Jaeson Santos Calla Choque

An increasing frequency of CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Escherichia coli has been documented among pediatric infections in recent decades. This study aimed to analyze the antibiotic susceptibility profiles of ESBL-producing and non-ESBL-producing E. coli isolates, to detect CTX-M groups 1, 2, and 9 among ESBL producers, and to assess the clonal relationships between both groups in pediatric patients. A total of 124 E. coli isolates were recovered from clinical specimens (urine, stool, peritoneal fluid, and tracheal aspirates) collected between April and September 2025 at the Pediatric Emergency Hospital (PEH), Lima, Peru. CTX-M groups were detected by multiplex Polymerase Chain Reaction (mPCR), and clonal relationships were assessed by BOX-PCR. ESBL production was identified in 27.4% of isolates, significantly more frequent in urine samples (p < 0.05) and associated with multidrug-resistant (MDR) or extensively drug-resistant (XDR) profiles (p < 0.001). CTX-M group 1 predominated among ESBL producers (91.3%), while groups 2 and 9 were sporadic (2.9% each). BOX-PCR revealed high genetic heterogeneity, resolving 113 patterns (Simpson’s Index of Discrimination = 0.9986) and 31 ESBL-associated clones. These findings indicate that ESBL-producing E. coli dissemination in this setting is driven by diverse lineages rather than a dominant clone, highlighting the need for continued resistance surveillance.

Rationale: Lepidoceras peruvianum Kuijt is an understudied hemiparasitic species endemic to the Peruvian Andes that lacks comprehensive phytochemical characterization. Investigating its metabolome is important for understanding its chemotaxonomic relevance and potential as a source of bioactive compounds. Methods: Leaves and fruits of L. peruvianum were subjected to untargeted metabolomic profiling using high‐resolution UHPLC–ESI‐Orbitrap‐MS/MS in positive and negative ionization modes. Metabolites were annotated based on accurate mass measurements, isotopic patterns, collision‐induced dissociation (CID) fragmentation data, and spectral matching with the Global Natural Products Social Molecular Networking (GNPS), METLIN, and MassBank databases. Structural elucidation employed diagnostic fragmentation pathways, including retro‐Diels–Alder (RDA), heterocyclic ring fission (HRF), quinone methide (QM), and benzofuran‐forming (BFF) cleavages. Antioxidant activity was evaluated using DPPH●, ABTS●+, and FRAP assays, while antimicrobial activity and toxicity were assessed through antibacterial testing and the Artemia salina lethality assay. Results: Leaves contained 34 metabolites, mainly flavan‐3‐ols, proanthocyanidins, flavonols, lignans, and isoquinoline alkaloids, whereas fruits were characterized by anthocyanins, organic acids, and polar lipids. Total phenolic content was higher in leaves (292.4 mg GAE/g) than in fruits (216.8 mg GAE/g), corresponding to stronger antioxidant activity. Leaves extracts exhibited greater antibacterial activity against Staphylococcus aureus , while fruit extracts showed stronger effects against Gram‐negative bacteria. LC ₅₀ values in the A. salina assay were 181.8 μg/mL for leaves and 475.9 μg/mL for fruits. Conclusions: This study provides the first comprehensive metabolomic characterization of L. peruvianum , revealing organ‐specific chemical diversity and notable bioactive properties. The findings demonstrate the utility of HRMS/MS fragmentation analysis for metabolite annotation and identify this endemic species as a promising source of bioactive natural products.

