Sex differences in neuronal activation in the cortex and midbrain during quinine-adulterated alcohol intake.

AIMS: Continued alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This is modeled in mice by pairing negative stimuli with alcohol, such as adulterating alcohol solution with quinine. Mice consuming alcohol under these conditions are considered to be engaging in aversion-resistant intake. Previously, we have observed sex differences in this behavior, with females more readily expressing aversion-resistant consumption. We also identified three brain regions that exhibited sex differences in neuronal activation during quinine-alcohol drinking: ventromedial prefrontal cortex (vmPFC), posterior insular cortex (PIC), and ventral tegmental area (VTA). Specifically, male mice showed increased activation in vmPFC and PIC, while females exhibited increased activation in VTA. In this study, we aimed to identify what specific type of neurons are activated in these regions during quinine-alcohol drinking. METHOD: We assessed quinine-adulterated alcohol intake using the two-bottle choice procedure. We also utilized RNAscope in situ hybridization in the three brain regions that previously exhibited a sex difference to examine colocalization of Fos, glutamate, GABA, and dopamine. RESULT: Females showed increased aversion-resistant alcohol consumption compared to males. We also found that males had higher colocalization of glutamate and Fos in vmPFC and PIC, while females had greater dopamine and Fos colocalization in the VTA. CONCLUSIONS: Collectively, these experiments suggest that glutamatergic output from the vmPFC and PIC may have a role in suppressing, and dopaminergic activity in the VTA may promote, aversion-resistant alcohol consumption. Future experiments will examine neuronal circuits that contribute to sex differences in aversion resistant consumption.

Sodium Bicarbonate Decreases Alcohol Consumption in Mice.

We previously reported that mice with low neuronal pH drink more alcohol, demonstrating the importance of pH for alcohol reward and motivation. In this study, we tested whether systemic pH affects alcohol consumption and if so, whether it occurs by changing the alcohol reward. C57BL/6J mice were given NaHCO3 to raise their blood pH, and the animals' alcohol consumption was measured in the drinking-in-the-dark and two-bottle free choice paradigms. Alcohol consumption was also assessed after suppressing the bitterness of NaHCO3 with sucrose. Alcohol reward was evaluated using a conditioned place preference. In addition, taste sensitivity was assessed by determining quinine and sucrose preference. The results revealed that a pH increase by NaHCO3 caused mice to decrease their alcohol consumption. The decrease in high alcohol contents (20%) was significant and observed at different ages, as well as in both males and females. Alcohol consumption was also decreased after suppressing NaHCO3 bitterness. Oral gavage of NaHCO3 did not alter quinine and sucrose preference. In the conditioned place preference, NaHCO3-treated mice spent less time in the alcohol-injected chamber. Conclusively, the results show that raising systemic pH with NaHCO3 decreases alcohol consumption, as it decreases the alcohol reward value.

Change in taste sensation after orthognathic surgery.

OBJECTIVE: The objective of this study was to evaluate the effect of orthognathic surgery on taste sensation. MATERIALS AND METHODS: Thirty-five patients scheduled to undergo Le Fort I osteotomy (LFIO), sagittal split ramus osteotomy (SSRO), and bimaxillary surgery (BMS) were evaluated by administering localized and whole-mouth taste tests preoperatively and postoperatively at months 1, 3, and 6. The patients were asked to identify the quality of four basic tastes applied to six locations on the palate and tongue and to rate the taste intensities they perceived. Taste recognition thresholds and taste intesity scores were evaluted according to operation groups and follow-ups. RESULTS: There were significant decreases in the quinine HCl recognition thresholds at the postoperative follow-ups compared to the preoperative in LFIO patients (p = 0.043). There were significant decreases in sucrose taste intensity scores in the right posterolateral part of the tongue at months 3 and 6 compared to preoperative in SSRO patients (p = 0.046), and significant increases in quinine HCL taste intensity scores in the right and left anterior parts of the tongue at month 6 compared to preoperative in LFIO patients (p < 0.05). CONCLUSION: Taste perception is affected due to potential damage to the chemosensory nerves during orthognathic surgical procedures. Generally, non-significant alterations have been observed in taste perception after orthognathic surgery, except for significant alterations in bitter and sweet taste perceptions. CLINICAL RELEVANCE: Maxillofacial surgeons should be aware of taste perception change after orthognathic surgery procedures and patients should be informed accordingly. THE TRIAL REGISTRATION NUMBER (TRN): NCT06103422/Date of registration: 10.17.2023 (retrospectively registered).

