A novel and highly sensitive colorimetric DNA sensor for determination of miRNA-155 at attomolar levelsis presented that combines the peroxidase-like activity of copper nanoparticles (CuNPs) with the hybridization chain reaction (HCR) . The utilization of CuNPs offers advantages such as strong interaction with double-stranded DNA, excellent molecular recognition, and mimic catalytic activity. Herein, a capture probe DNA (P1) was immobilized on carboxylated magnetic beads (MBs), allowing for amplified immobilization due to the 3D surface. Subsequently, the presence of the target microRNA-155 led to the formation of a sandwich structure (P2/microRNA-155/P1/MBs) when P2 was introduced to the modified P1/MBs. The HCR reaction was then triggered by adding H1 and H2 to create a super sandwich (H1/H2)n. Following this, Cu2+ ions were attracted to the negatively charged phosphate groups of the (H1/H2)n and reduced by ascorbic acid, resulting in the formation of CuNPs, which were embedded into the grooves of the (H1/H2)n. The peroxidase-like activity of CuNPs catalyzed the oxidation reaction of 3,3\',5,5\'-Tetramethylbenzidine (TMB), resulting in a distinct blue color measured at 630 nm. Under optimal conditions, the colorimetric biosensor exhibited a linear response to microRNA-155 concentrations ranging from 80 to 500 aM, with a detection limit of 22 aM, and discriminate against other microRNAs. It was also successfully applied to the determination of microRNA-155 levels in spiked human serum.

The Mu opioid receptor (MOR) has been the subject of intense research over the past decades, especially in the field of analgesic therapeutics. It is the primary target for both clinical and recreational opioids. Recently, camelid-derived nanobodies have received significant attention due to their applicability in stabilizing the crystal structure of activated MOR, via specific recognition of and binding to the active receptor conformation. In the present study, we developed and applied a novel bio-assay to monitor MOR activation, utilizing intracellular expression of one such nanobody, Nb39. The principle of functional complementation of a split nanoluciferase was used to assess recruitment of Nb39 to MOR, following activation by a set of five synthetic opioids. The obtained pharmacological parameters-negative logarithm of EC50 (pEC50, as a measure of potency) and maximal response provoked by a ligand (Emax, as a measure of efficacy; relative to hydromorphone)-were compared with those obtained using a G protein recruitment assay, in which a mini-Gi protein (engineered GTPase domain of Gαi subunit) is recruited to activated MOR. Similar EC50 but distinct Emax values were obtained with both bio-assays, with lower Emax values for the Nb-based bio-assay. Both bio-assays may assist to gain better insight into activation of the MOR. Graphical abstract.

The landscape of new psychoactive substances (NPS) is constantly evolving, with new compounds entering the illicit drug market at a continuous pace. Of these, opioid NPS form a threat given their high potency and prevalence. Whereas previously, the use of fentanyl and fentanyl derivatives was the main point of attention, legislations have reacted accordingly, which may have been a driving force towards the (ab)use of alternative µ-opioid receptor (MOR) agonists. In contrast to fentanyl (analogues), details on these novel non-fentanyl opioid NPS are scarce. We investigated the biological activity of a panel of 11 \'alternative\', newly emerging MOR agonists (2-methyl-AP-237, AP-237, bromadol, brorphine, butorphanol, isotonitazene, mitragynine, 7-OH-mitragynine, MT-45, piperidylthiambutene, and tianeptine) using two closely related in vitro MOR activation bio-assays, monitoring either G protein (mini-Gi), or β-arrestin2 (βarr2) recruitment. Activity profiles were obtained for all tested compounds, with values for potency (EC50) ranging from 1.89 nM (bromadol) to > 3 µM (AP-237 and tianeptine). Bromadol, brorphine, isotonitazene, piperidylthiambutene, and tianeptine had the highest efficacy (Emax) values, exceeding that of the reference compound hydromorphone ≥ 1.3-fold (βarr2 assay) and > 2.6-fold (mini-Gi assay). Information on the recruitment of two distinct signaling molecules additionally enabled evaluation of biased agonism; none of the evaluated opioids being significantly biased. Taken together, this study is the first to systematically investigate the in vitro biological activity of a diverse panel of emerging non-fentanyl opioid NPS at MOR. Given the known danger of (fatal) intoxications with many opioid NPS, it is important to continuously monitor and characterize newly emerging compounds.

