The temporomandibular joint (TMJ) is an articulation formed between the temporal bone and the mandibular condyle which is commonly affected. These affections are often so painful during fundamental oral activities that patients have lower quality of life. Limitations of therapeutics for severe TMJ diseases have led to increased interest in regenerative strategies combining stem cells, implantable scaffolds and well-targeting bioactive molecules. To succeed in functional and structural regeneration of TMJ is very challenging. Innovative strategies and biomaterials are absolutely crucial because TMJ can be considered as one of the most difficult tissues to regenerate due to its limited healing capacity, its unique histological and structural properties and the necessity for long-term prevention of its ossified or fibrous adhesions. The ideal approach for TMJ regeneration is a unique scaffold functionalized with an osteochondral molecular gradient containing a single stem cell population able to undergo osteogenic and chondrogenic differentiation such as BMSCs, ADSCs or DPSCs. The key for this complex regeneration is the functionalization with active molecules such as IGF-1, TGF-β1 or bFGF. This regeneration can be optimized by nano/micro-assisted functionalization and by spatiotemporal drug delivery systems orchestrating the 3D formation of TMJ tissues. The temporomandibular joint (TMJ) is an articulation covered by dense fibrocartilage formed between the mandibular condyle and the temporal bone. The temporal articular surface is large and consists of the mandibular fossa and the articular tubercle. Along this large articular temporal surface, each mandibular condyle has a wide motion range, consisting of both rotation and translation.Fibrocartilaginous disc cushions mechanical stresses that exist between the temporal and mandibular articular surfaces. The high collagen content of this disc provides great rigidity and durability. The TMJ disc has no direct vascularization or innervation by itself. However, its posterior attachment, known as retrodiscal tissue, features many vessels and nerves which are crucial during physio-pathological processes.TMJD (temporomandibular joint disorders) are very common, their prevalence being around 52% . TMJD isPain, malocclusion, limited range of motion, deviation, joint clicking and clenching are most of time associated with TMJD. These disorders are often so painful during basic oral activities (eating and speaking) that quality of life of patients is greatly impacted . Pain isOsteoarthritis-like degenerative joint disease belonging to TMJD is a destruction of bone and cartilage with a consecutive inflammation which enhances tissue destruction . TMJ degOnce the primary factors of TMJD have been identified and eliminated, the treatment can vary according to the severity of the disorder: non-invasive, minimally invasive and invasive procedures. However, permanent recovery is rarely obtained and therefore, follow-up therapies are required .Occlusal orthodontics, medications, physical therapy and acupuncture are the most common non-invasive treatments. Although occlusal orthodontics are widely used, their effectiveness remains questionable ,9. Non-iMinimally invasive treatments can target either extrinsic masticatory muscles or TMJ itself. Masticatory muscles  have been injected with botulinum toxin type A (Botox) for thirty years ,13. The Minimally invasive treatments of TMJ itself include intra-articular injections, arthrocentesis, and arthroscopy. Intra-articular injections of corticosteroids into either one or both joint compartments improve TMJD symptoms ,16. ArthInvasive treatment is the only option for patients suffering from ankylosis, neoplasia, dislocation, and developmental disorders ,23. It cRegarding invasive treatment of the TMJ disc, both its surgical repositioning and the removal of affected tissues have short-term efficacy . The surThe many options proposed for disc replacement have not shown satisfactory clinical outcomes: teflon-proplast or silicone synthetic implants ,29, searFor severe TMJ degeneration, the total surgical joint replacement is currently prescribed. The first experiments on regeneration using alloplastic TMJ implants showed a tendency for premature failure. Subsequent immune reactions were triggered, therefore resulting in catastrophic joint damage ,33. ThusTissue engineering of TMJ has been a primary concern in scientific discussion and practice. The limitations of current therapeutics for TMJD have led to an increased interest in regenerative strategies combining cells, implantable scaffolds and well-targeting bioactive molecules. The recent advances of regenerative medicine for orthopedics may contribute to meet the challenge of this specific complex regeneration.Meeting the demands for anatomic, structural, and functional regeneration of TMJ is very challenging. Innovative strategies and biomaterials are absolutely crucial because TMJ can be considered as one of the most difficult tissues to regenerate.The TMJ is an anatomic zone so narrow and dense that surgical access is not easy. Its limited blood supply and hence its very limited healing capacity do not facilitate regenerative strategies either.The mechanical and structural  properties of the mandibular condyle implant must match that of anatomic condyle for human clinical applications. Also, the implanted tissue-engineered mandibular condyle must have rapid and adequate remodeling potential allowing oral functions.Histologically, the TMJ cartilage differs a lot from the other hyaline articular cartilages. Thus, regenerating this unique articular cartilage with its complex structure and organization is very tricky. TMJ regeneration means to be able to engineer a mandibular condyle implant with its unique cartilage and its underlying bone in a single osteochondral construct. The challenge in TMJ regeneration is to promote matrix synthesis and tissue maturation of chondrogenic and osteogenic cells in suitable scaffolds containing active molecules which are able to separately orchestrate osteogenesis and chondrogenesis .The success of TMJ regenerative strategy is not only measured by the restoration of function but also by the long-term prevention of ossified or fibrous adhesions which are the main complications of engineered TMJ replacements . Thus, pThere is a continuous debate about the embryonic origin of mandibular articulating surface: blastemal or periosteal origin ,39. The The TMJ disc attached to the condyle and temporal bone by fibrous connective tissue measures 14 mm antero-posteriorly and 23 mm medico-laterally in humans . Its perTwo methods are possible in cartilage and bone engineering: (1) in situ tissue engineering, which involves an incorporation of an acellular scaffold matrix attracting local cells (cell homing) guiding the process of regeneration; (2) ex vivo cell seeding on the scaffold, which provides enough competent cells to orchestrate the regenerative mechanism . The secAutogenic cells are the ideal cell source for tissue regeneration. Fibrochondrocytes from mandibular condyle seeded on polyglycolic acid (PGA) scaffolds showed weaker regenerative capacities than chondrocytes from ankle joint. Notably, they produced less GAGS and collagens . In the To regenerate TMJ condylar cartilage, primary costal-chondrocytes or hyaline cartilage cells from all cartilages in the body can be used ,62. StemBone marrow mesenchymal stem cells (BMSCs) provide a high rate of cell growth and division. Their advantage is the important volume of cells available and the numerous kind of possible differentiation. They can promote bone and cartilage regeneration of TMJ. Their disadvantage is their tendency to endochondral ossification ,65,66,67Adipose stem cells (ADSCs) could be a potential cell source for TMJ engineering . They arDifferent tooth-derived stem cells are also potential competent cells for TMJ regeneration. periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAPs) similar to mesenchymal stem cells (MSCs) ,70 are aDental pulp stem cells (DPSCs) mesenchymal stem cells from dental pulp  are knowThe capacity of osteogenic differentiation of DPSCs is well-documented ,78,79. D8 cells/mL of scaffold volume enhanced fibrocartilaginous deposition but modified mechanical properties. The lowest seeding density that promotes functional properties close to in vivo conditions must be identified for each cell source to regenerate TMJ disc [For regeneration of discal fibrocartilage, dermal fibroblasts are promising. Easily available, these autologous cells seeded in quantity and treated with IGF-1 showed a high chondrogenic potential . For disTMJ disc .These stem cells able to undergo both chondrogenic and osteogenic differentiation are crucial for TMJ regeneration. The best strategy should be to use a unique stem cell type able to support in a unique scaffold biphasic osteochondral regeneration orchestrated by active pro-chondrogenic and pro-osteogenic molecules.Hyaluronic acid (HA) is a polysaccharide abundant in cartilaginous matrices, which constitutes an ideal chondrogenic microenvironment, ideal for cartilage regeneration . HA hydrAgarose is a polysaccharide extracted from seaweed, used as agar for cell culture. Its advantage is its adaptable stiffness, which allows an easy variation of mechanical features of the scaffold . AgarosePoly-vinyl alcohol (PVA) is a hydrophilic polymer which is also very appropriate for cartilage regeneration due to its high water content and its elastic properties . Its capl-lactic-coglycolic acid (PLGA) is a synthetic polymer approved by the FDA for clinical applications which is greatly interesting for cartilage regeneration. The versatility of its structure allows also a modulation of mechanical properties of the scaffold. PLGA scaffolds promote colonization and differentiation of MSCs in vivo [Poly- in vivo . PLGA in in vivo . Incorpo in vivo .l-Lactic acid (PLLA) is interesting for its slow degradation rate. PLLA scaffolds seeded with porcine TMJ cells and treated with TGF β-1 improved mechanicals properties and showed higher collagen and GAGS deposition as compared to PGA scaffolds [For the specific regeneration of TMJ disc, a variety of scaffolds have shown their efficacy in vitro and in vivo ,104. An caffolds . A mixedcaffolds . An