Diagnostic Positron Emission Tomography Imaging with Zirconium-89 Desferrioxamine B Squaramide: From Bench to Bedside.

ConspectusMolecular imaging with antibodies radiolabeled with positron-emitting radionuclides combines the affinity and selectivity of antibodies with the sensitivity of Positron Emission Tomography (PET). PET imaging allows the visualization and quantification of the biodistribution of the injected radiolabeled antibody, which can be used to characterize specific biological interactions in individual patients. This characterization can provide information about the engagement of the antibody with a molecular target such as receptors present in elevated levels in tumors as well as providing insight into the distribution and clearance of the antibody. Potential applications of clinical PET with radiolabeled antibodies include identifying patients for targeted therapies, characterization of heterogeneous disease, and monitoring treatment response.Antibodies often take several days to clear from the blood pool and localize in tumors, so PET imaging with radiolabeled antibodies requires the use of a radionuclide with a similar radioactive half-life. Zirconium-89 is a positron-emitting radionuclide that has a radioactive half-life of 78 h and relatively low positron emission energy that is well suited to radiolabeling antibodies. It is essential that the zirconium-89 radionuclide be attached to the antibody through chemistry that provides an agent that is stable in vivo with respect to the dissociation of the radionuclide without compromising the biological activity of the antibody.This Account focuses on our research using a simple derivative of the bacterial siderophore desferrioxamine (DFO) with a squaramide ester functional group, DFO-squaramide (DFOSq), to link the chelator to antibodies. In our work, we produce conjugates with an average ∼4 chelators per antibody, and this does not compromise the binding of the antibody to the target. The resulting antibody conjugates of DFOSq are stable and can be easily radiolabeled with zirconium-89 in high radiochemical yields and purity. Automated methods for the radiolabeling of DFOSq-antibody conjugates have been developed to support multicenter clinical trials. Evaluation of several DFOSq conjugates with antibodies and low molecular weight targeting agents in tumor mouse models gave PET images with high tumor uptake and low background. The promising preclinical results supported the translation of this chemistry to human clinical trials using two different radiolabeled antibodies. The potential clinical impact of these ongoing clinical trials is discussed.The use of DFOSq to radiolabel relatively low molecular weight targeting molecules, peptides, and peptide mimetics is also presented. Low molecular weight molecules typically clear the blood pool and accumulate in target tissue more rapidly than antibodies, so they are usually radiolabeled with positron-emitting radionuclides with shorter radioactive half-lives such as fluorine-18 (t1/2 ∼ 110 min) or gallium-68 (t1/2 ∼ 68 min). Radiolabeling peptides and peptide mimetics with zirconium-89, with its longer radioactive half-life (t1/2 = 78 h), could facilitate the centralized manufacture and distribution of radiolabeled tracers. In addition, the ability to image patients at later time points with zirconium-89 based agents (e.g. 4-24 h after injection) may also allow the delineation of small or low-uptake disease sites as the delayed imaging results in increased clearance of the tracer from nontarget tissue and lower background signal.

Real-time measurements of ATP dynamics via ATeams in Plasmodium falciparum reveal drug-class-specific response patterns.

Malaria tropica, caused by the parasite Plasmodium falciparum (P. falciparum), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of P. falciparum is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP2- levels in a ratiometric manner, thereby facilitating in cellulo measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in P. falciparum to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein in vitro and stably integrated the sensors into the genome of the P. falciparum NF54attB cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs' mode of action, with possible implications for target identification and drug development.

