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Regulated protein secretion is required for malaria parasite life cycle progression and transmission between the mammalian host and mosquito vector. During transmission from the host to the vector, exocytosis of highly specialised secretory vesicles, such as osmiophilic bodies, is key to the dissolution of the red blood cell and parasitophorous vacuole membranes enabling gamete egress. The positioning of adhesins from the TRAP family, from micronemes to the sporozoite surface, is essential for gliding motility of the parasite and transmission from mosquito to mammalian host. Here we identify a conserved role for the putative pantothenate transporter PAT in Plasmodium berghei in vesicle fusion of two distinct classes of vesicles in gametocytes and sporozoites. PAT is a membrane component of osmiophilic bodies in gametocytes and micronemes in sporozoites. Despite normal formation and trafficking of osmiophilic bodies to the cell surface upon activation, PAT-deficient gametes fail to discharge their contents, remain intraerythrocytic and unavailable for fertilisation and further development in the mosquito. Sporozoites lacking PAT fail to secrete TRAP, are immotile and thus unable to infect the subsequent rodent host. Thus, P. berghei PAT appears to regulate exocytosis in two distinct populations of vesicles in two different life cycle forms rather than acting as pantothenic transporter during parasite transmission.

Flaviviruses such as Yellow fever, Dengue, West Nile, and Zika generate disease-linked viral noncoding RNAs called subgenomic flavivirus RNAs. Subgenomic flavivirus RNAs result when the 5′–3′ progression of cellular exoribonuclease Xrn1 is blocked by RNA elements called Xrn1-resistant RNAs located within the viral genome’s 3′-untranslated region that operate without protein co-factors. Here, we show that Xrn1-resistant RNAs can halt diverse exoribonucleases, revealing a mechanism in which they act as general mechanical blocks that ‘brace’ against an enzyme’s surface, presenting an unfolding problem that confounds further enzyme progression. Further, we directly demonstrate that Xrn1-resistant RNAs exist in a diverse set of flaviviruses, including some specific to insects or with no known arthropod vector. These Xrn1-resistant RNAs comprise two secondary structural classes that mirror previously reported phylogenic analysis. Our discoveries have implications for the evolution of exoribonuclease resistance, the use of Xrn1-resistant RNAs in synthetic biology, and the development of new therapies. Subgenomic flavivirus RNAs are generated by a host exoribonuclease and play an important role in virus replication and pathogenesis. Here, the authors show the mechanism by which subgenomic flavivirus RNAs are generated and identify two structurally distinct sfRNA classes in flaviviruses.

Ookinetes, one of the motile and invasive forms of the malaria parasite, rely on gliding motility in order to establish an infection in the mosquito host. Here we characterize the protein PBANKA_0407300 which is conserved in the Plasmodium genus but lacks significant similarity to proteins of other eukaryotes. It is expressed in gametocytes and throughout the invasive mosquito stages of P. berghei, but is absent from asexual blood stages. Mutants lacking the protein developed morphologically normal ookinetes that were devoid of productive motility although some stretching movement could be detected. We therefore named the protein Ookinete Motility Deficient (OMD). Several key factors known to be involved in motility however were normally expressed and localized in the mutant. Importantly, the mutant failed to establish an infection in the mosquito which resulted in a total malaria transmission blockade.

UIS4 is a key protein component of the host-parasite interface in the liver stage of the rodent malaria parasite Plasmodium berghei and required for parasite survival after invasion. In the infectious sporozoite, UIS4 protein has variably been shown to be translated but also been reported to be translationally repressed. Here we show that uis4 mRNA translation is regulated by the P. berghei RNA binding protein Pumilio-2 (PbPuf2 or Puf2 from here on forward) in infectious salivary gland sporozoites in the mosquito vector. Using RNA immunoprecipitation we show that uis4 mRNA is bound by Puf2 in salivary gland sporozoites. In the absence of Puf2, uis4 mRNA translation is de-regulated and UIS4 protein expression upregulated in salivary gland sporozoites. Here, using RNA immunoprecipitation, we reveal the first Puf2-regulated mRNA in this parasite.

