New strategic insights into managing fungal biofilms

Fungal infections have dramatically increased in the last decades in parallel with an increase of populations with impaired immunity, resulting from medical conditions such as cancer, transplantation or other chronic diseases. Such opportunistic infections result from a complex relationship between fungi and host, and can range from self-limiting to chronic or life-threatening infections. Modern medicine, characterized by a wide use of biomedical devices, offers new niches for fungi to colonize and form biofilm communities. The capability of fungi to form biofilms is well documented and associated with increased drug tolerance and resistance. In addition, biofilm formation facilitates persistence in the host promoting a persistent inflammatory condition. With a limited availability of antifungals within our arsenal, new therapeutic approaches able to address both host and pathogenic factors that promote fungal disease progression, i.e. chronic inflammation and biofilm-formation, could represent an advantage in the clinical setting. In this paper we discuss the antifungal properties of Myriocin, Fulvic Acid and Acetylcholine in light of their already known anti-inflammatory activity and as candidate dual action therapeutics to treat opportunistic fungal infections

Roles for Intestinal Bacteria, Viruses, and Fungi in Pathogenesis of Inflammatory Bowel Diseases and Therapeutic Approaches

Intestinal microbiota are involved in the pathogenesis of Crohn’s disease, ulcerative colitis, and pouchitis. We review the mechanisms by which these gut bacteria, fungi, and viruses mediate mucosal homeostasis, via their composite genes (metagenome) and metabolic products (metabolome). We explain how alterations to their profiles and functions under conditions of dysbiosis contribute to inflammation and effector immune responses that mediate inflammatory bowel diseases (IBD) in humans and enterocolitis in mice. It could be possible to engineer the intestinal environment by modifying the microbiota community structure or function to treat patients with IBD— either with individual agents, via dietary management, or as adjuncts to immunosuppressive drugs. We summarize the latest information on therapeutic use of fecal microbial transplantation and propose improved strategies to selectively normalize the dysbiotic microbiome in personalized approaches to treatment

Granulocyte-Derived Cationic Peptide Enhances Homing and Engraftment of Bone Marrow Stem Cells after Transplantation

Current strategies to accelerate hematopoietic reconstitution after transplantation include transplantation of greater numbers of hematopoietic stem/progenitor cells (HSPCs) or ex vivo expansion of harvested HSPCs before transplant. However, the number of cells available for transplantation is usually low, and strategies to expand HSPCs and maintain equivalent engraftment capability ex vivo are limited. We noted that activated granulocyte-derived cationic peptides positively primed responsiveness of HSPCs to a CXCL12 gradient. Accordingly, we noted that accelerated homing/engraftment of β-defensin-2, a well-known antimicrobial cationic peptide, primed bone marrow nucleated cells (BMNCs) compared to normal BMNCs after transplantation into lethally irradiated recipients. We envision that small cationic peptides, which primarily possess antimicrobial functions and are harmless to mammalian cells, could be applied to prime HSPCs before transplantation. This novel approach would be particularly important in cord blood transplantation, where the number of HSPCs available for transplantation is usually limited

Glycan Engineering for Cell and Developmental Biology

Cell-surface glycans are a diverse class of macromolecules that participate in many key biological processes, including cell-cell communication, development, and disease progression. Thus, the ability to modulate the structures of glycans on cell surfaces provides a powerful means not only to understand fundamental processes but also to direct activity and elicit desired cellular responses. Here, we describe methods to sculpt glycans on cell surfaces and highlight recent successes in which artificially engineered glycans have been employed to control biological outcomes such as the immune response and stem cell fate

The Expanding Family of Bone Marrow Homing Factors for Hematopoietic Stem Cells: Stromal Derived Factor 1 Is Not the Only Player in the Game

The α-chemokine stromal derived factor 1 (SDF-1), which binds to the CXCR4 and CXCR7 receptors, directs migration and homing of CXCR4+ hematopoietic stem/progenitor cells (HSPCs) to bone marrow (BM) and plays a crucial role in retention of these cells in stem cell niches. However, this unique role of SDF-1 has been recently challenged by several observations supporting SDF-1-CXCR4-independent BM homing. Specifically, it has been demonstrated that HSPCs respond robustly to some bioactive lipids, such as sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), and migrate in response to gradients of certain extracellular nucleotides, including uridine triphosphate (UTP) and adenosine triphosphate (ATP). Moreover, the responsiveness of HSPCs to an SDF-1 gradient is enhanced by some elements of innate immunity (e.g., C3 complement cascade cleavage fragments and antimicrobial cationic peptides, such as cathelicidin/LL-37 or β2-defensin) as well as prostaglandin E2 (PGE2). Since all these factors are upregulated in BM after myeloblative conditioning for transplantation, a more complex picture of homing emerges that involves several factors supporting, and in some situations even replacing, the SDF-1-CXCR4 axis

Engineering physiological environments to advance kidney organoid models from human pluripotent stem cells