Molecular Targets of the 5-Amido-Carboxamide Bumped Kinase Inhibitor BKI-1748 in Cryptosporidium parvum and HCT-8 Host Cells.

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing.

A Case Report of Quinine-Induced Thrombotic Microangiopathy Successfully Treated With Eculizumab.

Drug-induced thrombotic microangiopathy (DITMA) is a life-threatening condition which may be immune or nonimmune mediated. Quinine is the most implicated drug in immune-mediated DITMA. However, the optimal treatment is unclear. Complement inhibition by eculizumab has demonstrated success in many DITMA (e.g., carfilzomib, gemcitabine, and tacrolimus), but there are limited data in DITMA, including quinine-associated cases. A 55-year-old female was diagnosed with quinine-associated thrombotic microangiopathy (TMA), as confirmed by a positive quinine-dependent platelet-associated antibody. This was successfully treated with eculizumab with complete resolution of thrombocytopenia and anemia by 1 and 6 weeks. She required hemodialysis for a month and gained full recovery of renal function. We discuss various challenges with the diagnosis and management of DITMA. We also review published data on the use of eculizumab in various DITMA. Our case demonstrates successful treatment of quinine-induced TMA with eculizumab. We recommend further studies to assess the efficacy of complement inhibition in quinine and other DITMA.

Alteration of Sweet and Bitter Taste Sensitivity with Development of Glucose Intolerance in Non-insulin-Dependent Diabetes Mellitus Model OLETF Rats.

Patients with diabetes exhibit altered taste sensitivity, but its details have not been clarified yet. Here, we examined alteration of sweet taste sensitivity with development of glucose intolerance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a model of non-insulin-dependent diabetes mellitus. Compared to the cases of Long Evans Tokushima Otsuka (LETO) rats as a control, glucose tolerance of OLETF rats decreased with aging, resulting in development of diabetes at 36-weeks-old. In brief-access tests with a mixture of sucrose and quinine hydrochloride, OLETF rats at 25 or more-weeks-old seemed to exhibit lower sweet taste sensitivity than age-matched LETO ones, but the lick ratios of LETO, but not OLETF, rats for the mixture and quinine hydrochloride solutions decreased and increased, respectively, aging-dependently. Expression of sweet taste receptors, T1R2 and T1R3, in circumvallate papillae (CP) was almost the same in LETO and OLETF rats at 10- and 40-weeks-old, while expression levels of a bitter taste receptor, T2R16, were greater in 40-weeks-old rats than in 10-weeks-old ones in both strains. There was no apparent morphological alteration in taste buds in CP between 10- and 40-weeks-old LETO and OLETF rats. Metagenomic analysis of gut microbiota revealed strain- and aging-dependent alteration of mucus layer-regulatory microbiota. Collectively, we concluded that the apparent higher sweet taste sensitivity in 25 or more-weeks-old OLETF rats than in age-matched LETO rats was due to the aging-dependent increase of bitter taste sensitivity in LETO rats with alteration of the gut microbiota.

Mosquitoes do not Like Bitter.

Chemical repellents play a crucial role in personal protection, serving as essential elements in reducing the transmission of vector-borne diseases. A biorational perspective that extends beyond the olfactory system as the classical target may be a promising direction to move. The taste system provides reliable information regarding food quality, helping animals to discriminate between nutritious and potentially harmful food sources, often associated with a bitter taste. Understanding how bitter compounds affect feeding in blood-sucking insects could unveil novel molecules with the potential to reduce biting and feeding. Here, we investigated the impact of two naturally occurring bitter compounds, caffeine and quinine, on the feeding decisions in female Aedes aegypti mosquitoes at two distinctive phases: (1) when the mosquito explores the biting substrate using external taste sensors and (2) when the mosquito takes a sip of food and tastes it using internal taste receptors. We assessed the aversiveness of bitter compounds through both an artificial feeding condition (artificial feeder test) and a real host (arm-in-cage test). Our findings revealed different sensitivities in the external and internal sensory pathways responsible for detecting bitter taste in Ae. aegypti. Internal detectors exhibited responsiveness to lower doses compared to the external sensors. Quinine exerted a more pronounced negative impact on biting and feeding activity than caffeine. The implications of our findings are discussed in the context of mosquito food recognition and the potential practical implications for personal protection.