Background Experimental research has reported beneficial effects of mesenchymal stromal cell (MSC) therapy in pulmonary arterial hypertension (PAH). However, these studies either were based on prophylactic protocols or assessed basic remodeling features without evaluating possible mechanisms. We analyzed the effects of MSC therapy on lung vascular remodeling and hemodynamics and its possible mechanisms of action in monocrotaline (MCT)-induced PAH. Methods Twenty-eight Wistar rats were randomly divided into two groups. In the PAH group, animals received MCT 60 mg/kg intraperitoneally, while a control group received saline (SAL) instead. On day 14, both groups were further randomized to receive 10 5 adipose-derived MSCs or SAL intravenously ( n  = 7/group). On day 28, right ventricular systolic pressure (RVSP) and the gene expression of mediators associated with apoptosis, inflammation, fibrosis, Smad-1 levels, cell proliferation, and endothelial–mesenchymal transition were determined. In addition, lung histology (smooth muscle cell proliferation and plexiform-like injuries), CD68 + and CD163 + macrophages, and plasma levels of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) were evaluated. Results In the PAH group, adipose-derived MSCs, compared to SAL, reduced mean RVSP (29 ± 1 vs 39 ± 2 mmHg, p  < 0.001), lung tissue collagen fiber content, smooth muscle cell proliferation, CD68 + macrophages, interleukin-6 expression, and the antiapoptotic mediators Bcl-2 and survivin. Conversely, expression of the proapoptotic mediator procaspase-3 and plasma VEGF increased, with no changes in PDGF. In the pulmonary artery, MSCs dampened the endothelial–mesenchymal transition. Conclusion In MCT-induced PAH, MSC therapy reduced lung vascular remodeling, thus improving hemodynamics. These beneficial effects were associated with increased levels of proapoptotic markers, mesenchymal-to-endothelial transition, reduced cell proliferation markers, and inflammation due to a shift away from the M1 phenotype.

During pneumonia, normal alveolar areas coexist adjacently with consolidated areas, and high inspiratory efforts may predispose to lung damage. To date, no study has evaluated different degrees of effort during Biphasic positive airway pressure (BIVENT) on lung and diaphragm damage in experimental pneumonia, though largely used in clinical setting. We aimed to evaluate lung damage, genes associated with ventilator-induced lung injury (VILI) and diaphragmatic injury, and blood bacteria in pressure-support ventilation (PSV), BIVENT with low and high inspiratory efforts in experimental pneumonia. Twenty-eight male Wistar rats (mean ± SD weight, 333±78g) were submitted Pseudomonas aeruginosa-induced pneumonia. After 24-h, animals were ventilated for 1h in: 1) PSV; 2) BIVENT with low (BIVENTLow-Effort); and 3) BIVENT with high inspiratory effort (BIVENTHigh-Effort). BIVENT was set at Phigh to achieve VT = 6 ml/kg and Plow at 5 cmH2O (n = 7/group). High- and low-effort conditions were obtained through anaesthetic infusion modulation based on neuromuscular drive (P0.1). Lung mechanics, histological damage score, blood bacteria, and expression of genes related to VILI in lung tissue, and inflammation in diaphragm tissue. Transpulmonary peak pressure and histological damage score were higher in BIVENTHigh-Effort compared to BIVENTLow-Effort and PSV [16.1 ± 1.9cmH2O vs 12.8 ± 1.5cmH2O and 12.5 ± 1.6cmH2O, p = 0.015, and p = 0.010; median (interquartile range) 11 (9-13) vs 7 (6-9) and 7 (6-9), p = 0.021, and p = 0.029, respectively]. BIVENTHigh-Effort increased interleukin-6 expression compared to BIVENTLow-Effort (p = 0.035) as well as expressions of cytokine-induced neutrophil chemoattractant-1, amphiregulin, and type III procollagen compared to PSV (p = 0.001, p = 0.001, p = 0.004, respectively). Tumour necrosis factor-α expression in diaphragm tissue and blood bacteria were higher in BIVENTHigh-Effort than BIVENTLow-Effort (p = 0.002, p = 0.009, respectively). BIVENT requires careful control of inspiratory effort to avoid lung and diaphragm damage, as well as blood bacteria. P0.1 might be considered a helpful parameter to optimize inspiratory effort.