acelcaffolds .Collagens are natural polymers very convenient for osteochondral regeneration and also for total TMJ disc reconstruction . CollageGelatin, derived from the lysis of collagen is also appropriate for osteochondral regeneration. Gelatin extracellular environment is favorable to the adhesion and colonization of chondrocytes . GelatinNanofibers constitute pro-regenerative biomimetic extracellular matrices very interesting for tissue regeneration. The electrospinning technique makes it possible to obtain different matrices made of synthetic and natural polymers whose nanofiber diameter is close to the size of the collagen nanofibers (50–500 nm). The network of electrospun nanofibers as well as the micropores formed (less than 100 μm in diameter) mimics the structure of the connective tissue matrix ,118. PolFibrin presents a great interest for osteochondral regeneration. Most of studies deal with fibrin scaffolds for culture and differentiation of stem cells . Fibrin-These scaffolds, able to support both cartilage and bone regeneration are crucial for TMJ regeneration. They give the opportunity to build an osteochondral construct in the same scaffold, i.e., a sole scaffold but biphasic due to its functionalization.Growth factors help tissue regeneration at different levels. They can promote the differentiation and proliferation of cells. They can support extracellular matrix synthesis and its mineralization . They caThe three key growth factors for TMJ regeneration are basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1) and transforming growth factor-β1 (TGF-β1). They are able to maintain disc-like tissue in culture ,128 and The effect of IGF-1 on stimulating chondrogenesis is well-documented in vitro and in vivo. In particular, it increases both GAG and collagen contents of the engineered cartilage ,132. TheTGF-β1 shows some positive effects on cellular proliferation and on the production of extracellular matrix in TMJ disc implants . It induThe stimulating effect of bFGF on cell proliferation and production of collagen is well-known. GAGS synthesis is also significantly stimulated by bFGF . bFGF anPlatelet derivative growth factor (PDGF) significantly increases the proliferation rate of the TMJ-disc derived cells, collagen and hyaluronic acid synthesis in engineered TMJ disc. It upregulates RNA levels of type I and II collagens, matrix metalloproteinases (MMPs), and their specific tissue inhibitors (TIMPs) . PDGF alOver-expression of some interesting growth factors for tissue regeneration has been evidenced in malignant tumors. The debate about their oncogenic capability still hounds their clinical employment for tissue regeneration of the oral and maxillofacial region . FinallyVarious technologies for incorporation of growth factors into scaffolds are possible. The release of growth factors must match the rate of healing and regeneration ,139. TheFunctionalization can otherwise be accomplished by gene therapy. Gene transfer can also be conducted by viral or non-viral transduction. For tissue regeneration, the most appropriate method for gene transfer uses retroviruses, adenoviruses or adeno-associated viruses ,142,143.Nanofunctionalization of scaffolds made of electrospun nanofibers is possible by different techniques: plasma or wet chemical treatment, surface graft polymerization and co-axial electrospinning . The co-Intra-articular drug delivery methods applied to the TMJ seem very attractive for both pain management and regenerative strategies. Benefits of current methods of intra-articular injection are controversial. Some alarming reports describing post-injection complications have discouraged their use for TMJ pain ,152. TheBone and cartilage regeneration occur in very different competing conditions. To engineer a biphasic osteochondral implant is therefore challenging. Ideal approaches for TMJ regeneration are a single scaffold functionalized by an osteochondral molecular gradient and a unique stem cell population associated using rapid and synchronized tissue engineering techniques . UnderstIn large osteochondral defects of goat condyles, PLGA composite implants seeded with Nel-related protein 1 (NRP1) modified-autologous BMSCs were able to regenerate bone and cartilage tissue after transplantation. The fibrocartilage was regenerated six weeks after transplantation and the subchondral bone native articular cartilage after 24 weeks .Promising results in mandibular condyle tissue regeneration were obtained after subcutaneous implantation of athymic mice with PGA and PLA scaffolds seeded with calf osteoblasts and chondrocytes in athymic mice. Analysis after 12 weeks of implantation evidenced the condylar shape of the neoformed bone and the formation of hyaline cartilage on the articular surface and of trabecular bone .Hyper-hydrated collagen gels seeded with MSCs preconditioned in two different media were proposed with one osteogenic and one chondrogenic medium at each extremity. After seven days of in vitro culture, distinct bone-like and cartilage-like areas were observed which resembled to primordial joint-like structure .The same strategy of gradient-based scaffolding was proposed with PLGA microspheres: implants were functionalized with TGF-β1 at the cartilaginous end and BMP-2 at the bony end. It promoted neoformed osteochondral tissue after six weeks of implantation in mandibular condyle defects of New Zealand rabbits ,160.A hybrid compartmented implant was proposed with cartilage-promoting alginate/HA hydrogel at the cartilaginous end and bone-promoting nanofibrous collagen membrane at the bony end. This biphasic scaffold promoted in vitro osteogenic and chondrogenic differentiation of a single stem cell population (human MSCs). A gradient of mineralization for articular cartilage and a natural ‘glue’ at the osteochondral interface were obtained in vitro .Whatever the strategies used, the regeneration of TMJ must match the anatomic, structural, and functional particularities of the mandibular condyle and its disc.A bone implant of biodegradable PLGA seeded with porcine bone marrow MSCs was designed as mandibular condyle . SimilarThe application of static uniaxial load on shape-specific TMJ disc engineered by co-culture of articular chondrocytes and meniscal fibrochondrocytes increased its functional properties. It optimized GAG synthesis and anisotropic properties resembling those of a TMJ disc .Current TMJ replacement is made by prostheses. These alloplastic strategies are constantly improving in order to obtain personalized 3D prosthesis. Personalized prostheses of TMJ fabricated by 3D-printing were designed and implanted in patients. Compared with stock devices, these personalized 3D prostheses present better biomechanical and clinical outcomes. This 3D-printing technique also improves the surgery. Indeed, the positioning of an implant is easier, due to its optimal shape and to the opportunity to have an optimal 3D surgical guide ,169,170.These crucial clinical advances of personalized 3D prosthesis benefit regenerative strategies. Indeed, personalized 3D scaffolds can be considered. In that direction, the computer-designed nanofibrous and microporous scaffolds proposed by Chen et al. are very attractive and lead the way of a personalized 3D bone regenerative nanomedicine .Currently, different techniques exist to produce 3D scaffolds such as phase separation, self-assembly, electrospinning and bioprinting. As seen previously, two points are necessary for the 3D scaffold to help and favor tissue regeneration: growth factors and living cells. Electrospinning can combine these three parameters. It allows 3D and porous structures constituted of nanofibers mimicking extracellular matrix. It can also be tuned to modulate biodegradability and resistance depending of the type of tissue to regenerate. Living cells can be added on the 3D scaffold for colonization of the matrix, or directly be included inside fibers using coaxial techniques ,173. 3D A personalized 3D polyamide implant coated by nanoscale hydroxyapatite was rapidly designed and manufactured by computer in replacement of mandibular condyle. Its implantation into a patient showed positive clinical outcomes .3D printed scaffolds were engineered with a spatiotemporal delivery of connective tissue growth factor (CTGF) and TGF-β3 encapsulated in microparticles in order to build a rabbit TMJ disc. Their implantation evidenced positive results. Significant improvement of regeneration was observed with the spatiotemporal gradient of functionalization . Same apPrevalence of affections of TMJ is important. Severe affections are preferentially concerned by regeneration. Currently, they are treated by arthrotomy and implantation of prostheses. The recent advances in regenerative medicine for orthopedics may provide solutions for TMJ regeneration. However, anatomic, structural, and functional regeneration of TMJ is very challenging and specific. The fibrocartilaginous property of the mandibular condyle and its tight link with its fibrocartilaginous disk contribute to modifying issues. The difficulty is not to obtain a pure hyaline cartilage with an underlying bone as for the other articulations. The main issue is to get a long-term fibrocartilage well-separated from its underlying bone without ossifications or fibrous adhesions which are dramatic for crucial oral functions of patients. At present, concrete progress of TMJ arthroscopy allows adequate visualization and manipulation of pathological intra-articular tissues and motivate the emergence of innovative and specific regenerative strategies of TMJ. Numerous proposals of interest have been presented focusing on suitable cells, scaffolds or active molecules for TMJ regeneration. Global strategies, able to support the entire mandibular condyle regeneration, are very attractive. So, the desired approach is a unique scaffold inducing an osteochondral molecular gradient containing a single stem cell population able to undergo osteogenic and chondrogenic differentiation such as BMSCs, ADSCs or DPSCs. The key to this complex regeneration is the functionalization by active molecules such as IGF-1, TGF-β1 or b-FGF. This regeneration can be optimized by nano/micro-assisted functionalization and by spatiotemporal drug delivery systems orchestrating the 3D formation of TMJ tissues.