Fentanyl and morphine are agonists of the Mu opioid receptor (MOR), which is a member of the GPCR family. Their analgesic effects are associated with unwanted side effects. On a signaling level downstream from MOR, it has been hypothesized that analgesia may be mediated through the G protein pathway, whereas the undesirable effects of opioids have been linked to the β-arrestin (βarr) pathway. Despite being an increasingly debated subject, little is known about a potential \'bias\' (i.e. the preferential activation of one pathway over the other) of the novel synthetic opioids (NSO) - including fentanyl analogs - that have emerged on the illegal drug market. We have therefore developed and applied a novel, robust bio-assay platform to study the activity of 21 NSO, to evaluate to what extent these MOR agonists show biased agonism and to investigate the potential correlation with their structure. In addition, we evaluated the functional selectivity of TRV130, a purported G protein-biased agonist. We applied newly established stable bio-assays in HEK293T cells, based on the principle of functional complementation of a split nanoluciferase, to assess MOR activation via recruitment of a mini-Gi protein (GTPase domain of Gαi subunit) or βarr2. All but two of the tested NSO demonstrated a concentration-dependent response at MOR in both bio-assays. The developed bio-assays allow to gain insight into the βarr2 or G protein recruitment potential of NSO, which may eventually help to better understand why certain opioids are associated with higher toxicity. Adding to the recent discussion about the relevance of the biased agonism concept for opioids, we did not observe a significant bias for any of the evaluated compounds, including TRV130.

Acinetobacter baumannii is a non-motile, gram-negative member of the gamma proteobacteria. A specific and sensitive approach was established for the detection of Acintobacter baumannii via DNA based bio-assay. In this study, gold nano-star was synthesized and used for bio-conjugation with pDNA toward the detection of target sequences. Synthesized probe (5\' TTG TGA ACT ATT TAC GTC AGC ATG C3\') of Acinetobacter baumannii was found with excellent sensitivity. After the hybridization of pDNA with cDNA, target DNA (5\' GCA TGC TGA CGT AAA TAG TTC ACA A 3\') was easily measured. According to ultra-sensitivity of the engineered optical DNA-based bio-assay, it is potentially applied in the bacterial detection of the environmental and clinical specimens. Here, the selection of engineered biosensor in the presence of two mismatch sequences was investigated. The results indicated an acceptable choice for DNA-based assays. The low limit of quantification (LLOQ) of genosensor was obtained as 1 fM. The present study is a very important diagnostic examination to recognize Acinetobacter baumannii, which can be a best alternative to the traditional methods.

Haemophilus influenza (H. influenza) is a gram negative coccobacillus pathogenic microorganism. H. influenza produces beta-lactamases, and it is also able to modify its penicillin-binding proteins, so it has gained resistance to the penicillin family of antibiotics. In this work, a novel sensitive approach was established for the monitoring of H. influenza using DNA based bio-assay. For the first time, specific sequence of thiolated probe of Haemophilus influenza (SH-5\'-AAT TTT CCA ACT TTT TCA CCT GCA T-3\') was immobilized on the surface of gold (Au) electrode. Square wave voltammetry (SWV) was carried out in toluidine blue (TB) solution for DNA hybridization and targeting of cDNA sequence of Haemophilus influenza. Field scanning electron microscope (FE-SEM) was applied to investigation of the electrode morphology and estimate of particle size. In the optimal conditions, the planned strategy could detect target DNA (5\'-ATG CAG GTG AAA AAG TTG GAA AAT T-3\') down to 1 ZM with a linear range from 1 μM to 1 ZM. Moreover, engineered geno-assay selectively differentiates the complementary sequence from target sequences with one, double and three base mismatch sequences.