Marine Coelenterazine is one of the most well-known chemi-/bioluminescent systems, and in which reaction the chemi-/bioluminophore (Coelenteramide) is generated and chemiexcited to singlet excited states (leading to light emission). Recent studies have shown that the bromination of compounds associated with the marine Coelenterazine system can provide them with new properties, such as anticancer activity and enhanced emission. Given this, our objective is to characterize the photophysical properties of a previously reported brominated Coelenteramide analog, by employing a combined experimental and theoretical approach. To better analyze the potential halogen effect, we have also synthesized and characterized, for the first time, two new fluorinated and chlorinated Coelenteramide analogs. These compounds show similar emission spectra in aqueous solution, but with different fluorescence quantum yields, in a trend that can be correlated with the heavy-atom effect (F > Cl > Br). A blue shift in emission in other solvents is also verified with the F–Cl–Br trend. More relevantly, the fluorescence quantum yield of the brominated analog is particularly sensitive to changes in solvent, which indicates that this compound has potential use as a microenvironment fluorescence probe. Theoretical calculations indicate that the observed excited state transitions result from local excitations involving the pyrazine ring. The obtained information should be useful for the further exploration of halogenated Coelenteramides and their luminescent properties.

Cancer is still a challenging disease to treat, both in terms of harmful side effects and therapeutic efficiency of the available treatments. Herein, to develop new therapeutic molecules, we have investigated the anticancer activity of halogenated derivatives of different components of the bioluminescent system of marine Coelenterazine: Coelenterazine (Clz) itself, Coelenteramide (Clmd), and Coelenteramine (Clm). We have found that Clz derivatives possess variable anticancer activity toward gastric and lung cancer. Interestingly, we also found that both brominated Clmd (Br-Clmd) and Clm (Br-Clm) were the most potent anticancer compounds toward these cell lines, with this being the first report of the anticancer potential of these types of molecules. Interestingly, Br-Clm possessed some safety profile towards noncancer cells. Further evaluation revealed that the latter compound induced cell death via apoptosis, with evidence for crosstalk between intrinsic and extrinsic pathways. Finally, a thorough exploration of the chemical space of the studied Br-Clm helped identify the structural features responsible for its observed anticancer activity. In conclusion, a new type of compounds with anticancer activity toward gastric and lung cancer was reported and characterized, which showed interesting properties to be considered as a starting point for future optimizations towards obtaining suitable chemotherapeutic agents.

Chemi- and bioluminescence are remarkable light-emitting phenomena, in which thermal energy is converted into excitation energy due to a (bio)chemical reaction. Among a wide variety of chemi-/bioluminescent systems, one of the most well-known and studied systems is that of marine imidazopyrazinones, such as Coelenterazine and Cypridina luciferin. Due to the increasing usefulness of their chemi-/bioluminescent reactions in terms of imaging and sensing applications, among others, significant effort has been made over the years by researchers to develop new derivatives with enhanced properties. Herein, we report the synthesis and chemiluminescent characterization of a novel dibrominated Coelenterazine analog. This novel compound consistently showed superior luminescence, in terms of total light output and emission lifetime, to natural imidazopyrazinones and commercially available analogs in aprotic media, while being capable of yellow light emission. Finally, this new compound showed enhanced chemiluminescence in an aqueous solution when triggered by superoxide anion, showing potential to be used as a basis for optimized probes for reactive oxygen species. In conclusion, bromination of the imidazopyrazinone scaffold appears to be a suitable strategy for obtaining Coelenterazines with enhanced properties.

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro , SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.