It is acknowledged that vaccines remain the best hope for eliminating the HIV-1 epidemic. However, the failure to produce effective vaccine immunogens and the inability of conventional delivery strategies to elicit the desired immune responses remains a central theme and has ultimately led to a significant roadblock in HIV vaccine development. Consequently, significant efforts have been applied to generate novel vaccine antigens and delivery agents, which mimic viral structures for optimal immune induction. Here, we review the latest developments that have occurred in the nanoparticle vaccine field, with special emphasis on strategies that are being utilized to attain highly immunogenic, systemic, and mucosal anti-HIV humoral and cellular immune responses. This includes the design of novel immunogens, the central role of antigen-presenting cells, delivery routes, and biodistribution of nanoparticles to lymph nodes. In particular, we will focus on virus-like-particle formulations and their preclinical uses within the HIV prophylactic vaccine setting. Human immunodeficiency virus-1 (HIV-1) and HIV-2 have emerged as the result of zoonotic lentiviral transmissions of simian immunodeficiency viruses (SIV), from chimpanzees/gorillas and sooty mangabeys, respectively, into the human population. First identified as the etiological agent behind acquired immunodeficiency syndrome (AIDS) in humans in the early 1980s, HIV has continued to spread into a global pandemic and major public health concern. At the end of 2015 alone, there were 36.7 million people infected with HIV with an additional 2.1 million new infections that same year. Excluding parenteral infections, HIV acquisition is almost entirely transmitted through sexual intercourse. Worldwide, approximately 80% of the 30 million people infected with HIV acquired the virus through heterosexual transmission, with receptive vaginal and anal intercourse accounting for 60–70 and 5–10%, respectively, of the global total.Human immunodeficiency virus infection is characterized by the catastrophic depletion of CD4 T cells—the very cells that orchestrate host immune responses against invading pathogens. The combination of viral cytopathic effects, cell suicide by caspase-1 mediated pyroptosis, and caspase-3-mediated apoptosis are the major mechanisms behind the depletion of CD4 T cells , HIV is no longer a death sentence. From 1995–2010, approximately 5.1 million AIDS-related deaths were averted in low- and middle-income countries due to greater accessibility of ART . Currentvia the intramuscular (IM), intradermal (I.D.), or subcutaneous (S.C.) routes, high titers of neutralizing antibodies against homologous viruses could be elicited.Initial HIV vaccine candidates to enter human clinical trials were subunit vaccines based on the surface Env glycoprotein. These were mainly soluble monomeric gp120 or gp160 constructs –7. Thesepost hoc analysis of the protection data, it was observed that should the study have stopped after the first year of implementation, vaccine efficacy would have been as high as 60%, indicating a non-sustained protection from acquisition was achieved. In terms of the immune correlates of protection a weak neutralizing antibody response was again observed; however, non-neutralizing plasma IgG binding antibodies to the top of the Env V1/V2 variable loop were associated with protection  infused neutralizing antibody protects against viral challenge in non-human primates (NHP) studies, (2) a percentage of individuals will generate neutralizing antibody responses against the autologous infecting virus, and that (3) highly potent neutralizing antibody can be generated in some individuals, solidifies the notion that an anti-HIV B cell response can be protective. As neutralizing antibodies are directed against the surface glycoprotein, presentation of natural Env structures, mimicking the native viral Env is deemed imperative to eliciting a neutralizing immune response. Resolving this scientific challenge has led to re-engineering of the Env itself to achieve a native conformation. As a result, numerous antigenic structures have been developed, including germline revertants targeting the N332 supersite and near native trimers candidates such as BG505 SOSIP.664 –27. ThesTo overcome these afore-mentioned issues in antigen design, Env trimers have been expressed in lipid membranes. This enables their conformations to more accurately resemble native and functional Env spikes found on infectious virus. Taking this into consideration, nanoparticle platforms such as VLPs provide the ideal technology to express HIV Env immunogens. VLPs are non-infectious and genomeless multiprotein structures that mimic the conformation of viral proteins in their natural environment. These self-assembling molecules can generate ordered arrays of polypeptides that come together to form the VLP. The resulting repetitive geometry offers maximal VLP–host interactions due to increased avidity . As a reThe use of VLPs as potential vaccine candidates offers numerous advantages over traditional strategies. VLPs have the unique ability to present viral Env spikes in their natural conformation and, therefore, the potential to elicit neutralizing antibody responses against HIV. This feature is often lost when antigens are purified from pathogens or when pathogens are rendered inert through chemical or heat inactivation. VLP vaccines are also cheaper than subunit vaccines since less of the vaccine can be administered without impairing the resulting immune response. Another attractive property of VLP vaccines are their particulate structure, which allows them to be efficiently taken up by antigen-presenting cells (APCs) and stimulate strong humoral and cellular immune responses , 33 sinc®. It mainly consists of Hepatitis B surface antigen that spontaneously assembles into ~20 nm lipid-containing particles , none exist for HIV Table . One of articles . The majarticles . Gardasi virions .Agrobacterium tumefaciens, a Gram-negative soil-based bacteria used to transform plant cells  and co-receptors (CCR5) whereas the gp41 anchors Env in the membrane. Taking the well-characterized influenza virus as a model system shows that the virus may require only 2–3 trimers to engage with its cellular receptors to create a pre-fusion structure. However, it is believed that 6–8 trimers may be necessary to build the stable fusion pore . This quin vitro using healthy donor CD4 T cell-DC co-cultures  viruses to see if transmission is through stochastic events or whether the genetic bottle neck selects for viruses with certain properties. Features associated with transmitted viruses include CCR5 tropism, short variable loops and less N-linked glycan residues. With such distinguishing features in mind, McClure et al. tested the antigenicity and immunogenicity of DNA and modified vaccinia Ankara expressed VLPs, displaying native forms of T/F clade C Envs . The hypAntigen delivery by liposomal vesicles was demonstrated over 30 years ago when unilamellar vesicles were generated that were composed of phosphatidylserine. These liposomes contained poliovirus and purified poliovirus RNA. They were found to be infectious and could be potentially utilized as a delivery agent for biological macromolecules . LiposomInterbilayer-cross-linked multilamellar vesicles (ICMV) with His-tagged gp140 trimers anchored onto the surface of the delivery agent using Ni-NTA functionalized lipids were recently explored . The aimvia non-covalent metal chelation. Furthermore, the liposomes-coupled antigens were better activators of B cells as determined through Ca2+ flux measurements. Finally, murine immunogenicity studies demonstrate that the covalent coupling of antigens enabled the expansion of B cell germinal center reactions, over and above what was seen with soluble antigens, and that they also blocked the access of B cells and antibody to the c-terminal end of the exposed Env spike, suggesting that the trimers were intact at the point of immune recognition and adaptive immune activation , TLR9, or STING Interestingly, intrastructural help through helper epitopes promoted IgG responses to MPER without increasing B cell responses against the help sequence  of Env. This gp41 region is the target for several neutralizing antibodies and is attractive as a vaccine candidate due to its conserved and linear nature. A significant drawback to MPER immunogens is its inherent lack of immunogenicity. Recently Hanson et al. utilized 150 nm liposomes as membrane display vehicles to promote a more native vaccine antigen conformation . In thissequence .Polymeric nanoparticles are very promising candidate delivery systems and adjuvants for a range of vaccine antigens. Their favorability stems from their ease of synthesis, biocompatibility, their biodegradable nature, the fact they are non-immunogenic, non-toxic and fairly inexpensive. Numerous different polymeric nanoparticle exist but the most frequently encountered types include chitosan, poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) Table .H1 immune responses and to also act as both delivery system and adjuvant when applied to mucosal surfaces.This cationic polysaccharide, polymeric nanoparticle is derived from the deacetylation of chitin, a naturally occurring polymer found in the cuticles of insect species and crustaceans, such as crabs and shrimp. Due to its strong immune-stimulatory properties and low immunogenicity, chitosan has frequently been utilized as an antigen delivery system. A significant advantage of chitosan to conventional adjuvants such as alum is its ability to promote TIn 2016, Cosgrove et al., published the first comparative Phase 1 clinical trial investigating the safety and immunogenicity of three HIV-1 clade C gp140 vaccinations delivered by either the IM, intranasal (IN), and intravaginal routes in women . The vacChitosan has also served as a formulation adjuvant by promoting penetration enhancement of formulations, enabling their uptake and increasing the bioavailability of vaccine antigens when applied topically to mucosal surfaces. This was clearly demonstrated by Klein et al., who compared various polymeric penetration enhancers to promote trans-mucosal delivery of trimeric CN54gp140 protein . In thisThe use of PLGA as a nanoparticle vaccine delivery system has been studied comprehensively over the last two decades, resulting in numerous publications. However, it is the ability of PLGA nanoparticles to entrap bacterial toxoids/antigen or surface display antigens and induce long-lasting immune responses in small animal models which makes them an attractive delivery system –71. Earld,l-lactide-co-glycolide) (PLGA), and VLPs are known to elicit both arms of the immune response. Understanding the cellular events involved in such vaccines are critical for their development as vaccine strategies and advancing test formulations to human clinical trials. Specifically, the molecular and cellular interactions occurring between nanoparticles and the vaccinated