The widespread use of pharmaceuticals has led to their frequent detection in aquatic environments, raising concerns about adverse effects on non-target organisms. This study examined behavioral and endocrine responses to acute repetitive exposure (4 days, 15 min day-1) to environmentally relevant concentrations of azithromycin (AZT, 12.5 μg mL-1), nimesulide (NIME, 0.4 μg mL-1), and their combination in zebrafish (Danio rerio). Behavior was assessed using the Novel Tank Test, Social Preference Test, and Light/Dark Test. Whole-body cortisol, acetylcholinesterase (AChE) and catalase (CAT) activities, lipid peroxidation (TBARS), and non-protein thiols (NTP) were measured as stress biomarkers. NIME significantly reduced locomotion (~30%) and time spent in upper zones (p < 0.05), indicating hypolocomotion and anxiety-like behavior. AZT elevated cortisol (p < 0.05) but caused minimal behavioral changes. Co-exposure intensified behavioral impairments and further increased cortisol (p < 0.01), suggesting synergistic effects. NIME increased TBARS, indicating oxidative damage. AChE activity was inhibited in the brain by NIME, while AZT and the combination increased systemic AChE. CAT activity rose in the combination group, possibly reflecting a compensatory response to reactive oxygen species. These findings demonstrate that short-term, repeated exposure to mixtures of commonly detected pharmaceuticals, even at environmentally relevant concentrations, can disrupt behavioral, biochemical, and endocrine homeostasis in aquatic organisms. This highlights the ecological risks posed by multi-compound contamination in aquatic environments.

It was first hypothesized in 2009 that endogenous host reverse transcriptase (eRT) arising from retrotransposon elements in arthropod genomes would be critical for defense against viral pathogens in crustaceans and insects. This was not proven until 2016 in insects. In the succeeding 20 years, insect studies have confirmed the eRT role in antiviral defenses that result in tolerated, persistent infections via viral accommodation mechanisms (VAM). In insects, eRT transcribes viral RNA fragments into viral copy DNA (vcDNA) in both linear (lvcDNA) and circular (cvcDNA) forms. These, in turn, give rise to small interfering RNA (siRNA) fragments to feed the host RNA interference (RNAi) mechanism. At the same time, some vcDNA fragments are integrated into the host genome as endogenous viral elements (EVE), that can also give rise to siRNA. Studies with shrimp have progressed more slowly but results so far have mimicked those revealed with insects. Here we describe the presence and functional activity of a 5383 bp-RT-related gene from a retrotransposon in the black tiger shrimp Penaeus monodon upon challenge with white spot syndrome virus (WSSV). It encompasses four conserved domains of protease (retropepsin), reverse transcriptase (RT-RNase H), transposase, and integrase as a single ORF of 1248 deduced amino acids. The recombinant protein of a conserved 408-amino acid RT-RNase H domain, designated as Pm-eRT was expressed and found to possess reverse transcriptase activity. During WSSV infection, Pm-eRT was upregulated in shrimp hemocytes and pleopods and DNA/RNA hybrids in hemocytes were detected. The DNA/RNA hybrid signals were translocated from the plasma membrane to the nuclear membrane in the shrimp that survived WSSV infection with low viral loads. Pharmacological inhibition of Pm-eRT activity using azidothymidine (AZT) resulted in enhanced viral replication and accelerated shrimp mortality. Collectively, these findings support those described for insects, paving the way to further work on details of VAM in shrimp with the ultimate aim of developing genetically viral-tolerant breeding stocks for shrimp cultivation.

