in vitro antifungal assay demonstrated that compound 34 exhibited superior activity against Rhizoctonia solani with an EC50 value of 0.04 µM, outperforming commercial fluxapyroxad (EC50 = 0.18 µM) and boscalid (EC50 = 3.07 µM). Furthermore, compound 34 showed similar effects to fluxapyroxad on other pathogenic fungi such as Sclerotinia sclerotiorum (EC50 = 1.13 µM), Monilinia fructicola (EC50 = 1.61 µM), Botrytis cinerea (EC50 = 1.21 µM), and also demonstrated protective and curative efficacies in vivo on rapeseed leaves and tomato fruits. Enzyme activity experiments and protein-ligand interaction analysis by surface plasmon resonance revealed that compound 34 had a stronger inhibitory effect on succinate dehydrogenase compared to fluxapyroxad. Additionally, molecular docking and DFT calculation confirmed that the fluoroalkenyl unit in compound 34 could enhance its binding capacity with the target protein through p-π conjugation and hydrogen bond interactions.

Non-destructive visual detection of fresh food spoilage.

Two coumarin derivatives SWJT-33 and SWJT-34 were synthesized as probes for the detection of cadaverine (Cad). Among them, SWJT-34 exhibits high selectivity and low detection limits for Cad using fluorescence or UV-vis spectrum. The detection limits for Cad were calculated to be 0.018 µM by fluorescence spectrum and 0.34 µM by UV-vis spectrum, which was much lower than those of most of reported probes. The probe could realize colorimetric and ratiometric detection of Cad because an aza-Michael addition reaction occurred between SWJT-34 and Cad to form the Schiff base compound as a product. In addition, SWJT-34 could be fabricated as portable test strips for the selective detection of Cad in vapor, and the corresponding detection limits were calculated to be 1.88 ppm in the fluorescent mode and 57 ppm in UV-vis mode, respectively. Additionally, these test strips could be used to test the freshness of Rottweiler shrimp and pork at different temperatures.