host immune system  will be essential. Although many alternative vaccination routes have been explored in animal models and in humans, most vaccine studies employ IM vaccination. Intradermal (ID) vaccination demonstrates strong immune reactions to vaccinating antigens in a dose sparing manner compared to IM vaccination; however, ID vaccination is associated with a higher frequency of undesired side effects. Thus, we will concentrate on nanoparticle vaccines administered via the IM route.The immune response to nanoparticle formulations such as liposomes, poly elicited TH1 biased responses , into early endosome-like phagosomes, whereas the smaller particles (155 nm) and soluble antigen were rapidly localized to endo-lysosomes  the recognition of exogenous signals by innate immune cells through pathogen-associated molecular patterns (PAMPS) and (2) being activated in response to recognition of danger-associated molecular patterns, which are endogenous molecules released by damaged or perturbed tissues/cells . Such imin vitro within APC population, such as DCs and macrophages via NLRP3 inflammasome activation  capable of adsorbing subunit vaccines onto their surface, thereby increasing BCR crosslinking, presentation to APCs, and can act to form an antigen depot at the vaccination site . Criticativation . While AThe use of alum to augment antiviral immunity has been evaluated in the context of therapeutic HIV vaccines. Although therapeutic vaccinations are now aimed at a sterilizing or functional cure, initial attempts in the 1990s and early 2000s were made to therapeutically “modulate” immune responses to curb CD4 T cell decline and lower the rate of AIDS progression , 104. AsAlum has also been used as an adjuvant for VLPs used for prophylactic vaccine strategies for prevention of HIV acquisition. Insect cell expression systems have been used to generate chimeric VLPs that express the V3 domain and a linear section of the discontinuous CD4-binding domain of gp120 within gag . SubsequCollectively, the literature presented suggests that despite Alum’s positive influence in some preclinical experiments and in some clinically approved VLP formulations, its inappropriate use can diminish the effects of test vaccines, resulting in ineffectual immune responses. Thus, inclusion of alum as a VLP adjuvant in any preclinical or clinical trial must be scrutinized.via MHC. The MHC presentation to T cells occurs in the context of several signals, including, co-stimulatory molecules, cytokines, and by TLR ligation. Collectively, this stimulates T cells to surpass the minimal activation thresholds for naïve T cells to partake in immune responses. Due to their abilities to activate innate signaling pathways, PRR ligands are an expanding field of study for HIV vaccine design. This includes the use of TLR4 (MPL) and 7/8 (R848) agonists encapsulated in PLGA particles admixed with VLPs expressing SIV gp160 and Gag  are the best-defined families of PRRs. They are expressed by a plethora of mammalian cells as either endosomal or plasma membrane bound receptors. Cell surface TLRs  detect conserved microbial patterns present on the cell surface such as LPS and flagellin, while endosomal TLRs  detect microbial genomic material such as double-stranded RNA and DNA and single-stranded RNA . Detecti and Gag , the inc and Gag  and the  and Gag . Normall and Gag . Therefo and Gag . However and Gag . In anot and Gag . This suO-deacyl-4′-monophosphoryl lipid A (MPLA), is a lipopolysaccharide (LPS)-derived, heterogeneous blend of varying length MPL molecules from Salmonella minnesota R595. The MPLA extraction and treatment procedure resulted in three distinct modifications compared to the parent molecule: (1) the removal of the core polysaccharide containing the O-antigen, (2) the removal of one phosphate group, and (3) one fatty acid chain  responses  adjuvant. The original formula was a mix of saponin, cholesterol, phospholipid, and other hydrophobic compounds . The majH1 and TH2 responses as noted by rapid IL-5 and IFN-g production following immunization  and also monocultures of Caco-2 cells. Oral application of the RGD-nanoparticles was also shown to be immunogenic in some mice, with some animals having elevated anti-OVA IgG antibody responses and splenic IFN-γ T cell responses , Garinot et al. were able to significantly increase the transport of particles using an esponses . Additiovaccines –156. Altvaccines . TherefoThe last three decades has seen tremendous advancements in our understanding of HIV acquisition, treatment, and prevention. During this time, several vaccine efficacy trials have been completed and all have failed to prevent HIV infections apart from the modestly effective RV144. A major impediment to a successful vaccine has been antigen design and delivery. Crucial to overcoming this roadblock has been the development of nanoparticles to stabilize and orientate candidate vaccine immunogens for optimal immune triggering. Within this review we have covered the basis for vaccine development and the promising nanoparticle-based approaches to answer some of the very complicated issues surrounding HIV vaccine progress. Nanoparticle-based vaccines have demonstrated themselves to be versatile, and depending on which antigen, adjuvant and delivery route used, can have significant impacts on prevailing immune responses. While nanoparticle vaccines hold great promise, a significant number of unknowns still exist, and when answered could greatly advance our understanding on how to maximally deliver promising vaccine immunogens. These include a better understanding of the effects of charge, size, structure, and potential toxic side effects with various nanoparticle platforms.While the major focus of this nanoparticle review has been on the development of neutralizing antibody, nanoparticle formulations targeting the V1/V2 region of Env by non-neutralizing antibody is a significant area of interest and needs to be explored further. Justification for this is drawn from the correlates of protection from the RV144 study, which is being further evaluated and validated by on-going clinical trials in Thailand and Africa. In addition, although most vaccines are delivered systemically, HIV is predominantly transmitted through mucosal surfaces and as such, it is imperative that vaccine candidates be developed that can either augment mucosal immunity or be designed to draw protective systemic responses into the mucosal environment. The latter point would enable the provision of protective frontline responses within tissues at risk of infection. The only caveat being that mucosal tissue activation and vaccine-induced inflammation be carefully managed, to not increase the likelihood of transmission by growing the number of potential target cells.Finally, the use of nanoparticle technology will also undoubtedly have a significant role to play in therapeutic HIV vaccines. The thought of an HIV-1 cure was once regarded a dream. Now, growing evidence suggests that a cure could be a reality and the generation of vaccine induced antiviral immunity will take center stage. There are numerous cure technologies that currently being evaluated, however it is the “Shock and Kill” tactic that has garnered significant attention. Several latency reversal agents have been devised to reactivate (shock) latent virus into transcriptional activity; however, vaccines that can prime for protective antibody and cytotoxic T and NK cell responses will be essential for subsequent viral eradication (kill).We are clearly entering an exciting time for HIV vaccine development and nanoparticle formulations will have a lot to offer. As long as there is continued support for the nanoparticle vaccine effort, prophylactic and therapeutic HIV/AIDS interventions will continue to advance until such time as a vaccine becomes a reality.YG, CW, and JM wrote sections of the manuscript and YG, CW, and JM edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Transcranial direct current stimulation (tDCS) is a non-invasive, plasticity-inducing brain stimulation technique that can induce long-lasting excitability changes in the motor cortex and has been discussed as an alternative treatment option for patients with schizophrenia. Therefore, the aim of the present study was to detect electrophysiological correlates after motor cortical and prefrontal tDCS in order to improve the understanding of tDCS-mechanisms. Anodal and cathodal tDCS was applied over motor cortex (M1) and dorsolateral prefrontal cortex (DLPFC), which is known as one major region of interest considering neurobiology of psychosis. Thus, we looked for tDCS-induced source-localized activity changes in resting EEG by using sLORETA (standardized low-resolution brain electromagnetic tomography) and compared the effects of motor and prefrontal cortex.A total of 20 healthy volunteers were examined within five sessions (within-subject design). Anodal tDCS  and cathodal tDCS  were applied over M1 and respectively DLPFC. In addition, there was a sham tDCS of DLPFC. Transcranial magnetic stimulation (TMS) was performed before and after motor cortical tDCS in order to generate motor evoked potentials (MEP) as periphery indicators of motor cortical plasticity. A 6-minute resting EEG was performed before and after each tDCS treatment. EEG data was then investigated by sLORETA for source-localized brain activity changes.After tDCS over M1, the expected increase of MEP amplitude after anodal tDCS and reduction after cathodal tDCS could be measured. Following anodal tDCS over M1 an increased activity was found in the area of precuneus in EEG frequency band alpha. After cathodal tDCS over M1 an activity decrease was seen in frequency band alpha, beta and total power, which could be localized in insula and temporal gyrus.After anodal as well as cathodal tDCS over DLPFC decreased activities could be measured in most frequency bands . Most of these changes were found in frontal lobe, anterior cingulate or insula. Unexpectedly there were also significant changes after sham tDCS in all frequency bands. However, these were measured mostly in right-sided temporal lobe, which could be due to jaw muscle artefacts.The polarity-specific tDCS effects, which can be demonstrated in motor cortex, cannot be seen in prefrontal cortex; instead we detected a polarity-independent frontal modulation. This lack of prefrontal polarity specificity may be explained by a more complex mode of action in frontal cortex. This is consistent with the variable results of prefrontal tDCS in other publications. As the effects from motor cortical studies cannot easily be transferred to the frontal system in healthy subjects, one could speculate that in schizophrenic patients the responses to prefrontal tDCS might be even more difficult to predict. Further investigations are required to evaluate the heterogeneity of source-localized tDCS-effects and to understand prefrontal mechanisms, so that frontal tDCS may be used as a future treatment in schizophrenia patients.


