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Abstract Background Breast cancer, the most diagnosed cancer, remains the second leading cause of cancer death in the United States, and excessive Progesterone (PRG) or Mifepristone (MIF) exposure may be at an increased risk for developing breast cancer. PRG exerts its cellular responses through signaling cascades involving classic, non-classic, or combined responses by binding to either classic nuclear PRG receptors (nPRs) or non-classic membrane PRG receptors (mPRs). Currently, the intricate balance and switch mechanisms between these two signaling cascades remain elusive. Three genes, CCM1-3, form the CCM signaling complex (CSC) which mediates multiple signaling cascades. Methods Utilizing molecular, cellular, Omics, and systems biology approaches, we analyzed the relationship among the CSC, PRG, and nPRs/mPRs during breast cancer tumorigenesis. Results We discovered that the CSC plays an essential role in coupling both classic and non-classic PRG signaling pathways by mediating crosstalk between them, forming the CmPn (CSC-mPRs-PRG-nPRs) signaling network. We found that mPR-specific PRG actions (PRG + MIF) play an essential role in this CmPn network during breast cancer tumorigenesis. Additionally, we have identified 4 categories of candidate biomarkers (9 intrinsic, 2 PRG-inducible, 1 PRG-repressive, 1 mPR-specific PRG-repressive, and 2 mPR-responsive) for Luminal-A breast cancers during tumorigenesis and have confirmed the prognostic application of RPL13 and RPL38 as intrinsic biomarkers using a dual validation method. Conclusions We have discovered that the CSC plays an essential role in the CmPn signaling network for Luminal-A breast cancers with identification of two intrinsic biomarkers. Video Abstract

Through the applications of recycling sewage sludge to soils as nutrients, bisphenol A (BPA) and titanium dioxide nanoparticles (TiO2-NPs) are commonly found in the agricultural environment. Previous studies have reported that BPA and nanoparticles are harmful to the environment. However, the combined toxicity of both compounds is not yet understood. This work presented an in-depth proteomic analysis of Arabidopsis thaliana exposed to BPA and TiO2-NPs concurrently at environmentally relevant levels. Seeds were simultaneously treated with varying concentrations of BPA (0, 10, 100, and 1000 µg·kg−1) and TiO2-NPs (0, 1, 10 and 100 mg·kg−1). In treatment of 1000 µg·kg−1 BPA and 100 mg·kg−1 TiO2-NPs, highest seed germination rate (87.97%, p

Giardia lamblia, a protozoan parasite, is a major cause of waterborne infection, worldwide. While the trophozoite form of this parasite induces pathological symptoms in the gut, the cyst form transmits the infection. Since Giardia is a noninvasive parasite, the actual mechanism by which it causes disease remains elusive. We have previously reported that Giardia assembles cholesterol and GM1 glycosphingolipid-enriched lipid rafts (LRs) that participate in encystation and cyst production. To further delineate the role of LRs in pathogenesis, we isolated LRs from Giardia and subjected them to proteomic analysis. Various cellular proteins including potential virulence factors—e.g., giardins, variant surface proteins, arginine deaminases, elongation factors, ornithine carbomyltransferases, and high cysteine-rich membrane proteins—were found to be present in LRs. Since Giardia secretes virulence factors encapsulated in extracellular vesicles (EVs) that induce proinflammatory responses in hosts, EVs released by the parasite were isolated and subjected to nanoparticle tracking and proteomic analysis. Two types of EV—i.e., small vesicles (SVs;

Chagas disease is emerging in countries to which it is not endemic. Biomarkers for earlier therapeutic response assessment in patients with chronic Chagas disease are needed. We profiled plasma-derived extracellular vesicles from a heart transplant patient with chronic Chagas disease and showed the potential of this approach for discovering such biomarkers.

ABSTRACT In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation in vitro. By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence. IMPORTANCE The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.

ABSTRACT Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) extensively N-glycosylates its spike proteins, which are necessary for host cell invasion and the target of both vaccines and immunotherapies. These N-glycans are predicted to modulate spike binding to the host receptor by stabilizing its open conformation and host immunity evasion. Here, we investigated the essentiality of both the host N-glycosylation pathway and SARS-CoV-2 N-glycans for infection. Ablation of host N-glycosylation using RNA interference or inhibitors, including FDA-approved drugs, reduced the spread of the infection, including that of variants B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Under these conditions, cells produced fewer virions and some completely lost their infectivity. Furthermore, partial enzymatic deglycosylation of intact virions showed that surface-exposed N-glycans are critical for cell invasion. Altogether, we propose protein N-glycosylation as a targetable pathway with clinical potential for treatment of COVID-19. IMPORTANCE The coronavirus SARS-CoV-2 uses its spike surface proteins to infect human cells. Spike proteins are heavily modified with several N-glycans, which are predicted to modulate their function. In this work, we show that interfering with either the synthesis or attachment of spike N-glycans significantly reduces the spread of SARS-CoV-2 infection in vitro, including that of several variants. As new SARS-CoV-2 variants, with various degrees of resistance against current vaccines, are likely to continue appearing, halting virus glycosylation using repurposed human drugs could result in a complementary strategy to reducing the spread of COVID-19 worldwide.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6–7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galβ. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.

