A simple polystyrene microfluidic device for sensitive and accurate SERS-based detection of infection by malaria parasites.

Early and accurate detection of infection by pathogenic microorganisms, such as Plasmodium, the causative agent of malaria, is critical for clinical diagnosis and ultimately determines the patient's outcome. We have combined a polystyrene-based microfluidic device with an immunoassay which utilises Surface-Enhanced Raman Spectroscopy (SERS) to detect malaria. The method can be easily translated to a point-of-care testing format and shows excellent sensitivity and specificity, when compared to the gold standard for laboratorial detection of Plasmodium infections. The device can be fabricated in less than 30 min by direct patterning on shrinkable polystyrene sheets of adaptable three-dimensional microfluidic chips. To validate the microfluidic system, samples of P. falciparum-infected red blood cell cultures were used. The SERS-based immunoassay enabled the detection of 0.0012 ± 0.0001% parasitaemia in a P. falciparum-infected red blood cell culture supernatant, an ∼7-fold higher sensitivity than that attained by most rapid diagnostic tests. Our approach successfully overcomes the main challenges of the current Plasmodium detection methods, including increased reproducibility, sensitivity, and specificity. Furthermore, our system can be easily adapted for detection of other pathogens and has excellent properties for early diagnosis of infectious diseases, a decisive step towards lowering their high burden on healthcare systems worldwide.

Unveiling a family of spiro-ß-lactams with anti-HIV and antiplasmodial activity via phosphine-catalyzed [3+2] annulation of 6-alkylidene-penicillanates and allenoates.

The molecular architecture of spirocyclic compounds has been widely explored within the medicinal chemistry field to obtain new compounds with singular three-dimensional pharmacophoric features and improved bioactivity. Herein, the synthesis of 68 new spirocyclopentene-ß-lactams is described, resulting from a rational drug design and structural modulation of a highly promising lead compound BSS-730A, previously identified as having dual antimicrobial activity associated with a novel mechanism of action. Among this diverse library of new compounds, 22 were identified as active against HIV-1, with eight displaying an IC50 lower than 50 nM. These eight compounds also showed nanomolar activity against HIV-2, and six of them displayed micromolar antiplasmodial activity against both the hepatic and the blood stages of infection by malaria parasites, in agreement with the lead molecule's bioactivity profile. The spirocyclopentene-ß-lactams screened also showed low cytotoxicity against TZM-bl and Huh7 human cell lines. Overall, a family of new spirocyclopentene penicillanates with potent activity against HIV and/or Plasmodium was identified. The present structure-activity relationship open avenues for further development of spirocyclopentene-ß-lactams as multivalent, highly active broad spectrum antimicrobial agents.

An Internal Promoter Drives the Expression of a Truncated Form of CCC1 Capable of Protecting Yeast from Iron Toxicity.

In yeast, iron storage and detoxification depend on the Ccc1 transporter that mediates iron accumulation in vacuoles. While deletion of the CCC1 gene renders cells unable to survive under iron overload conditions, the deletion of its previously identified regulators only partially affects survival, indicating that the mechanisms controlling iron storage and detoxification in yeast are still far from well understood. This work reveals that CCC1 is equipped with a complex transcriptional structure comprising several regulatory regions. One of these is located inside the coding sequence of the gene and drives the expression of a short transcript encoding an N-terminally truncated protein, designated as s-Ccc1. s-Ccc1, though less efficiently than Ccc1, is able to promote metal accumulation in the vacuole, protecting cells against iron toxicity. While the expression of the s-Ccc1 appears to be repressed in the normal genomic context, our current data clearly demonstrates that it is functional and has the capacity to play a role under iron overload conditions.

Yeast AP-1 like transcription factors (Yap) and stress response: a current overview.

Yeast adaptation to stress has been extensively studied. It involves large reprogramming of genome expression operated by many, more or less specific, transcription factors. Here, we review our current knowledge on the function of the eight Yap transcription factors (Yap1 to Yap8) in Saccharomyces cerevisiae, which were shown to be involved in various stress responses. More precisely, Yap1 is activated under oxidative stress, Yap2/Cad1 under cadmium, Yap4/Cin5 and Yap6 under osmotic shock, Yap5 under iron overload and Yap8/Arr1 by arsenic compounds. Yap3 and Yap7 seem to be involved in hydroquinone and nitrosative stresses, respectively. The data presented in this article illustrate how much knowledge on the function of these Yap transcription factors is advanced. The evolution of the Yap family and its roles in various pathogenic and non-pathogenic fungal species is discussed in the last section.

Ace1 prevents intracellular copper accumulation by regulating Fet3 expression and thereby restricting Aft1 activity.