Modulation between sleep and wake states is controlled by a number of heterogeneous neuron populations. Due to the topological proximity and genetic co-localization of the neurons underlying sleep-wake state modulation optogenetic methods offer a significant improvement in the ability to benefit from both the precision of genetic targeting and millisecond temporal control. Beginning with an overview of the neuron populations mediating arousal, this review outlines the progress that has been made in the investigation of arousal circuits since the incorporation of optogenetic techniques and the first in vivo application of optogenetic stimulation in hypocretin neurons in the lateral hypothalamus. This overview is followed by a discussion of the future progress that can be made by incorporating more recent technological developments into the research of neural circuits. Before the development of optogenetic tools researchers used methods such as electrophysiological stimulation and recording of neural activity, pharmacological stimulation or inhibition of targeted neuron populations, or the manipulation of gene expression through use of knockout animals to investigate the function and activation patterns of different neural populations. While these methods have undoubtedly produced an immense body of literature and insight into neural functions, structures, and mechanisms across multiple areas of neuroscience research, as new technologies have developed, the weaknesses of these techniques have naturally become highlighted. While electrophysiology allows the temporal precision that is lacking in pharmacological and genetic manipulation, it lacks the ability to target specific neuron populations using their gene expression. Pharmacological and genetic manipulations allow this neuron targeting specificity but lack the temporal flexibility of electrophysiological recordings, either requiring time for the pharmacological agent to peak or washout, or, in genetically manipulated mice, lacking the ability to observe the effect of the gene knockout during specific development periods, or even during specific behaviors.The development of optogenetic tools has provided researchers with a method to genetically target specific neuron populations with millisecond precision, either for recording (fiber-photometry) or stimulation (optogenetic activation) of neural activity. The advances provided by this technique have allowed researchers to combine the gene-targeting specificity of pharmacological and genetic manipulations with the temporal flexibility of electrophysiological recordings. While the use of electrophysiological recording and stimulation methods is still very useful for applications where the genetic profile of the target neurons are not yet known or are not uniform, research areas in which it is known which neurotransmitters and cell types are involved can benefit greatly from this specificity. One area of research that has benefited greatly from this precision and flexibility is the investigation of neural circuits underlying arousal and transitions between sleeping and waking states. This review will focus on the neural correlates of sleep-wake state modulation, giving an overview of the neural populations involved, and presenting the progress that has been made following the incorporation of optogenetic technologies into the investigation of individual neuron populations, as well as circuit integration.Considering the ubiquity of sleep across the animal kingdom, the precise function and purpose of sleep remains relatively elusive; however, it is apparent that effectively regulating arousal is crucial for survival. As well as maintaining an appropriate sleep/wake cycle we must also be able to respond appropriately to unexpected stimuli, whether they be an unanticipated reward or the sudden presentation of a stressor. The ability to focus and react can mean the difference between life and death. In states of disordered arousal we may exhibit hyper-arousal—the inability to drop out of a state of heightened vigilance resulting in anxiety disorders, insomnia, etc. or if we are unable to successfully maintain stable sleep-wake states this can lead to either an inability to maintain prolonged sleep or prolonged wakefulness. In order to understand what is going wrong in sleep disorders, researchers first had to understand what normal, healthy sleep-wake states look like.Active/motivated wakefulness—θ 5–9 Hz) ,4 and γ –9 Hz ,4 Quiet wakefulness—slower EEG frequencies such as α (8–14 Hz) and β (15–24 Hz)  and muscNREM—high amplitude, low frequency Δ oscillations (0.5–4 Hz) ,9,10,11 REM—dominated by θ oscillations and strong suppression of muscular tone ,16,17,18The first step away from a binary “sleep” versus “wake” definition of sleep-wake states came with the observation of what is now called Rapid Eye Movement (REM) sleep, in which a sleeping subject elicits eye movements and electroencephalogram (EEG) activity that is similar to those elicited from an awake subject. REM sleep was first observed by Aserinsky and Kleitman , and thiAlongside these sleep-wake states, further categorizations are possible to perform more fine-grained analysis of different sleep states, for example in humans NREM sleep can be further categorized into four different stages (I–VI) of increasing depth of sleep . HoweverResearchers studying sleep to wake transitions have used many different methods to uncover the numerous neuron populations that are involved in modulating wakefulness and sleep states. Following the influenza epidemic of 1918, Economo ,21, a sl1 receptor antagonists) and their sedative side effects implicated histamine as a wake-promoting neuropeptide [Pharmaceuticals have also highlighted potential candidate systems; particularly the development of first-generation antihistamines  neurons throughout the brainstem . AdminisThe role of dopamine in modulating wakefulness came into the spotlight due to the side effect of increased alertness following the consumption of certain drugs of abuse that are categorized as stimulants. Stimulants such as amphetamine and modafinil interact with other neuromodulatory systems, as well as dopaminergic neurons they also enhance the release of other wake-promoting neuropeptides serotonin and norepinephrine. In order to determine the main mechanism inducing increased alertness Wisor, et al.  used a dDA neurons [DA neurons may play a role in the directing of attention. The case for DA modulation of arousal is also supported by lesion studies in the vPAG, which showed a ~20% reduction in waking duration [DA neurons are performing separate functions to the consistently active VTADA neurons that elicit increased burst activity during waking and REM states.Dopaminergic (DA) neurons in both the ventral tegmental area (VTA) and the ventral periaqueductal gray matter (vPAG) have been investigated to observe their activity patterns across different sleep/wake states. Fos studies have shown that DA neurons in the vPAG are active during waking but not sleep states , however neurons . Additio neurons  showed tduration . The varNeuropeptide S (NPS) precursor mRNA (messenger ribonucleic acid) is most strongly expressed around the locus coeruleus (LC), in the the principal sensory 5 nucleus, and in the the lateral parabrachial nucleus, and its receptor mRNA has highest expression levels in the cortex, thalamus, hypothalamus, and amygdala . NPS has1 receptors [1 receptor antagonists, as well as subsequent studies showing that pharmacological inhibition of the histamine system produces drowsiness whereas pharmacological activation of the histamine system promotes wakefulness [1 receptor knockout mice exhibited similar sleep/wake durations to controls, only showing fewer micro-arousals (<16 s brief awakening), and fewer transitions between NREM sleep and REM sleep, but no difference in overall sleep/wake duration [The link between histamine and wake-promotion was first suggested due to the side effect of drowsiness caused by first-generation antihistamines, which antagonized histamine Heceptors ,24. A waeceptors ,46,47. Defulness ,99, otheefulness  and the efulness  producedduration . Investiduration ,104, sugduration .Hypocretin (Hcrt) neurons are found in the lateral hypothalamus and were first linked to sleep function due to a significant loss of Hcrt neurons in the brains of patients with narcolepsy ,21 and lStudies investigating the role of Hcrt in sleep modulation in non-diseased mouse models have found evidence for a wake-promoting role for Hcrt; Hcrt 1 administration (i.c.v.) increases wakefulness in a dose-dependent manner in rats . ConversWhile considering the role of different neuropeptides in modulating sleep-wake states across the day/night cycle, researchers must also consider how outside influences on sleep behaviors interact with these neurotransmitters. The two strongest determinants of sleeping behaviors are time of day and food availability, humans generally adhere to an active light-period and a quiet dark-period. EEG rhythms that are used to distinguish between sleep-wake states can oscillate according to these 24 h light–dark cycles, for example hippocampal θ rhythm, most often associated with waking states, is modulated on a circadian rhythm and can be entrained by food availability . ConsideMelanin-concentrating hormone (MCH) is another sleep-relevant neuron population located in the hypothalamus. MCH appears to have an opposing function to the other hypothalamic population of Hcrt neurons, while Hcrt promotes wakefulness it appears that MCH plays a role in promoting sleep, particularly REM sleep. Fos studies have shown that MCH neurons are most responsive after REM sleep ,58. ThisAlongside the other neuropeptides mentioned researchers must also consider the contribution of glutamatergic neurons and γ-amino butyric acidergic (GABAergic) neurons to sleep-wake states. These neuron types are found in all of the brain areas previously discussed ,130,131,A receptors [GABA neurons in particular may be involved in directing arousal linked to the reward system following evidence that their firing increases before intracranial self-stimulation in the medial forebrain bundle [Within the hypothalamus it has been shown that a population of wake-active neurons will increase their firing rate in response to antagonism of GABAeceptors , whereaseceptors ,137. It n bundle . GABAergn bundle . Addition bundle . These on bundle . The hetThe development of optogenetics has revolutionized neuroscience research. It has been particularly useful for studying heterogeneous neuron populations in areas like the hypothalamus where many neuropeptides may be co-expressed within the same cell. Within the hypothalamus investigation of Hcrt and MCH neurons were made particularly difficult using traditional research methods due to their proximity and their apparent duality of function . The genThe first illustration of in vivo optogenetic manipulation was an investigation of Hcrt activation in the lateral hypothalamus (LH), which showed that optogenetic activation (5–30 Hz) of Hcrt neurons increases the probability of a sleep-wake transition from either NREM or REM sleep . This efMCH neurons at the start of the dark phase (waking period) produces a dramatic increase in sleep duration, NREM sleep by 60% and REM sleep by 95% across the first 6 h [Optogenetic techniques have also been used to investigate MCH neurons in the hypothalamus showing that optogenetic activation of LHirst 6 h . Whereasirst 6 h ,146. In irst 6 h  also useDA neurons over 6 h is sufficient to maintain wakefulness and inhibit sleep-relevant behaviors such as nest building [+ VTA neurons, Eban-Rothschild, Rothschild, Giardino, Jones and de Lecea [DA neurons appeared to have increased activity during REM sleep compared to waking states [DA showed higher activation during waking states compared to REM sleep, especially during the early stages of waking, showing a gradual decrease in activity across the waking phase [DA neurons promoting wakefulness in the earlier stages of the waking period, and Hcrt neurons taking over modulation of arousal as the wake-phase progresses.Optogenetic investigation of DA neurons in the VTA has shown that semi-chronic stimulation of VTAbuilding . Conversbuilding . Additiode Lecea  observedde Lecea . In linede Lecea . Similarde Lecea . Similarde Lecea . Interesg states , whereasng phase . Consideng phase , it is pWake- and REM-active cholinergic neurons are found in the basal forebrain ,26,30 anAs previously stated, GABAergic neurons are located throughout the