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Ramirez, J. C.

Publications and source records attributed to Ramirez, J. C..

4 recordsLinked to original sources

Illusory cold desensitizes touch

In Jack Londons To Build a Fire, the doomed protagonist notes that "the tremendous cold had already driven the life out of his fingers... dead fingers could neither touch nor clutch". Indeed, our hands are numbed when exposed to cold, a desensitization process traditionally attributed to biophysical and physiological effects on mechanoreceptor and afferent responses1. Yet, perception is not based solely on sensory inputs and often reflects prior beliefs and expectations2. Accordingly, the mere experience of coldness, given its association with tactile desensitization, conceivably modulates touch independent of physical effects. We dissociated the physical and subjective influences of cooling on touch by leveraging the Thermal Referral Illusion3-7, in which cooling of the index and ring fingers induces an illusory perception of coldness that is referred to the middle finger which did not experience local cooling. We found that both veridical and illusory cold elevated tactile detection thresholds. Threshold shifts scaled with subjective ratings of cold intensity which varied with thermal adaptation. Thermal referral induction did not result in changes to skin temperature, ruling out indirect physical effects on the tested finger. To account for cold effects on touch, we implemented a computational model incorporating both physical and subjective factors by simulating changes in tactile afferent spiking activity8 and modulation of decision computations, respectively. The model reproduced empirical cooling effects on afferent responses9 and recapitulated psychophysical detection patterns. Our results reveal that subjective thermal perception contributes to cold-induced attenuation of touch, highlighting how multimodal experiences and expectations shape somatosensation.

neuroscience↗

Identification of antifungal agents AR-12 and Fosmanogepix as anti-Trypanosoma cruzi drugs through an enhanced fluorogenic β-galactosidase phenotypic screening assay

Phenotypic screening remains essential for identifying and characterizing bioactive compounds or their combinations against human parasitic pathogens. In the case of Trypanosoma cruzi, the etiological agent of Chagas disease, transgenic parasites expressing the reporter enzyme {beta}-galactosidase have been extensively used to this end. Here, we replaced the traditional chromogenic substrate chlorophenol red-{beta}-D-galactopyranoside (CPRG) with the fluorogenic 4-methylumbelliferyl-{beta}-D-glucopyranoside (MUG) to derive a highly sensitive, continuous enzymatic assay to obtain a quantitative surrogate of parasite growth in T. cruzi cultures. The assay detects as few as 3x103 trypomastigotes/well, tracks linearly with the parasite load in a two-order range (3x103-2x105 trypomastigotes/well), takes 1 hour, and has a similar cost per assay as its colorimetric counterpart. To demonstrate its convenience and versatility, we used this assay to estimate the half-maximal inhibitory concentration (IC50) of six emerging antifungal compounds, not targeting CYP-51 and novel for T. cruzi. Finally, the assay was adapted to a semi-automatic methodology and used to explore dual combinations of the active antifungals in the primary screening and with benznidazole. The multitarget compound AR-12 (IC50 = 1.9 M) and the Gwt1 inhibitor Fosmanogepix (IC50 = 7.2 M) resulted in bona fide hits, inhibiting parasite replication with only low-to-moderate toxicity on Vero host cells, thus suggesting potential for repurposing to Chagas disease.

microbiology↗

In vitro and In vivo Genetic Disease Modelling via NHEJ precise deletions using CRISPR/Cas9

Development of advanced gene and cell therapies for the treatment of genetic diseases requires confident animal and cellular models to test their efficacy and is crucial in the cases where no primary samples from patients are available. CRISPR/Cas9 technology, has become one of the most spread endonuclease tools for editing the genome at will. Moreover, it is known that the use of two guides tends to produce the precise deletion between the guides via NHEJ. Different distances between guides were tested (from 8 to 500 base pairs). We found that distances between the two cutting sites and orientation of Cas9 protein-DNA interaction are important for the efficiency within the optimal range (30-60 bp), we could obtain new genetically reproducible models for two rare disease, a Pyruvate Kinase Deficiency model, using human primary cells, and a (for in vivo primary hyperoxaluria therapeutic mouse model. We demonstrate that the use of a 2-guideRNAs at the optimal distance and orientation is a powerful strategy for disease modelling in those diseases where the availability of primary cells is limited.

bioengineering↗

Real Time PCR for the Evaluation of Treatment Response in Clinical Trials of Adult Chronic Chagas Disease: Usefulness of Serial Blood Sampling and qPCR Replicates.

This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real time PCR (qPCR) for baseline detection and quantification of parasitic loads and post-treatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely DNDi-CH-E1224-001 (NCT01489228) and MSF-DNDi PCR sampling optimization study (NCT01678599). Patients from Cochabamba (N= 294), Tarija (N = 257), and Aiquile (N= 220) were enrolled. Three serial blood samples were collected at each time-point, and qPCR triplicates were tested per sample. The first two samples were collected during the same day and the third one seven days later.\n\nA patient was considered PCR positive if at least one qPCR replicate was detectable. Cumulative results of multiple samples and qPCR replicates enhanced the proportion of pre-treatment sample positivity from 54.8 to 76.2%, 59.5 to 77.8%, and 73.5 to 90.2% in Cochabamba, Tarija, and Aiquile cohorts, respectively and increased cumulative detection of treatment failure from 72.9 to 91.7%, 77.8 to 88.9%, and 42.9 to 69.1% for E1224 low, short, and high dosage regimes, respectively; and from 4.6 to 15.9% and 9.5 to 32.1% for the benznidazole (BZN) arm in the DNDi-CH-E1224-001 and MSF-DNDi studies, respectively. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasional non-detectable results. This serial sampling strategy enhanced PCR sensitivity to detecting treatment failure during follow-up and has the potential for improving recruitment capacity in Chagas disease trials which require an initial positive qPCR result for patient admission.

microbiology↗