Quinine and chloroquine: Potential preclinical candidates for the treatment of tegumentary Leishmaniasis.

Leishmaniasis is an endemic disease in more than 90 countries, constituting a relevant public health problem. Limited treatment options, increase in resistance, and therapeutic failure are important aspects for the discovery of new treatment options. Drug repurposing may accelerate the discovery of antiLeishmanial drugs. Recent tests indicating the in vitro potential of antimalarials Leishmania resulted in the design of this study. This study aimed at evaluating the susceptibility of Leishmania (L.) amazonensis to chloroquine (CQ) and quinine (QN), alone or in combination with amphotericin B (AFT) and pentamidine (PTN). In the in vitro tests, first, we evaluated the growth inhibition of 50 % of promastigotes (IC50) and cytotoxicity for HepG2 and THP-1 cells (CC50). The IC50 values of AFT and PNT were below 1 µM, while the IC50 values of CQ and QN ranged between 4 and 13 µM. Concerning cytotoxicity, CC50 values ranged between 7 and 30 µM for AFT and PNT, and between 22 and 157 µM for the antimalarials. We also calculated the Selectivity Index (SI), where AFT and PTN obtained the highest values, while the antimalarias obtained values between 5 and 12. Both antimalarials were additive (Æ©FIC 1.05-1.8) in combination with AFT and PTN. For anti-amastigote activity, the drugs obtained the following ICA50 values: AFT (0.26 µM), PNT (2.09 µM), CQ (3.77 µM) and QN (24.5 µM). In the in vivo tests, we observed that the effective dose for the death of 50 % of parasites (ED50) of AFT and CQ were 0.63 mg/kg and 27.29 mg/kg, respectively. When combining CQ with AFT, a decrease in parasitemia was observed, being statistically equal to the naive group. For cytokine quantification, it was observed that CQ, despite presenting anti-inflammatory activity was effective at increasing the production of IFN-γ. Overall, our data indicate that chloroquine will probably be a candidate for repurposing and use in drug combination therapy.

Can humans smell tastants?

Although studies have shown that olfaction may contribute to the perception of tastant, literature is scarce or circumstantial, especially in humans. This study aims to (i) explore whether humans can perceive solutions of basic prototypical tastants through orthonasal and retronasal olfaction and (ii) to examine what volatile odor compounds (VOCs) underlie this ability. Solutions of 5 basic tastants (sucrose, sodium chloride, citric acid, monosodium glutamate [MSG], quinine) dissolved in water, and 2 fatty acids (oleic and linoleic acid) dissolved in mineral oil were prepared. Triangle discrimination tests were performed (n = 41 in duplicate) to assess whether the tastant solutions can be distinguished from blanks (solvents) through ortho- and retronasal olfaction. Participants were able to distinguish all tastant solutions from blank through orthonasal olfaction. Only sucrose, sodium chloride, oleic acid, and linoleic acid were distinguished from blank by retronasal olfaction. Ethyl dichloroacetate, methylene chloride, and/or acetone were identified in the headspace of sucrose, MSG, and quinine solutions but not in the headspace of water, sodium chloride, and citric acid solutions. Fat oxidation compounds such as alcohols and aldehydes were detected in the headspace of the oleic and linoleic acid solutions but not the mineral oil. We conclude that prototypical tastant solutions can be discriminated from water and fatty acid solutions from mineral oil through orthonasal olfaction. Differences in the volatile headspace composition between blanks and tastant solutions may have facilitated the olfactory discrimination. These findings can have methodological implications for future studies assessing gustatory perception using these prototypical taste compounds.

Chiral zwitterionic stationary phases based on Cinchona alkaloids and dipeptides - design, synthesis and application in chiral separation.