central nervous system and are co-localized with many different sleep-relevant neuropeptides. Optogenetic activation of GABAergic neurons found in the ventral medulla has been shown to rapidly and reliably induce REM sleep, or extend REM sleep episodes when stimulated during an already initiated REM sleep episode, whereas pharmacogenetic inhibition of these neurons results in dose-dependently reduced REM sleep . GeneticA recent optogenetic investigation of the basal forebrain studied the role of two GABAergic neuron populations in sleep/wake states. It was observed that parvalbumin-positive GABAergic neurons are primarily wake- and REM-active, and optogenetic activation of these neurons results in increased NREM-wake transitions, and an overall increase in the duration of waking states and decrease in the duration of NREM sleep . ConversWhile DA, Hcrt, MCH, and GABAergic neurons are well accepted as being part of the sleep-wake state machine, recent studies have also taken advantage of optogenetic tools to investigate possible roles in sleep regulation in other non-neuron cell types in the brain. Pelluru, et al.  used theWhile optogenetic tools have provided insight into each of these neuron populations individually, the most beneficial quality of these tools is the possibilities they present for circuit investigation. By combining optogenetic methods with other pharmacological and genetic approaches such as DREADDs, chemogenetic inhibition/excitation, or genetic mouse models researchers can observe how manipulating one part of the sleep–wake state circuitry affects the relevant upstream/downstream targets. The majority of studies using optogenetic methods to investigate these neuropeptides have focused on the wake promoting neurons, with a particular focus on interactions with Hcrt neurons.1 receptor function either by application of an H1 receptor antagonist [1 receptor knockout mice [Hcrt and histamine are both wake-active neuron populations and the possibility of some kind of sleep-wake state mediating circuit was suggested by in vitro evidence that Hcrt neurons excite histamine neurons , and reptagonist ,158 or atagonist , and patout mice , optogenout mice . These fTo observe Hcrt activation of histamine neurons more closely Schone, et al.  combinedThe densest afferent projections from Hcrt neurons project to the wake-promoting locus coeruleus  and in vWhile the majority of arousal and sleep–wake state research focuses on transitions between waking, NREM, and REM sleep due to their clear delineations, there is also the possibility of subcategorizing wake-states. Although there are not currently widely accepted criteria to define categories of wakefulness, states of quiet-waking and active-waking are discussed frequently as well as the state following presentation of salient stimuli to induce either exploratory behaviors (rewards) or stress responses (aversive/fearful) stimuli. Hypocretin has been linked to states of hyper-arousal such as stress in a study by Bonnavion, et al. , which sHcrt is inhibited following food intake  and by bA receptor blocker (10 μm gabazine), suggesting a possible role for GABA in mediating interactions between MCH and Hcrt neurons [A receptors.Optogenetic methods have also been used to investigate the apparent opposing functions of wake-promoting Hcrt neurons  and REM- neurons . Indeed, neurons ) and thi neurons . Taken tOptogenetic stimulation dramatically improved the ability of researchers to stimulate specific populations of neurons without unintentionally stimulating neighboring neuron populations. This specificity can also be achieved for the recording of neural activity of targeted neurons populations without inadvertently recording activity from other adjacent populations by using fiber-photometry ,179. TheThe main improvement that optogenetic tools provide over traditional electrophysiological recording and stimulating methods is the ability to target specific neurons based on their gene-expression. However, this does not help if the population of neurons being studied is not yet genetically defined. Therefore, in order for researchers to receive the maximum benefit from these tools it is important to carry out a systematic characterization of the target population. Recent developments in next-generation sequencing methods now allow researchers to investigate gene-expression profiles of single neurons . These mWhile the optogenetic investigations discussed here have all taken great steps toward understanding the mechanisms underlying how the brain modulates between REM, NREM, quiet waking, active waking, and hyper-arousal states, there is still much to be learned. Each of the neural populations discussed here hold their own interest for the function of sleep and wake states, however, no behavior as complex as the modulation of sleep-wake transitions occurs in a single-structure vacuum, and it will only be by investigating circuits that researchers can finally comprehend the mechanisms via which animals can modulate sleep behaviors. Optogenetic technologies have vastly improved the ability of researchers to investigate the complex circuitry underlying sleep behaviors and these technologies will be fundamental in increasing our current knowledge of the sleep functions carried out in the brain. While much attention has been paid to optogenetic stimulation in particular, without first observing the endogenously occurring neuronal activation it would be impossible to successfully mimic neural activity using optogenetic stimulation. Therefore preliminary studies using optogenetic techniques such as fiber photometry should be taken advantage of to first observe the naturally occurring neural patterns researchers wish to recreate in the neurons expressing their neuropeptide of interest.


Background: Breast cancer is intrinsically heterogeneous and is commonly classified into four main subtypes associated with distinct biological features and clinical outcomes. However, currently available data resources and methods are limited in identifying molecular subtyping on protein-coding genes, and little is known about the roles of long non-coding RNAs (lncRNAs), which occupies 98% of the whole genome. lncRNAs may also play important roles in subgrouping cancer patients and are associated with clinical phenotypes. Methods: The purpose of this project was to identify lncRNA gene signatures that are associated with breast cancer subtypes and clinical outcomes. We identified lncRNA gene signatures from The Cancer Genome Atlas (TCGA )RNAseq data that are associated with breast cancer subtypes by an optimized 1-Norm SVM feature selection algorithm. We evaluated the prognostic performance of these gene signatures with a semi-supervised principal component (superPC) method. Results: Although lncRNAs can independently predict breast cancer subtypes with satisfactory accuracy, a combined gene signature including both coding and non-coding genes will give the best clinically relevant prediction performance. We highlighted eight potential biomarkers (three from coding genes and five from non-coding genes) that are significantly associated with survival outcomes. Conclusion: Our proposed methods are a novel means of identifying subtype-specific coding and non-coding potential biomarkers that are both clinically relevant and biologically significant. Molecularly targeted therapies significantly contribute to efforts toward personalized approaches for the treatment of breast cancer, one of the most aggressive and prevalent diseases in women . In 2017Clinically, breast cancer has main four subtypes: Luminal A, Luminal B, Her2 positive, and Basal ,4,5. TheRecent studies have shown long non-coding RNAs to be involved in breast cancer progression via certain biological mechanisms and to display characteristics typical of cancer subtypes ,12,13. GOne approach to identify patient subgroups/clusters uses an unsupervised learning method based on expression data. However, the drawback of this method is that it is usually not necessarily related to clinical outcomes. Another approach is the supervised learning method, which aims to identify molecular targets associated with cancer subtypes supervised in terms of clinical outcomes. The most famous supervised learning methods are support vector machines (SVMs) ,22 and rTo assist comprehensive studies of all types of transcripts and their role in breast tumorigenesis, we propose a novel procedure that will be able to identify cancer subtype-specific biomarkers by a supervised multi-class feature selection method using the 1-Norm Support Vector Machine (SVM)  with recn = 1092) RNAseq and clinical data from the UCSC Xena database (http://xena.ucsc.edu/). Among 1092 samples, 839 of them are labeled the intrinsic subtypes, and 253 are missing subtype information. The raw TCGA RNAseq data (Illumina HiSeq 2000 RNA Sequencing platform) was re-processed with UCSC’s Xena Toil . A combined gene list (n = 36) from both lncRNAs and PCGs exhibited a slightly improved prediction accuracy of 88.5%: , which is an improvement over PCGs or lncRNAs alone. The prognostic risk scores trained by superPC were used to compare the associations of various gene signatures with overall survival (OS) and recurrence-free survival (RFS). The Cox proportional regression analysis for each gene signature risk score showed that the 36-gene combined list had the best performance for both OS and RFS. We also applied the 36 genes to TCGA 253 unlabeled samples to predict their subtypes. Results showed that 92% of the predicted Basal subgroup was associated with TNBC, and 95.8% of the Luminal subgroup (LumA: 80.9% and LumB: 14.9%) was associated with the ER+/PR+ positive group. Since the above findings are consistent with clinical acceptance, our methods are useful and will provide more prediction options for determining cancer subtypes. In addition to identifying the gene signatures for prediction of breast cancer subtypes, we also identified eight new prognostic markers. Among these prognostic biomarkers, three of them are PCGs, and five of them are lncRNAs. These identified lncRNAs were validated in the laboratory through CRISPR technology. Compared with the currently clinically accepted 50 gene signatures, our eight genes will decrease diagnostic expenses and are easy to validate in biological experiments.Our results further showed that lncRNAs could independently predict breast cancer subtypes without any help from the PAM50 classifier. Our two-step recursive 1-Norm SVM feature selection method identified a smaller number of lncRNAs (n = 3851), whose functions are relatively clear. Though the majority of non-coding genes  annotated by GENCODE v23 database are ambiguous, they might be functionally important and so are worth following up on. For example, one of our lncRNA prognostic biomarkers, CTD-2616J11, has also been reported as a significant prognostic marker for gastric cancer [We have provided all types of gene signatures as references for further investigation. Many lncRNAs had previously never reported before. This is partly because we have an insufficient knowledge of lncRNAs and many new lncRNAs have been recently updated. Another reason is that most current lncRNA experimental studies focus on the HGNC-database-approved lncRNAs  The other papers only focus on the study of functional known long non-coding genes, thereby ignoring the vast majority of long non-coding genes that have unknown functions. Our results provide more information for further biological validation. (2) Although other papers have identified lncRNAs, none of these papers have evaluated the subtype-specific gene signatures from a prognostic perspective, though the prognosis of individual lncRNA has been evaluated ,42,43. (Despite its preliminary character, our future efforts could be directed toward developing feature selection and visualization software for any multi-class phenotypes across different “omics” platforms. Biological determinations of these identified biomarkers can be further investigated through experimental validations.Our novel feature method identified protein-coding and non-coding gene signatures for the prediction of breast cancer subtypes. Our results show that prediction accuracies are improved after RNAseq is integrated with the PAM50 classifier from the microarray. Our results also showed that a 36-gene subtype-specific signature combining coding and non-coding genes exhibited the best prognostic performance and classification accuracy. We propose that eight important lncRNAs be investigated as potential biomarkers in the future.