Currently, the increasing isolation rates of New Delhi metallo-beta-lactamase (NDM) positive Klebsiella pneumoniae and the lack of a standard algorithm for the treatment of infections caused by these isolates necessitate the search for effective treatment options. This study aimed to investigate the efficacy of the combination of ceftazidime-avibactam (CZA), frequently used in the treatment of carbapenem-resistant Enterobacterales (CRE) infections in recent years with aztreonam (AZT), which is expected to become available in our country soon and the combination of meropenem (MEM) with colistin (COL), which is frequently preferred in our hospital against NDM-positive K.pneumoniae isolates using in vitro synergy tests and to determine the susceptibility of these isolates to cefiderocol (FDC), which is recommended as an alternative treatment option in CRE infections. A total of 63 K.pneumoniae isolates, isolated from clinical samples in the Bacteriology Laboratory of the Department of Medical Microbiology at Süleyman Demirel University Faculty of Medicine and identified as NDM positive by real-time polymerase chain reaction, were included in the study. The susceptibility of the isolates to CZA, AZT, MEM and COL was tested by broth microdilution method and FDC susceptibility was tested by Kirby-Bauer disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing criteria. The efficacy of CZA-AZT and MEM-COL combinations against NDM positive K.pneumoniae isolates was tested using the checkerboard method. Time-kill assays were performed to investigate the bactericidal activity of CZA-AZT and MEM-COL combinations at different concentrations and time points in five isolates harboring different carbapenemase genes (two NDM+OXA-48, one NDM, one NDM+OXA-48+KPC, and one NDM+OXA-48/VIM/IMP). All isolates included in our study were found as resistant to CZA, AZT and MEM, while 77.8% were resistant to FDC and 54% were resistant to COL. According to the checkerboard test results, the CZA-AZT combination showed synergistic interaction against all isolates, while the MEM-COL combination exhibited synergistic interaction against 57.1% of the isolates, additive interaction against 25.4% and undefined interaction against 17.5%. In time-kill assays, synergistic activity was observed at various time points in all isolates with the CZA-AZT combination and in four isolates with the MEM-COL combination, while bactericidal activity was detected at certain time points. Although an undefined interaction was determined in the checkerboard test against one isolate (NDM+OXA-48 positive) with the MEM-COL combination, synergistic activity and bactericidal effect were observed at the 12th and 24th hours with the 1xMIC MEM + 1xMIC COL combination in the time-kill assay against the same isolate. In conclusion, the combination of CZA-AZT seems to be a promising treatment option in infections caused by NDM positive K.pneumoniae isolates. In cases where CZA-AZT combination therapy cannot be applied, MEM-COL combination therapy may be considered as an alternative treatment option. In situations where both treatment options cannot be used, cefiderocol monotherapy may be considered as an alternative in selected cases, given the low and heterogeneous susceptibility rates among NDM-positive isolates, provided that close clinical and microbiological monitoring is ensured. However, careful monitoring is required during cefiderocol therapy and treatment modification should be considered in cases of clinical non-response.

Faithful DNA replication in Escherichia coli requires the DNA polymerase III holoenzyme (DNA pol III HE) and its clamp loader complex, which couples processive DNA synthesis with β -clamp loading. The clamp loader accessory subunits χ and ψ link single-stranded DNA-binding protein (SSB) to the replisome, stabilizing replication on SSB-coated templates through interactions with the SSB C-terminal tail. Chi has also been implicated in tolerance to the chain-terminating nucleoside analog azidothymidine (AZT), though whether this function depends on χ within DNA pol III HE or on its independent interaction with the YoaA helicase remains unclear. To address this, we engineered ψ - χ fusion proteins with flexible glycine-serine linkers to tether the two subunits while preserving folding and activity. Both fusions were biochemically competent, supporting ATP hydrolysis and clamp loading on SSB-coated DNA. In vivo, however, neither the ψ -GS12- χ fusion nor expression of a ψχ operon restored AZT tolerance in ΔholC cells, whereas expression of χ alone was sufficient. Fusion expression impaired growth in both wild-type (WT) and ΔholC backgrounds, a phenotype alleviated by disrupting χ -SSB binding. These findings support a model in which χ must dynamically engage SSB and YoaA outside of the clamp loader to promote AZT tolerance, highlighting the importance of regulated χ -SSB interactions in genome maintenance.

In 2008 it was revealed that C6/36 cells challenged with Dengue virus serotype 2 (DENV-2 strain NGC) show cytopathic effects (CPE) but can recover via 2-day serial passaging leading to morphologically normal cell cultures despite persistent infection with DENV-2. In 2009 it was hypothesized that ability to accommodate DENV-2 was dependent on host reverse-transcriptase (HRT) activity. This was proven in 2016 using the RT inhibitor azidothymidine (AZT, a nucleoside analogue and antiretroviral drug) to treat C6/36 cells challenged with DENV-1. Here these results are confirmed using a different RT inhibitor tenofovir disoproxil fumarate (TDF) with different C6/36 cell line and DENV-2. In addition, we confirm earlier work from 2021 that persistent HRT activity is also required to maintain persistent tolerance to DENV-2 infections. Specifically, TDF treatment (0.1 mM) blocked viral accommodation and returned cells to the lethal infection pathway characterized by CPE. This phenomenon has practical applications for screening grossly healthy, living commercial crustaceans and insects for viral pathogens and for research on the development of viral tolerant breeding stocks for commercial crustaceans and insects.

