Background: Fungal extracellular vesicles (EVs) have been implicated in host-pathogen and pathogen-pathogen communication in some fungal diseases. In depth research into fungal EVs has been hindered by the lack of specific protein markers such as those found in mammalian EVs that have enabled sophisticated isolation and analysis techniques. Despite their role in fungal EV biogenesis, ESCRT proteins such as Vps23 (Tsg101) and Bro1 (ALIX) are not present as fungal EV cargo. Furthermore, tetraspanin homologs are yet to be identified in many fungi including the model yeast S. cerevisiae. Objective: We performed de novo identification of EV protein markers for the major human fungal pathogen Candida albicans with adherence to MISEV2018 guidelines. Materials and methods: EVs were isolated by differential ultracentrifugation from DAY286, ATCC90028 and ATCC10231 yeast cells, as well as DAY286 biofilms. Whole cell lysates (WCL) were also obtained from the EV-releasing cells. Label-free quantitative proteomics was performed to determine the set of proteins consistently enriched in EVs compared to WCL. Results: 47 proteins were consistently enriched in C. albicans EVs. We refined these to 22 putative C. albicans EV protein markers including the claudin-like Sur7 family (Pfam: PF06687) proteins Sur7 and Evp1 (orf19.6741). A complementary set of 62 EV depleted proteins was selected as potential negative markers. Conclusions: The marker proteins for C. albicans EVs identified in this study will be useful tools for studies on EV biogenesis and cargo loading in C. albicans and potentially other fungal species and will also assist in elucidating the role of EVs in C. albicans pathogenesis. Many of the proteins identified as putative markers are fungal specific proteins indicating that the pathways of EV biogenesis and cargo loading may be specific to fungi, and that assumptions made based on studies in mammalian cells could be misleading. Abbreviations: A1-ATCC10231; A9-ATCC90028; DAY B-DAY286 biofilm; DAY Y-DAY286 yeast; EVextracellular vesicle; Evp1extracellular vesicle protein 1 (orf19.6741); GOgene ontology; Log 2 (FC)log 2 (fold change); MCCmembrane compartment of Can1; MDSmultidimensional scaling; MISEVminimal information for studies of EVs; sEVssmall EVs; SPsignal peptide; TEMstetraspanin enriched microdomains; TMtransmembrane; VDMvesicle-depleted medium; WCLwhole cell lysate ARTICLE HISTORY

No abstract

Portal hypertension and bleeding from gastroesophageal varices is the major cause of morbidity and mortality in patients with cirrhosis. Portal hypertension is initiated by increased intrahepatic vascular resistance and a hyperdynamic circulatory state. The latter is characterized by a high cardiac output, increased total blood volume and splanchnic vasodilatation, resulting in increased mesenteric blood flow. Pharmacological manipulation of cirrhotic portal hypertension targets both the splanchnic and hepatic vascular beds. Drugs such as angiotensin converting enzyme inhibitors and angiotensin II type receptor 1 blockers, which target the components of the classical renin angiotensin system (RAS), are expected to reduce intrahepatic vascular tone by reducing extracellular matrix deposition and vasoactivity of contractile cells and thereby improve portal hypertension. However, these drugs have been shown to produce significant off-target effects such as systemic hypotension and renal failure. Therefore, the current pharmacological mainstay in clinical practice to prevent variceal bleeding and improving patient survival by reducing portal pressure is non-selective -blockers (NSBBs). These NSBBs work by reducing cardiac output and splanchnic vasodilatation but most patients do not achieve an optimal therapeutic response and a significant proportion of patients are unable to tolerate these drugs. Although statins, used alone or in combination with NSBBs, have been shown to improve portal pressure and overall mortality in cirrhotic patients, further randomized clinical trials are warranted involving larger patient populations with clear clinical end points. On the other hand, recent findings from studies that have investigated the potential use of the blockers of the components of the alternate RAS provided compelling evidence that could lead to the development of drugs targeting the splanchnic vascular bed to inhibit splanchnic vasodilatation in portal hypertension. This review outlines the mechanisms related to the pathogenesis of portal hypertension and attempts to provide an update on currently available therapeutic approaches in the management of portal hypertension with special emphasis on how the alternate RAS could be manipulated in our search for development of safe, specific and effective novel therapies to treat portal hypertension in cirrhosis.

Although β-amyloid (Aβ) may be the primary driver of Alzheimer’s disease (AD) pathology, accumulation of pathological tau correlates with dementia in AD patients. Thus, the prevention/inhibition of AD may require vaccine/s targeting Aβ and tau simultaneously or sequentially. Since high antibody titers are required for AD vaccine efficacy, we have decided to generate vaccines, targeting Aβ (AV-1959R), Tau (AV-1980R) or Aβ/tau (AV-1953R) B cell epitopes, based on immunogenic MultiTEP platform and evaluate the immunogenicity of these vaccines formulated with AdvaxCpG, delta inulin, Alhydrogel®, Montanide-ISA51, Montanide-ISA720, MPLA-SM pharmaceutical grade adjuvants. Formulation of AV-1959R in AdvaxCpG induced the highest cellular and humoral immune responses in mice. The dual-epitope vaccine, AV-1953R, or the combination of AV-1959R and AV-1980R vaccines formulated with AdvaxCpG induced robust antibody responses against various forms of both, Aβ and tau pathological molecules. While anti-Aβ antibody titers after AV-1953R immunization were similar to that in mice vaccinated with AV-1959R or AV-1959R/AV-1980R combination, anti-tau titers were significantly lower after AV-1953R injection when compared to the AV-1980R or AV-1959R/AV-1980R. In silico 3D-modeling provided insight into the differences in immunogenicity of these vaccine constructs. In sum, AV-1959R and AV-1980R formulated with AdvaxCpG adjuvant were identified as promising immunogenic vaccines for ongoing pre-clinical assessment and future human clinical trials.