Drosophila females for instance decrease their sexual receptivity following starvation. Insulin signaling, which regulates many aspects of insect physiology and behavior, also affects reproduction in females. We show that insulin signaling is involved in the starvation-induced reduction in female receptivity. More specifically, females mutant for the insulin-like peptide 5 (dilp5) were less affected by starvation compared to the other dilp mutants and wild-type flies. Knocking-down the insulin receptor, either in all fruitless-positive neurons or a subset of these neurons dedicated to the perception of a male aphrodisiac pheromone, decreased the effect of starvation on female receptivity. Disrupting insulin signaling in some parts of the brain, including the mushroom bodies even abolished the effect of starvation. In addition, we identified fruitless-positive neurons in the dorso-lateral protocerebrum and in the mushroom bodies co-expressing the insulin receptor. Together, our results suggest that the interaction of insulin peptides determines the tuning of female sexual behavior, either by acting on pheromone perception or directly in the central nervous system.Many animals adjust their reproductive behavior according to nutritional state and food availability.  Drosophila melanogaster, feeding, and mating are strongly interconnected. Mating modifies food preference in females  have been characterized in fruitless (fru) gene are necessary for females to be sexually receptive  regulates receptivity, independently of fru  expressing the odorant receptors Or67d and Or47b  while Or47b responds to methyl laurate, a compound produced by flies of both sexes  were then kept in 30-ml plastic tubes with fresh diet for 3 days before behavioral experiments. For starvation, females were transferred to a humidified piece of cotton for 1, 2, or 3 days before being tested. Flies starved for 3 days were allowed to feed up to 6 h following adult emergence, before they were collected for experiments.For behavioral experiments, the Dalby strain was used as a wild-type strain . Mutant and transgenic females were tested fed or 2-day starved. For mutant and transgenic flies, 33–50 couples were tested for each genotype and feeding condition in order to obtain 30–35 replicates of successful courtship.One random wild-type  3d-old fed male was introduced together with a virgin female  under a small round inverted plastic cup  placed on a clean glass plate. Wild-type females were either fed or starved for 1, 2, or 3 days , mating latency (time between the beginning of courtship and copulation initiation) and mating duration were recorded. To estimate female sexual receptivity, only cases where courtship from males was observed were taken into account. The percentage of females accepting to mate with a courting male within the 60-min period was then calculated for each 5-min interval.2-test. Courtship latency, mating latency and mating duration were analyzed using a Generalized Linear Model (GLM) with a Gamma family.Percentages of courting males were analyzed using a χFor each wild-type, mutant and transgenic line, an estimated decrease of sexual receptivity was calculated. For this purpose, the area under curve (AUC) was used as a proxy for sexual receptivity (R: auc function in kulife package). The percentage of decrease between AUC of fed and starved females was then calculated.Regarding the percentage of mating, repeated measurements on the same flies over time were treated as pseudo-replicates and therefore analyzed using a Linear Mixed-effect Model  with a binomial distribution and the “time” factor as a random effect Crawley, . SimilarStatistical analyses were performed in R .Standard immunohistochemical methods were used as earlier described in detail  and stacked images were processed using ZEN 2011 software (Zeiss) and edited for intensity and contrast in Adobe Photoshop CS6.We found that female sexual receptivity was significantly reduced after a period of starvation. When fed, 96% of virgin females mated within 1 h of being paired with a wild type male. However, this percentage dropped to 70, 37, and 16% after a starvation period of 1, 2, and 3 days, respectively Figure . AlthougThe drop in sexual receptivity observed in starved females was likely not due to changes in male courtship or mating behavior. First, neither the percentage of courting males Figure  nor theidilp mutant females.Given the preponderant role of insulin signaling in response to changes in nutritional states, we then tested whether it could be involved in the regulation of sexual receptivity during starvation. We therefore investigated the mating behavior of seven dilp genes showed significantly reduced sexual receptivity after 2 days of starvation .Similar to wild type flies, females carrying a mutation in either one or multiple n Figure . Althougs Figure , 66%, evs Figure . InteresIn conclusion, among all flies deficient for one or several DILPs, only the lack of DILP5 significantly reduced the effect of starvation.dilp5 mutants, the sexual receptivity of all dilp mutants was significantly reduced after 2 days of starvation, suggesting that insulin signaling is not involved. However, compensatory mechanisms exist among DILPs , disrupting InR and therefore all DILP signaling could reveal hidden effects. We therefore knocked-down InR in specific parts of the body, using specific Gal4 drivers. These drivers were chosen because they target specific sites known to regulate different aspects of Drosophila behavior. Fruitless-positive neurons  are involved in sexually dimorphic behavior  sexual receptivity was negatively affected by starvation Figure . HoweverFruitless-positive neurons diminished the effect of starvation on female receptivity. We therefore performed immunostaining on fly brains in order to visualize which Fruitless-positive neurons express the insulin receptor. A large number of Fruitless-positive Kenyon cells were also immunoreactive to the insulin receptor antibody as can be observed in Figure Fruitless-positive anterior dorso-lateral neurons (Figure Fru are co-expressed.Our behavioral analysis revealed that knocking down InR in s Figure . Thus, aDrosophila females need nutrients to produce eggs and a nutrient rich substrate to lay their eggs (Drummond-Barbosa and Spradling, dilps is differentially affected by food quality or food deprivation (Bai et al., dilp5 but increase the expression of dilp6, while the expression of dilp2 is not affected by either condition (Bai et al., dilp5 mutant females were less affected by starvation than other dilp mutants. The effect of the lack of DILP5 was no longer observed in the simultaneous absence of DILP2 and DILP3. Although, we cannot completely rule out background mutation effects, this suggests that DILP5 might interact with other DILPs to finely tune female sexual receptivity.Insulin is known to act on the olfactory system to modulate odor sensitivity after feeding (Root et al., Fruitless-positive cells other than pheromone-sensing neurons can also be involved. We found different Fruitless-positive cells in the protocerebrum that strongly express InR. First of all, a large number of Kenyon cells in the calyx of the mushroom bodies express both Fruitless and the insulin receptor. Additionally, we found one pair of neurons with somata located in the anterior dorso-lateral protocerebrum. We could not trace any processes from these somata and do thus not know what neuropils they innervate. However, the fact that InR immunostaining was observed in Fruitless neurons, most of which were Kenyon cells, corroborate our behavioral results. Indeed, the sexual receptivity of females in which insulin signaling was knocked down in the mushroom bodies was not affected by starvation. Interestingly, the mushroom bodies are not required for virgin females to be receptive (Neckameyer, Insulin signaling not only modulates neuronal activity in adults but also shapes neuronal networks during development (Song et al., In contrast with Fruitless neurons and the mushroom bodies, we did not observe any effect of the corpora allata in the insulin-dependent control of sexual receptivity, whereas these structures have been linked to the development of receptivity in virgin females Manning, , 1967. TDrosophila flies adjust their sexual behavior to match their nutritional state. Together with other hormonal pathways (Lebreton et al., Taken together, All the experiments described in the manuscript were performed with laboratory-reared insects. No recommendations from an ethical committee were required.SL and PW conceived the project. SL designed, performed and analyzed the behavioral experiments. MC performed the immunostaining and the acquisition of confocal microscopy images. SL wrote the first draft of the manuscript. All authors contributed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer ML and handling Editor declared their shared affiliation.