Background. The worldwide emergence of multi-drug resistant bacteria has become a health crisis, as fewer or sometimes no antimicrobial agents are effective against these bacteria. The Rio Grande River is the natural boundary between the United States (US) and Mexico. It spans a border region between Texas, New Mexico and Mexico. Underserved populations on the Mexican side use the river for recreational purposes, while on the US side, the river is used for irrigation and as a source of drinking water. Objectives. The purpose of the present study was to evaluate the concentration of antibiotic residues, to determine the presence of genetic elements conferring antibiotic resistance and to characterize multi-drug resistant bacteria in the waters of the Rio Grande River. Methods. Water samples were obtained from the Rio Grande River. Deoxyribonucleic acid (DNA) was extracted from both isolated bacteria and directly from the water. Amplification of selected genetic elements was accomplished by polymerase chain reaction. Identification and isolation of bacteria was performed through MicroScan autoSCAN-4. Fecal contamination was assessed by IDEXX Colilert. Antibiotic residues were determined by liquid chromatography and mass spectrometry. Results. Antibiotics were found in 92% of both water and sediment samples. Antibiotic concentrations ranged from 0.38 ng/L - 742.73 ng/L and 0.39 ng/l - 66.3 ng/g dry weight in water and sediment samples, respectively. Genetic elements conferring resistance were recovered from all collection sites. Of the isolated bacteria, 91 (64.08%) were resistant to at least two synergistic antibiotic combinations and 11 (14.79%) were found to be resistant to 20 or more individual antibiotics. Fecal contamination was higher during the months of April and July. Conclusions. The 26 km segment of the Rio Grande River from Sunland Park NM to El Paso, TX and Juarez, Mexico is an area of concern due to poor water quality. The presence of multidrug resistant bacteria, antibiotics and mobile genetic elements may be a health hazard for the surrounding populations of this binational border region. Policies need to be developed for the appropriate management of the environmental natural resources in this border region. Competing Interests. The authors declare no competing financial interests.

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients’ blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method’s limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal β-galactofuranosyl (β-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients’ sera react specifically with synthetic β-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfβ1,3Manpα-(CH2)3SH (glycan G29SH) and Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα-(CH2)3SH (glycan G32SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.

Extracellular vesicles (EVs) shed by trypomastigote forms of Trypanosoma cruzi have the ability to interact with host tissues, increase invasion, and modulate the host innate response. In this study, EVs shed from T. cruzi or T.cruzi-infected macrophages were investigated as immunomodulatory agents during the initial steps of infection. Initially, by scanning electron microscopy and nanoparticle tracking analysis, we determined that T. cruzi-infected macrophages release higher numbers of EVs (50–300 nm) as compared to non-infected cells. Using Toll-like-receptor 2 (TLR2)-transfected CHO cells, we observed that pre-incubation of these host cells with parasite-derived EVs led to an increase in the percentage of infected cells. In addition, EVs from parasite or T.cruzi-infected macrophages or not were able to elicit translocation of NF-κB by interacting with TLR2, and as a consequence, to alter the EVs the gene expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and STAT-1 and STAT-3 signaling pathways. By proteomic analysis, we observed highly significant changes in the protein composition between non-infected and infected host cell-derived EVs. Thus, we observed the potential of EVs derived from T. cruzi during infection to maintain the inflammatory response in the host.

Giardia lamblia, a single-celled eukaryote, colonizes and thrives in the small intestine of humans. Because of its compact and reduced genome, Giardia has adapted a “minimalistic” life style, as it becomes dependent on available resources of the small intestine. Because Giardia expresses fewer sphingolipid (SL) genes—and glycosphingolipids are critical for encystation—we investigated the SL metabolic cycle in this parasite. A tandem mass spectrometry (MS/MS) analysis reveals that major SLs in Giardia include sphingomyelins, sphingoid bases, ceramides, and glycosylceramides. Many of these lipids are obtained by Giardia from the growth medium, remodeled at their fatty acyl chains and end up in the spent medium. For instance, ceramide-1-phosphate, a proinflammatory molecule that is not present in the culture medium, is generated from sphingosine (abundant in the culture medium) possibly by remodeling reactions. It is then subsequently released into the spent medium. Thus, the secretion of ceramide-1-phospate and other SL derivatives by Giardia could be associated with inflammatory bowel disease observed in acute giardiasis. Additionally, we found that the levels of SLs increase in encysting Giardia and are differentially regulated throughout the encystation cycle. We propose that SL metabolism is important for this parasite and, could serve as potential targets for developing novel anti-giardial agents.