Chiral resolution of polar organic compounds such as amino acids and peptides represents an important chromatographic task due to increasing significance of natural species, which play important signaling and regulatory roles in the living organisms. Despite the number of available chiral stationary phases, this task remains challenging, since not many of the commercially available systems are capable to resolve non-derivatized zwitterionic species. In this study, we present a target-oriented design of a new class of chiral selectors. Pursuing the goal to separate amino acids, and especially short peptides, we have combined Cinchona alkaloids - quinine and quinidine - with three different biogenic dipeptides. We have synthesized six different chiral stationary phases, with selector loading of ∼200 µmol g-1, and tested their chiral recognition capabilities for acidic, basic and zwitterionic analytes using various mobile phases. We have observed that all chiral stationary phases retain the chiral anion exchange capability known for commercially available Cinchona-based columns leading to baseline or partial resolution of six out of ten analytes. The performance in chiral resolution of basic analytes is not optimum due to the weak cation exchange character of the peptidic residue. However, we report on encouraging results in the chiral resolution of short peptides, for which, depending on their structure, we see the chiral resolution of up to three stereoisomers (from four possible) in a preliminary screening.

Salivary proteins rescue within-session suppression and conditioned avoidance in response to an intragastric quinine infusion.

A subset of salivary proteins (SPs) upregulates in response to a quinine-containing diet. The presence of these SPs then results in decreased bitter taste responding and taste nerve signaling. Bitter taste receptors in the oral cavity are also found in the stomach and intestines and contribute to behaviors that are influenced by post-oral signaling. It has been previously demonstrated that after several pairings of post-orally infused bitter stimuli and a neutral flavor, animals learn to avoid the flavor that was paired with gastric bitter, this is referred to as conditioned avoidance. Furthermore, animals will decrease licking of a neutral solution within a test session, when licking is paired with an intragastric bitter infusion; this has been described as within-session suppression. We used these paradigms to test the role of SPs in behaviors influenced by post-oral signaling. In both paradigms, the animal is given a test solution directly into the stomach (with or without quinine, and with or without SPs), and the infusions are self-administered by licking to a neutral solution (Kool-Aid). Quinine successfully conditioned a flavor avoidance, but, in a separate trial, we were unable to detect conditioning in the presence of SPs from donor animals. Likewise, quinine was able to suppress licking within the conditioned suppression paradigm, but the effect of the bitter was blocked in the presence of saliva containing SPs. Together, these data suggest that behaviors driven by post-oral signaling can be altered by SPs.

The temporal-spatial expression and functional analysis of three gustatory receptor genes in Solenopsis invicta using sweet and bitter compounds.

The insect gustatory system participates in identifying potential food sources and avoiding toxic compounds. During this process, gustatory receptors (GRs) recognize feeding stimulant and deterrent compounds. However, the GRs involved in recognizing stimulant and deterrent compounds in the red imported fire ant, Solenopsis invicta, remain unknown. Therefore, we conducted a study on the genes SinvGR1, SinvGR32b, and SinvGR28a to investigate the roles of GRs in detecting feeding stimulant and deterrent compounds. In this current study, we found that sucrose and fructose are feeding stimulants and the bitter compound quinine is a feeding deterrent. The fire ant workers showed significant behavior changes to avoid the bitter taste in feeding stimulant compounds. Reverse transcription quantitative real-time polymerase chain reaction results from developmental stages showed that the SinvGR1, SinvGR32b, and SinvGR28a genes were highly expressed in fire ant workers. Tissue-specific expression profiles indicated that SinvGR1, SinvGR32b, and SinvGR28a were specifically expressed in the antennae and foreleg tarsi of workers, whereas SinvGR32b gene transcripts were also highly accumulated in the male antennae. Furthermore, the silencing of SinvGR1 or SinvGR32b alone and the co-silencing of both genes disrupted worker stimulation and feeding on sucrose and fructose. The results also showed that SinvGR28a is required for avoiding quinine, as workers with knockdown of the SinvGR28a gene failed to avoid and fed on quinine. This study first identified stimulant and deterrent compounds of fire ant workers and then the GRs involved in the taste recognition of these compounds. This study could provide potential target gustatory genes for the control of the fire ant.