Antimicrobial resistance (AMR) poses a major global health threat, with carbapenem-resistant and extended-spectrum β-lactamase (ESBL)-producing Enterobacterales causing widespread infections and deaths. Much of this resistance spreads through conjugative plasmids, autonomously replicating mobile genetic elements that transfer between bacteria and carry multiple AMR genes. Targeting plasmid conjugation could therefore help curb the spread of AMR. In this study, we tested whether clinically approved nucleoside analogues (NAs) inhibited the transfer of the GFP-tagged ESBL-encoding IncK plasmid pCTgfp in Escherichia coli and the carbapenemase-encoding IncF plasmid pKpQILgfp in Klebsiella pneumoniae using flow cytometry. Alongside the known inhibitor azidothymidine (AZT), didanosine, stavudine, and trifluridine reduced plasmid conjugation in both species without affecting growth. Conversely, famciclovir and zalcitabine promoted pCTgfp conjugation in E. coli, while aciclovir and valaciclovir enhanced pKpQILgfp conjugation in K. pneumoniae. Mechanistic studies showed that plasmid conjugation-promoting NAs altered intracellular ATP levels. RNA sequencing revealed that AZT downregulated the expression of genes linked to motility in E. coli. Genetic inactivation of motility in E. coli mirrored the decrease in pCTgfp conjugation, like AZT. In K. pneumoniae, AZT upregulated genes linked to DNA damage and the SOS response, but downregulated methionine biosynthesis and metabolism genes. The exogenous addition of zinc acetate to inhibit RecA or the end product of methionine metabolism, S-adenosyl-methionine, restored pKpQILgfp conjugation in K. pneumoniae. Overall, our results indicated that existing NAs, including AZT, represent structural scaffolds for the development of potent conjugation inhibitors and highlight motility, DNA repair, and methionine metabolism as potential key factors in plasmid conjugation.

In patients with severe surgical and gastrointestinal complications, standard oral combination antiretroviral therapy (ARV) may be temporarily impossible. Long-acting cabotegravir/rilpivirine (CAB/RPV LA) is approved for virologically suppressed individuals pretreated with oral therapy, but data on its use in treatment-naïve, critically ill patients with absent enteral absorption are extremely limited.

Neglected tropical diseases (NTDs) remain a major global health challenge, particularly in low- and middle-income countries. Kinetoplastid parasites causing Chagas disease, leishmaniasis, and African trypanosomiasis rely on host purine salvage pathways, making nucleoside analogues attractive therapeutic candidates. However, their clinical utility is limited by poor cellular uptake and rapid metabolism. Herein, we report the application of the ProTide prodrug technology, a clinically validated approach that enhances the intracellular delivery of nucleoside monophosphates for the treatment of kinetoplastids infections. As a proof of concept, a focused library of zidovudine (AZT) and cordycepin ProTide prodrugs was designed, synthesized, and evaluated for antiparasitic activity against T. b. rhodesiense, T. cruzi, and L. donovani, as well as for cytotoxicity in L6 rat myoblasts. Out of these, compound 16 exhibited substantial serum stability and potent activity (IC50 = 5 nM; selectivity index, SI = 2,560) against T. b. rhodesiense with robust activity also observed against T. cruzi and L. donovani. These findings establish the ProTide prodrug technology as a promising strategy for optimizing nucleoside analogues against kinetoplastid parasites and provide a framework for the development of new therapeutics for NTDs.

HIV-1 reverse transcriptase (RT) has been a key target for HIV treatment, and several RT inhibitors have been utilized in the clinic. However, RT inhibitors are incapable of eradicating reverse-transcribed DNA, and drug resistance has greatly compromised drug efficacy. Here, we have presented a nucleoside-autophagosome-tethering compound (ATTEC) aiming to inhibit HIV by inducing viral cDNA degradation during replication. 5,7-Dihydroxy-4-phenylcoumarin (DP) derivatives with varied linker lengths were designed and synthesized as LC3-binding ligands and further conjugated with 3'-azidothymidine (AZT) via a bioorthogonal click reaction to form nucleoside-ATTECs. After comparing and optimizing the linker length, our developed nucleoside-ATTEC can effectively inhibit HIV replication and infection in human cells without interfering with cell proliferation. Moreover, DP-AZT has been shown to have the capacity for the prolonged inhibition of HIV-1 replication. Therefore, this work provides a good example of developing a nucleoside-ATTEC approach to inhibit HIV replication and infection.

Optimizing nucleoside reverse transcriptase inhibitor (NRTI) backbones is vital for pediatric and adolescent HIV treatment, yet real-world comparative evidence is limited. This study assessed backbone regimen effectiveness and factors influencing viral suppression (VLS) in Uganda.