In the yeast Saccharomyces cerevisiae Aft1, the low iron-sensing transcription factor is known to regulate the expression of the FET3 gene. However, we found that a strain-lacking FET3 is more sensitive to copper excess than a strain-lacking AFT1, and accordingly, FET3 expression is not fully compromised in the latter. These findings suggest that, under such conditions, another regulator comes into play and controls FET3 expression. In this work, we identify Ace1, the regulator of copper detoxification genes, as a regulator of FET3. We suggest that the activation of FET3 by Ace1 prevents the hyper activation of Aft1, possibly by assuring the adequate functioning of mitochondrial iron-sulfur cluster biogenesis. While reinforcing the link between iron and copper homeostasis, this work unveils a novel protection mechanism against copper toxicity mediated by Ace1, which relies in the activation of FET3 and results in the restriction of Aft1 activity as a means to prevent excessive copper accumulation.

Inhibition of Yap2 activity by MAPKAP kinase Rck1 affects yeast tolerance to cadmium.

Yap2 is a cadmium responsive transcription factor that interacts with MAPK-activated protein (MAPKAP) kinase Rck1. We show that Rck1 deletion confers protection against cadmium toxicity and that the mechanism underlying this observation relies on Yap2. Rck1 removal from the yeast genome potentiates Yap2 activity by increasing protein half-life and delaying its nuclear export. As a consequence, several Yap2 antioxidant targets are over-activated by a mechanism that also requires Yap1. Several genes of the cell wall integrity (CWI) pathway are upregulated under cadmium stress in a Yap2 dependent way. We showed that deletion of CWI genes renders yeast cells more sensitive to cadmium. These findings led us to suggest that in response to cadmium stress Yap2 may serve a dual purpose: oxidative stress attenuation and cell wall maintenance.

Repression of the Low Affinity Iron Transporter Gene FET4: A NOVEL MECHANISM AGAINST CADMIUM TOXICITY ORCHESTRATED BY YAP1 VIA ROX1.

Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, causing several cellular damages and eventually leading to death. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1 plays a key role in the regulation of several genes involved in metal stress response. We have previously shown that Yap1 represses the expression of FET4, a gene encoding a low affinity iron transporter able to transport metals other than iron. Here, we have studied the relevance of this repression in cell tolerance to cadmium. Our results indicate that genomic deletion of Yap1 increases FET4 transcript and protein levels. In addition, the cadmium toxicity exhibited by this strain is completely reversed by co-deletion of FET4 gene. These data correlate well with the increased intracellular levels of cadmium observed in the mutant yap1. Rox1, a well known aerobic repressor of hypoxic genes, conveys the Yap1-mediated repression of FET4. We further show that, in a scenario where the activity of Yap1 or Rox1 is compromised, cells activate post-transcriptional mechanisms, involving the exoribonuclease Xrn1, to compensate the derepression of FET4. Our data thus reveal a novel protection mechanism against cadmium toxicity mediated by Yap1 that relies on the aerobic repression of FET4 and results in the impairment of cadmium uptake.

Yap1 mediates tolerance to cobalt toxicity in the yeast Saccharomyces cerevisiae.

BACKGROUND: Cobalt has a rare occurrence in nature, but may accumulate in cells to toxic levels. In the present study, we have investigated how the transcription factor Yap1 mediates tolerance to cobalt toxicity. METHODS: Fluorescence microscopy was used to address how cobalt activates Yap1. Using microarray analysis, we compared the transcriptional profile of a strain lacking Yap1 to that of its parental strain. To evaluate the extent of the oxidative damage caused by cobalt, GSH was quantified by HPLC and protein carbonylation levels were assessed. RESULTS: Cobalt activates Yap1 under aerobiosis and anaerobiosis growth conditions. This metal generates a severe oxidative damage in the absence of Yap1. However, when challenged with high concentrations of cobalt, yap1 mutant cells accumulate lower levels of this metal. Accordingly, microarray analysis revealed that the expression of the high affinity phosphate transporter, PHO84, a well-known cobalt transporter, is compromised in the yap1 mutant. Moreover, we show that Yap1 is a repressor of the low affinity iron transporter, FET4, which is also known to transport cobalt. CONCLUSIONS: Cobalt activates Yap1 that alleviates the oxidative damage caused by this metal. Yap1 partially controls cobalt cellular uptake via the regulation of PHO84. Although FET4 repression by Yap1 has no effect on cobalt uptake, it may be its first line of defense against other toxic metals. GENERAL SIGNIFICANCE: Our results emphasize the important role of Yap1 in mediating cobalt-induced oxidative damages and reveal new routes for cell protection provided by this regulator.

The role of the Yap5 transcription factor in remodeling gene expression in response to Fe bioavailability.

The budding yeast Saccharomyces cerevisiae has developed several mechanisms to avoid either the drastic consequences of iron deprivation or the toxic effects of iron excess. In this work, we analysed the global gene expression changes occurring in yeast cells undergoing iron overload. Several genes directly or indirectly involved in iron homeostasis showed altered expression and the relevance of these changes are discussed. Microarray analyses were also performed to identify new targets of the iron responsive factor Yap5. Besides the iron vacuolar transporter CCC1, Yap5 also controls the expression of glutaredoxin GRX4, previously known to be involved in the regulation of Aft1 nuclear localization. Consistently, we show that in the absence of Yap5 Aft1 nuclear exclusion is slightly impaired. These studies provide further evidence that cells control iron homeostasis by using multiple pathways.