Regulation of Ras p21 and RalA GTPases activity by quinine in mammary epithelial cells.

Quinine, a bitter compound, can act as an agonist to activate the family of bitter taste G protein-coupled receptor family of proteins. Previous work from our laboratory has demonstrated that quinine causes activation of RalA, a Ras p21-related small G protein. Ral proteins can be activated directly or indirectly through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using normal mammary epithelial (MCF-10A) and non-invasive mammary epithelial (MCF-7) cell lines, we investigated the effect of quinine in regulating Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF-10A and MCF-7 cells; however, RalA was inhibited in MCF-10A cells, and no effect was observed in the case of MCF-7 cells. MAP kinase, a downstream effector for Ras p21, was activated in both MCF-10A and MCF-7 cells. Western blot analysis confirmed the expression of RalGDS in MCF-10A cells and MCF-7 cells. The expression of RalGDS was higher in MCF-10A cells in comparison to the MCF-7 cells. Although RalGDS was detected in MCF-10A and MCF-7 cells, it did not result in RalA activation upon Ras p21 activation with quinine suggesting that the Ras p21-RalGDS-RalA pathway is not active in the MCF-10A cells. The inhibition of RalA activity in MCF-10A cells due to quinine could be as a result of a direct effect of this bitter compound on RalA. Protein modeling and ligand docking analysis demonstrated that quinine can interact with RalA through the R79 amino acid, which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change that results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that regulate Ral activity in mammary epithelial cells.

A simple and inexpensive method to monitor and minimize exposure from manipulation of cytotoxic drugs.

Pharmacy personnel that manipulate cytotoxic drugs are under continuous exposure risk. Therefore, training and strict adherence to recommended practices should always be promoted. The main objective of this study was to develop and apply a safe, effective and low-cost method for the training and assessment of the safe handling of cytotoxic drugs, using commercially available tonic water. To evaluate the potential of tonic water as a replacement marker for quinine hydrochloride, deliberate spills of 1 mL of four different tonic waters (one coloured and three non-coloured) were analysed under ultraviolet light (300-400 nm). The pigmented sample did not produce fluorescence under ultraviolet (UV) light. The three commercially available tonic waters that exhibited fluorescence were further analysed by UV/Vis spectrophotometry (300-500 nm). Afterwards, a protocol of simulated manipulation of cytotoxic drugs was developed and applied to 12 pharmacy technicians, that prepared 24 intravenous bags according to recommended routine procedures using tonic water. Participants responded to a brief questionnaire to evaluate the adequacy and applicability of the activity. Seven of the participants had spillages during manipulation, the majority of which recorded during manipulation with needles. All participants scored the tonic water manipulation simulation with 4 or 5 points for simplicity, efficiency and feasibility. The obtained results suggest that tonic water can be used to simulate the manipulation of cytotoxic drugs in training and assessment programs. By using this replacement marker for quinine hydrochloride, it is possible to perform a more cost-effective, yet equally effective, assessment.

Physiopathology and clinical management of blackwater fever: a scoping review.

BACKGROUND: Blackwater fever (BWF) is a severe syndrome occurring in patients with malaria upon antimalarial treatment, characterized by massive intravascular haemolysis and haemoglobinuria. BWF is a neglected condition and management recommendations are unavailable. OBJECTIVES: We performed a scoping review to appraise available data on clinical picture, treatment and physiopathology of BWF, which could guide rationally its clinical management. METHODS: MEDLINE, EMBASE, LILACS, Web of Science, and Scopus databases, and the reference list of relevant publications, were searched. Papers reporting original data on BWF cases or investigating the physiopathology of BWF were eligible. Data regarding case characteristics, trigger event, clinical management and outcome were extracted. For papers investigating the physiopathology of BWF, study design and principal findings were extracted. No quality assessment was performed. Data are presented as numbers and percentages, and summary of findings, grouped by paper focus (clinical description or physiopathology). RESULTS: 101 papers were included. The majority of BWF cases were observed in autochthonous children (75.7%) and adults (15.3%), in contrast with historical perception that BWF patients were typically expatriates. Clinical management was described for 794 cases; corticosteroids were used in 23. Outcome was reported for 535 patients, with 18.1% mortality. The trigger was reported for 552 (47.5%) cases; in 70.4% identified as quinine. However, two RCT comparing artesunate and quinine for falciparum malaria treatment did not find significant difference in BWF occurrence after their administration. Two case-control studies did not find significant difference in G6PDH deficiency between malaria patients with and without BWF. CONCLUSIONS: The physiopathology and optimal treatment of BWF remain similarly unknown as they were over a century ago. Empirical supporting treatment approach seems reasonable, while change of antimalarial drug and use of corticosteroids remain object of debate.