According to the European Pharmacopoeia monograph 1038 (Praeparationes homoeopathicae), homeopathic preparations are produced by successive dilution and succussion steps. Dilution levels beyond Avogadro\'s limit, however, render specific effects implausible according to standard scientific knowledge. Accordingly, we were interested in a critical empirical investigation of preparations produced according to this monograph. Within a precursor study we developed a bioassay based on a fingerprint metabolomic analysis of Lepidium sativum seeds germinated in vitro in either homeopathic preparations or controls in a blinded and randomized assignment. Results of the precursor study were not consistent with the hypothesis that the effects of a Stannum metallicum 30x preparation are identical to placebo. In the present study we investigated the reproducibility of these effects after scrutinizing and optimizing experimental procedures. Ten independent experiments were performed in a blinded and randomized assignment in two independent laboratories. Additionally, 10 systematic negative water control experiments were performed in both laboratories to critically assess the stability of the experimental set-up. The effects of the Stannum metallicum 30x treatment were reproduced. The systematic negative control experiments did not yield false-positive results, indicating a stable experimental set-up. We thus repeatedly observed biological effects conflicting with the assumption that Stannum metallicum 30x is identical to placebo. We therefore wish to discuss whether these findings are to be considered a scientific anomaly or whether they might stimulate further investigations to clarify whether application of the European Pharmacopoeia monograph 1038 may result in pharmaceutical preparations with specific effects.

Today, the emergence of the phenomenon of drug or multidrug-resistance for community-associated diseases represents a major concern in the world. In these contexts, the chronic infectious disease, leprosy, grounded by a slow-growing bacterium called Mycobacterium leprae or Mycobacterium lepromatosis is a leadingcause of severe disfiguring skin sores and nerve damage in the arms, legs, and skin areas around the body. Even, over 200,000 new leprosy cases are being accounted every year along with the relapsed leprosy cases. Nonetheless, this has been considered a curable disease with a higher dose of multidrug therapy (MDT) for a long period of time. The prolonged action of a high dose of combination drugs administration may cause an adverse reaction that can significantly affect patient compliance, particularly the outbreak of multidrug-resistance in the infected person. To overcome these shortfalls or prevent the resistance-associated problems, researchers are diligently involved in the structural modifications of the clinically used anti-leprosy drugs or the allied compounds for the structure-antimycobacterial activity relationship study. This review article described the detailed synthesis and biological assays of different anti-leprosy compounds reported by several research groups.

Indole- and indazole-based synthetic cannabinoid receptor agonists (SCRAs), featuring valine or tert-leucine substituents, are commonly abused new psychoactive substances (NPS). A major metabolic pathway for these SCRAs is hydrolysis of the terminal amide or methylester functionalities. Although these hydrolysis products were already detected as main ingredients in some \"legal highs,\" these metabolites are often poorly characterized. Here, we report a systematic investigation of the activity of 7 common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L-valine or L-tert-leucine in direct comparison to their parent compounds. An activity-based cannabinoid receptor 1 (CB1 ) bio-assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293T cell system. The recruitment of β-arrestin2 to the activated CB1 (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96-well plate-reader. The major hydrolysis metabolites of 5F-AB-PINACA, ADB-CHMICA, ADB-CHMINACA, ADB-FUBICA, and their methyl- and ethylester derivatives showed no detectable CB1 activation at concentrations up to 1 μM. On the other hand, metabolites of 5F-ADB-PINACA, AB-CHMINACA, and ADB-FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC50 values >100 nM). Activity-based characterization of SCRAs and their metabolites at CB1 may not only allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications, but may also allow application of the concept of \"activity equivalents\" present in biological fluids or, alternatively, in confiscated materials.

Metal colloids in 2 propanol using nanoparticles (NPs) of copper, nickel and cobalt were prepared by Chemical Liquid Deposition (CLD) method. The resulting colloidal dispersions were characterized by Transmission Electron Microscopy (TEM). The colloids were supported in chitosan. Then, microbiological assays were performed using E. coli and S. aureus in order to determine the bactericide/bacteriostatic activity of nanoparticles (NPs) trapped or chelated with chitosan. Finally, the toxicity of the metal colloids Cu, Ni and Co was tested. Bio-assays were conducted in three different animal species. First of all on earth warms (Eisenia foetida) to evaluate the toxicity and the biocompatibility of chitosan in lactic acid (1% and 0.5%). Secondly bio-assay done in fishes (rainbow trout), the liver toxicity of NPs in vivo was evaluated. Finally, a bio-assay was conducted in Sprange-Dawley rats of 100g weight, which were injected intraperitoneally with different solutions of chitosan metal colloids. Then, the minimum and maximum concentration were determined for copper, nickel and cobalt. The purpose of the use of chitosan was acting as a carrier for some magnetic NPs, which toxicity would allow to obtain new polymeric materials with potential applications as magnet future drugs carrier.