During embryogenesis, the mammalian kidney arises because of reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM), driving UB branching and nephron induction. These morphogenetic processes involve a series of cellular rearrangements that are tightly controlled by gene regulatory networks and signaling cascades. Here, we discuss how kidney developmental studies have informed the definition of procedures to obtain kidney organoids from human pluripotent stem cells (hPSCs). Moreover, bioengineering techniques have emerged as potential solutions to externally impose controlled microenvironments for organoid generation from hPSCs. Next, we summarize some of these advances with major focus On recent works merging hPSC-derived kidney organoids (hPSC-kidney organoids) with organ-on-chip to develop robust models for drug discovery and disease modeling applications. We foresee that, in the near future, coupling of different organoid models through bioengineering approaches will help advancing to recreate organ-to-organ crosstalk to increase our understanding on kidney disease progression in the human context and search for new therapeutics

CRISPR/Cas9 as a Therapeutic Approach to Duchenne Muscular Dystrophy

Transhumanism, designer babies, gene therapy, and super-soldiers are founded upon the same concept—genetic engineering. Clustered Regularly-Interspersed Short Palindromic Repeats (CRISPR) is a natural bacterial immune response method that takes advantage of gene manipulation to prevent an infection from mobile genetic elements. Since Mojica et al. (2005) first suggested the relationship between the CRISPR/Cas system and prokaryotic immunity, significant advancements have been made in understanding the mechanism and subsequent applications of CRISPR. CRISPR, has three main subtypes based on unique proteins and interference pathways and serves as an accurate and effective method for gene editing. Its mechanism consists of spacer acquisition, crRNA production, and interference. This highly dynamic form of genetic modification generates significant CRISPR sequence differences in species that are almost identical when comparing the rest of their genome. CRISPR/Cas9 demonstrates the simultaneous alteration of multiple gene loci in individual cells with a high degree of specificity and precision. Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder characterized by progressive muscle loss and eventual death in the late teens to early twenties. DMD affects calcium homeostasis, vasculature, genetic regulation, muscle movement, glycosylation, tissue remodeling, and inflammatory response mechanisms. Current treatments include antifibrotic pharmaceuticals, calcium maintenance, myostatin inhibitors, upregulation of uthrophin, nonsense suppression drugs, vector-mediated gene therapy, and cell transplantation. This review describes the mechanism of CRISPR/Cas9 and its application as a therapeutic approach to treating Duchenne muscular dystrophy

Nitric oxide formation from inorganic nitrate and nitrate : Contribution from eukaryotic and prokaryotic pathways

Nitric oxide (NO) is an essential signaling molecule that plays a central role in a broad range of physiological functions. Classically, NO is synthesized from L-arginine and molecular oxygen by NO synthases. Once formed, it is rapidly oxidized to nitrite and nitrate. These two inorganic anions were previously considered to be inert end products but this view is now being seriously challenged by research revealing that nitrite can be physiological reduced to again generate NO. The reduction of nitrite in vivo seems particularly enhanced during hypoxia and acidosis; conditions when the oxygendependent NO-synthase pathway is dysfunctional. Besides the endogenous formation of nitrate and nitrite by NO synthase, these anions are also ingested naturally via the diet. The first step in bioactivation of nitrate is formation of the more reactive nitrite anion; a reaction suggested to involve oral nitrate reducing bacteria. It has been generally viewed that mammalian cells cannot metabolize the stable nitrate anion. In the present thesis, we intended to further characterize NO generation from the nitrate-nitrite-NO pathway. In particular we have studied the importance of commensal bacteria in nitrate metabolism and attempted to explore if mammalian tissues are also capable of nitrate reduction. We also studied possible interactions between the classical NO synthase pathway and the nitrate-nitrite-NO pathway. We show that bacteria in the gastrointestinal tract play an interesting role in mammalian NO biology. Besides the bioactivation of nitrate in the oral cavity to form nitrite, bacteria in the small and large intestine can catalyze the same reaction and also the subsequent reduction of nitrite to form NO. NO formation in the gut can be stimulated in vivo by supplementation with dietary nitrate and probiotic bacteria. In further studies involving also germ-free mice, we surprisingly find that inorganic nitrate is enzymatically reduced to nitrite in tissues and we identify the enzyme xanthine oxidoreductase (XOR) as the dominant nitrate reductase. Mammalian nitrate reductase activity is enhanced during hypoxic conditions but is also active during normoxia. The functional consequences of this nitrate reductase activity were studied after nitrate administration in vivo. Nitrate attenuated the increased blood pressure caused by an NO synthase inhibitor and prevented the severe decline in blood flow during post-ischemic reperfusion. The expression of XOR is enhanced in tissues of germ free mice, which may reflect a feedback response to the absence of bacterial nitrate reduction in these animals. Such crosstalk is further supported in a study of long-term dietary nitrate supplementation in rats, in which expression of phosphorylated eNOS in aortic tissue and eNOS activity was down-regulated after nitrate supplementation. All together these data suggest a crosstalk between NOS-independent and NOS-dependent pathways in control of NO vascular homeostasis. In summary, the present thesis helps to draw a new picture of mammalian NO generation which occurs by serial reductions of the supposedly inert anions nitrate and nitrite. In this pathway both eukaryotic and prokaryotic pathways contribute to formation of NO and maintenance of homeostasis. Intriguingly, NO formation from nitrate in the gastrointestinal tract, the cardiovascular system and elsewhere, can be controlled by simple dietary interventions with resulting physiological effects