Bed Nucleus of the Stria Terminalis (BNST) neurons containing the serotonin 5HT2c receptor modulate operant alcohol self-administration behavior in mice.

The serotonin 5HT2c receptor has been widely implicated in the pathophysiology of alcohol use disorder (AUD), particularly alcohol seeking and the affective consequences of chronic alcohol consumption. However, little is known about the brain sites in which 5HT2c exerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT2c receptor system in the BNST on operant alcohol self-administration behaviors in adult mice of both sexes, including the acquisition and maintenance of fixed-ratio responding, motivation for alcohol (progressive ratio), and quinine-adulterated responding for alcohol on a fixed-ratio schedule (punished alcohol seeking). Knockdown of 5HT2c in the BNST did not affect the acquisition or maintenance of operant alcohol self-administration, nor did it affect progressive ratio responding for alcohol. This manipulation had only a subtle effect on responding for quinine alcohol selectively in females. On the other hand, chemogenetic inhibition of BNST 5HT2c-containing neurons (BNST5HT2c) increased operant alcohol self-administration behavior in both sexes on day 2, but not day 9, of testing. It also increased operant responding for 1000 µM quinine-adulterated alcohol selectively in males. Importantly, chemogenetic inhibition of BNST5HT2c did not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST5HT2c project to similar regions in males and females, many of which have been previously implicated in AUD. We next used chemogenetics and quantification of the immediate early gene cFos to characterize the functional influence of BNST5HT2c inhibition on vlPAG activity. We show that chemogenetic inhibition of BNST5HT2c reduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST5HT2c neurons, and to a small extent the BNST 5HT2c receptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.

Identification and structural validation of purine nucleoside phosphorylase from Plasmodium falciparum as a target of MMV000848.

About 247 million cases of malaria occurred in 2021 with Plasmodium falciparum accounting for the majority of 619,000 deaths. In the absence of a widely available vaccine, chemotherapy remains crucial to prevent, treat, and contain the disease. The efficacy of several drugs currently used in the clinic is likely to suffer from the emergence of resistant parasites. A global effort to identify lead compounds led to several initiatives such as the Medicine for Malaria Ventures (MMV), a repository of compounds showing promising efficacy in killing the parasite in cell-based assays. Here, we used mass spectrometry coupled with cellular thermal shift assay to identify putative protein targets of MMV000848, a compound with an in vitro EC50 of 0.5 µM against the parasite. Thermal shift assays showed a strong increase of P. falciparum purine nucleoside phosphorylase (PfPNP) melting temperature by up to 15 °C upon incubation with MMV000848. Binding and enzymatic assays returned a KD of 1.52 ± 0.495 µM and an IC50 value of 21.5 ± 2.36 µM. The inhibition is competitive with respect to the substrate, as confirmed by a cocrystal structure of PfPNP bound with MMV000848 at the active site, determined at 1.85 Å resolution. In contrast to transition states inhibitors, MMV000848 specifically inhibits the parasite enzyme but not the human ortholog. An isobologram analysis shows subadditivity with immucillin H and with quinine respectively, suggesting overlapping modes of action between these compounds. These results point to PfPNP as a promising antimalarial target and suggest avenues to improve inhibitor potency.