Trypanosoma cruzi, the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi, which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture-derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans-sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin-like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle-free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC-MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain-derived EVs caused higher infection of LLC-MK2 cells than Y strain-derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host–parasite interaction.

Background Cardiovascular disease is the main cause of death in the United States, with smoking being the primary preventable cause of premature death, and thrombosis being the main mechanism of cardiovascular mortality in smokers. Due to the perception that electronic/e‐cigarettes are “safer/less harmful” than conventional cigarettes, their usage—among a variety of ages—has increased tremendously during the past decade. Notably, there are limited studies regarding the negative effects of e‐cigarettes on the cardiovascular system, which is also the subject of significant debate. Methods and Results We employed a passive e‐VapeTM vapor inhalation system and developed an in vivo whole‐body e‐cigarette mouse exposure protocol that mimics real‐life human exposure scenarios/conditions and investigated the effects of e‐cigarettes and clean air on platelet function and thrombogenesis. Our results show that platelets from e‐cigarette–exposed mice are hyperactive, with enhanced aggregation, dense and α granule secretion, activation of the αIIbβ3 integrin, phosphatidylserine expression, and Akt and ERK activation, when compared with clean air–exposed platelets. E‐cigarette–exposed platelets were also found to be resistant to inhibition by prostacyclin, relative to clean air. Furthermore, the e‐cigarette–exposed mice exhibited a shortened thrombosis occlusion and bleeding times. Conclusions Taken together, our data demonstrate for the first time that e‐cigarettes alter physiological hemostasis and increase the risk of thrombogenic events. This is attributable, at least in part, to the hyperactive state of platelets. Thus, the negative health consequences of e‐cigarette exposure should not be underestimated and warrant further investigation.

Cerebral cavernous malformations (CCMs) are characterized by abnormally dilated intracranial capillaries that result in increased susceptibility to stroke. Three genes have been identified as causes of CCMs; KRIT1 (CCM1), MGC4607 (CCM2) and PDCD10 (CCM3); one of them is disrupted in most CCM cases. It was demonstrated that both CCM1 and CCM3 bind to CCM2 to form a CCM signaling complex (CSC) to modulate angiogenesis. In this report, we deployed both RNA-seq and proteomic analysis of perturbed CSC after depletion of one of three CCM genes to generate interactomes for system-wide studies. Our results demonstrated a unique portrait detailing alterations in angiogenesis and vascular integrity. Interestingly, only in-direct overlapped alterations between RNA and protein levels were detected, supporting the existence of multiple layers of regulation in CSC cascades. Notably, this is the first report identifying that both β4 integrin and CAV1 signaling are downstream of CSC, conveying the angiogenic signaling. Our results provide a global view of signal transduction modulated by the CSC, identifies novel regulatory signaling networks and key cellular factors associated with CSC., (© 2019 Published by Elsevier Ltd.)

Children with special health care needs (CSHCN) require comprehensive care with high levels of community and government assistance. Medical-legal partnerships may be particularly suited to address needs for this population. To explore this, we conducted in-depth telephone interviews of families of CSHCN cared for in the primary care practice of our tertiary care children's hospital. The majority of the sample (N = 46) had been late on housing payments and 17% of homeowners had been threatened with foreclosure. Families frequently reported denial of public benefits. Approximately 10% had executed advance planning documents such as guardianship plans for the children or wills for the parents. A minority of families had sought help from community agencies or lawyers. Less than one third had ever discussed any of the issues with health care providers, but two thirds were likely or very likely to in the future. CSHCN may especially benefit from the social support of a medical-legal partnership., (© The Author(s) 2015.)

Background: Absent or reversed end-diastolic flow velocity (ARED) in the umbilical arteries is typically associated with severe intrauterine growth restriction (IUGR), although there may be other causes, such as aneuploidy., Case: A healthy 30-year-old gravida 2, para 0 woman with an uneventful pregnancy and normal prenatal screening test results presented with sudden cessation of fetal movement at 26 weeks' gestation. Ultrasound demonstrated unexplained ARED with normal fetal growth and normal amniotic fluid volume. The fetus died during transfer to the labour and delivery unit. The cause of fetal demise was initially assumed to be feto-maternal hemorrhage, but postmortem examination revealed non-compaction of the ventricular myocardium., Conclusion: Intrinsic myocardial disease should be considered in the differential diagnosis when ARED is discovered in the umbilical arteries, especially when fetal growth is normal. Postmortem examination should be performed even if a prior 18- to 20-week anatomical ultrasound has been reported as normal.