Gene expression analysis in malaria parasites has been used to define transcriptional regulatory networks but has been used less frequently to characterize parasite response to drug treatment or to show how parasites may evade killing. Here, we applied single-cell RNA sequencing (scRNA-seq) to hundreds of thousands of individually infected asynchronous red blood cells to evaluate the parasite’s response to treatment with three chemotypes that can be used for treatment (artemisinin) or prophylaxis and treatment (atovaquone, ganaplacide). We found that each treatment gave rise to different cell populations with different transcriptional profiles. Comparing single cell transcription patterns in compound-treated cells, to transcript patterns observed previously with synchronized cells showed an enrichment of cells expressing gametocyte-associated genes after artemisinin treatment but fewer lifecycle perturbations after treatment with the two other compounds. In contrast, bulk analysis showed an enrichment of pyrimidine biosynthesis transcripts for atovaquone treatment. Our results show that scRNA-seq may be used to profile diverse drug responses across many lifecycle stages and to potentially classify drug classes. Importance: Determining the mechanism of action (MOA) of compounds with antimalarial activity remains a key activity in both drug development and drug resistance studies but remains challenging for some chemotypes. Here we highlight the potential of single cell transcriptional sequencing to augment the process of MOA deconvolution. We develop a new analytical pipeline that involves comparing single cell transcription patterns to existing profiles from synchronized parasites to comprehensively characterize life cycle stage enrichments that may be observed after chemical perturbations. We also show that transcriptional feedback regulation may be present for some drug classes.

Farfan AB, et al. Infect Chemother. 2025 Jan;57:e2. https://doi.org/10.3947/ic.2024.0101

Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3′-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3′-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis.

Here, we present remarkable epoxyketone‐based proteasome inhibitors with low nanomolar in vitro potency for blood‐stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000‐fold greater selectivity for erythrocytic‐stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D‐amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171‐fold more potent at inhibiting the β5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti‐malarial drugs.

A bstract: Changes in host cell morphology and transcription after apicomplexan parasite infection have long been noted, but there have been few studies of the functional consequences of host cell remodeling. Here we show, using time-dependent immunofluorescence microscopy of multiple human cell lines (HepG2, HC-04, Huh7.5.1 and primary human hepatocytes), infected with multiple Plasmodium species ( Plasmodium berghei, P. falciparum and P. vivax (hypnozoites and schizonts)), and antibodies to multiple human proteins (HsNR4A3, HsMUC13, HsGOLGA8A, HsCGA, HsBiP, HsCXCL2), that human protein trafficking is extensively modified in Plasmodium infected cells. Using conventional as well as ultrastructure expansion microscopy we show that newly-synthesized human proteins are trafficked to the parasitophorous vacuole instead of the infected-cell plasma membrane, nucleus or extracellular space. Universal redirection of human signaling proteins cells the parasitophorous vacuole may provide a mechanistic explanation for how apicomplexan parasites can block host cells response to infection.

Here we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,600-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 cells, which is largely driven by the accommodation of the parasite proteasome for a d -amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.

Potent and durable vaccine responses will be required for control of malaria caused by Plasmodium falciparum (Pf). RTS,S/AS01 is the first, and to date, the only vaccine that has demonstrated significant reduction of clinical and severe malaria in endemic cohorts in Phase 3 trials. Although the vaccine is protective, efficacy declines over time with kinetics paralleling the decline in antibody responses to the Pf circumsporozoite protein (PfCSP). Although most attention has focused on antibodies to repeat motifs on PfCSP, antibodies to other regions may play a role in protection. Here, we expressed and characterized seven monoclonal antibodies to the C-terminal domain of CSP (ctCSP) from volunteers immunized with RTS,S/AS01. Competition and crystal structure studies indicated that the antibodies target two different sites on opposite faces of ctCSP. One site contains a polymorphic region (denoted α-ctCSP) and has been previously characterized, whereas the second is a previously undescribed site on the conserved β-sheet face of the ctCSP (denoted β-ctCSP). Antibodies to the β-ctCSP site exhibited broad reactivity with a diverse panel of ctCSP peptides whose sequences were derived from field isolates of P. falciparum whereas antibodies to the α-ctCSP site showed very limited cross reactivity. Importantly, an antibody to the β-site demonstrated inhibition activity against malaria infection in a murine model. This study identifies a previously unidentified conserved epitope on CSP that could be targeted by prophylactic antibodies and exploited in structure-based vaccine design.