Introduction. According to the 2016 World Health Organization classification of Tumors of the Central Nervous System, the term Primitive Neuroectodermal Tumor has been replaced by the term Embryonal Tumor (ET). We present a case of disseminated cerebrospinal ET presenting in an adult patient. Illustrative Case. A 49-year-old male presenting with low back pain, dysuria, and hypoesthesia of the lower extremities referred to our emergency department. Brain and whole spine contrast-enhanced MRI documented a diffusively disseminated heterogeneous neoplasm with intradural extra- and intramedullary involvement of the cervicothoracic tract and cauda equina. A primary biopsy of the lumbosacral localization was performed through L5 bilateral laminectomy. Histologic diagnosis was Embryonal Tumor Not Otherwise Specified. The patient underwent chemotherapy with postoperative adjuvant alternating Vincristine-Doxorubicin-Ifosfamide (VAI) and Ifosfamide-Etoposide (IE). Discussion. Spinal ETs are exceedingly rare especially when presenting in the adult patient. Neurosurgical and oncologic management is still unclear. When feasible, surgical removal should always be performed to obtain a histologic diagnosis. Postoperative adjuvant therapy might entail both chemo- and radiotherapy; however a consensus on this matter is still lacking. According to the 2016 World Health Organization (WHO) Tumors of the Central Nervous System (CNS) classification, the term “Primitive Neuroectodermal Tumors (PNETs)” has been removed from the diagnostic lexicon. A newly defined category of CNS tumors, including PNETs among other nosological entities, according to the old nomenclature, has been introduced under the term Embryonal Tumors (ETs) .Intraspinal ETs are rapid-growing soft tissue masses, which cause symptoms of nerve compression, ranging from radiculopathy to sphincter dysfunction, and pain. They are highly malignant and invasive, with a high rate of relapse, and retain a poor prognosis. The five-year survival rate is 30–40% and the median survival ranges from 12 to 24 months; prognosis of these tumors in adults is somewhat worse when diagnosed in adults .We report one case of a diffuse ET with brain and spinal dissemination in a 49-year-old male presenting with low back pain, numbness of the lower extremities, and sphincter dysfunction which represented not only a diagnostic dilemma given its aspecific clinical presentation and peculiar radiological features but also, and most importantly, a therapeutic challenge given the lack of consensus regarding its management.A 49-year-old man presented to our emergency department in October 2015, describing numbness of the lower extremities and low back pain developed during the previous two weeks. Furthermore, he complained of dysuria during the last few days and progressive subjective wasting of lower limbs muscles over the last months. Patient family history and general physical examination did not provide any contributive information. Neurologic examination confirmed a diffuse hypotrophy of lower limbs muscles, without any focal motor deficit and associated with hypoesthesia of the sciatic territory bilaterally.A brain MRI  showed mA cerebrospinal fluid (CSF) sample was collected; however no neoplastic cells were documented on cytological examination.The patient underwent a primary biopsy on the lumbosacral localization which appeared to enlarge the S1-S2 posterior sacral foramen causing compression of the right S2 nerve root. Using a posterior approach, a central L5 laminectomy along with median durotomy was performed exposing a grey, soft, hemorrhagic mass which completely enveloped the roots of the cauda equina.Tissues samples were collected for histological examination.Patient was discharged on the ninth postoperative day and referred to our oncologic unit. Chemotherapy with alternating Vincristine-Doxorubicin-Ifosfamide (VAI) and Ifosfamide-Etoposide (IE) was the adjuvant treatment protocol of choice which the patient is currently undertaking with a satisfying profile of tolerability.Intraoperative smear cytopathological examination showed a “small blue cell” neoplasm, composed of sheets of discohesive undifferentiated, hyperchromatic cells, with occasional evidence of Homer Wright rosette formation. Histological examination on formalin-fixed, paraffin-embedded sections revealed a highly cellular neoplasm composed of small- to medium-sized cells with little perinuclear cytoplasm and hyperchromatic nuclei with granular chromatin. Mitoses were frequent, as was necrosis of single tumor cells. Immunohistochemistry showed an intense and diffuse positivity for Neural Cell Adhesion Molecule (N-CAM) and a slightly less intense, but almost equally diffuse positivity for glial fibrillary acid protein (GFAP). Expression of protein INI1 was detected while LIN28 was not detected; moreover no amplification of 19q13.42 was detected. These features allowed excluding both an Atypical Teratoid Rhabdoid Tumor (AT/RT) and Embryonal Tumor with Multilayered Rosettes (ETMR), respectively. Proliferation index was high (Ki-67 over 40%). Reactions with antibodies against synaptophysin, keratin, and epithelial membrane antigen (EMA) were negative in all tumor cells .According to the old WHO classification of Tumors of CNS, this tumor could be categorized as a PNET of central origin; however, since the introduction of the 2016 classification, this lesion could not be defined neither as a PNET (the latter being completely removed from current classifications) nor as the newly defined Embryonal Tumors with Multilayered Rosettes (ETMR), leaving the Embryonal Tumor Not Otherwise Specified (NOS) as the sole diagnostic option.The term Primitive Neuroectodermal Tumor (PNET) was created in 1973 by Hart and Earle to describe predominantly undifferentiated tumors of the cerebrum which contained 90–95% of undifferentiated cells not meeting diagnostic criteria for other tumor entities . First cPrimary spinal ETs are more frequently observed in infants, children, and young adults . The duration of symptoms, initially entailing radiculopathy and paresthesia, may range from one week to six months. Most cases of primary spinal ETs, according to the old classification, retain a peripheral origin (expression of CD99) and rarely present a central origin (CD99 not expressed). Despite being extremely useful for differentiating between ETs of central or peripheral origin, the expression of CD99 should always be associated with the research of   translocation, which is diagnostic for ETs of peripheral origin typically related to Ewing sarcoma .Imaging features are nonspecific, including intradural intra- and/or extramedullary more or less diffuse lesions showing heterogeneous patterns of contrast enhancement , central necrosis, and hemorrhage. The mainstay of treatment is surgical resection; however, this is uncommonly feasible due to the multifocal extensive dissemination of the disease which is not uncommon at presentation. The adjuvant treatment protocol should include a combination of chemo- and radiotherapy; however a consensus on this subject is still lacking due to the exceeding rarity of the disease preventing drawing meaningful conclusions on the basis of the currently available short case-series or single case reports in the literature.Most cases of ETs involving the spinal cord are “drop” metastases from primary intracranial tumors. Our patient showed an extremely diffuse spinal disease with few intracranial nodules Figures  and 2, aAccording to our experience, an open-field biopsy of the lumbosacral spine localization was the least risky method to obtain a histological diagnosis in this specific case considering both the reduced size of brain multifocal nodules and the unforeseeable risk of hemorrhagic complications of any needle biopsy procedure.Case reporting, for such rare nosological entity, may be extremely useful not only for further understanding of Embryonal Tumors but also for providing evidences for future reviews aiming to define a consensus regarding the therapeutic management of this malignant disease.


Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises several species inhabiting the animal and human gastrointestinal tract (GIT). They match the pathobiont description, are potential zoonotic agents and technological organisms in fermented foods. SBSEC members are associated with multiple diseases in humans and animals including ruminal acidosis, infective endocarditis (IE) and colorectal cancer (CRC). Therefore, this review aims to re-evaluate adhesion and colonization abilities of SBSEC members of animal, human and food origin paired with genomic and functional host-microbe interaction data on their road from colonization to infection. SBSEC seem to be a marginal population during GIT symbiosis that can proliferate as opportunistic pathogens. Risk factors for human colonization are considered living in rural areas and animal-feces contact. Niche adaptation plays a pivotal role where Streptococcus gallolyticus subsp. gallolyticus (SGG) retained the ability to proliferate in various environments. Other SBSEC members have undergone genome reduction and niche-specific gene gain to yield important commensal, pathobiont and technological species. Selective colonization of CRC tissue is suggested for SGG, possibly related to increased adhesion to cancerous cell types featuring enhanced collagen IV accessibility. SGG can colonize, proliferate and may shape the tumor microenvironment to their benefit by tumor promotion upon initial neoplasia development. Bacteria cell surface structures including lipotheichoic acids, capsular polysaccharides and pilus loci  govern adhesion. Only human blood-derived SGG contain complete pilus loci and other disease-associated surface proteins. Rumen or feces-derived SGG and other SBSEC members lack or harbor mutated pili. Pili also contribute to binding to fibrinogen upon invasion and translocation of cells from the GIT into the blood system, subsequent immune evasion, human contact system activation and collagen-I-binding on damaged heart valves. Only SGG carrying complete pilus loci seem to have highest IE potential in humans with significant links between SGG bacteremia/IE and underlying diseases including CRC. Other SBSEC host-microbe combinations might rely on currently unknown mechanisms. Comparative genome data of blood, commensal and food isolates are limited but required to elucidate the role of pili and other virulence factors, understand pathogenicity mechanisms, host specificity and estimate health risks for animals, humans and food alike.The  Streptococcus bovis/Streptococcus equinus complex (SBSEC) bacteria are Gram-positive species that inhabit the gastrointestinal tract (GIT) of animals and humans. Most SBSEC have been described as commensal bacteria, but some cause serious infections such as bacteremia and infective endocarditis (IE) in humans and animals and match the pathobiont description , Streptococcus infantarius subsp. infantarius (SII), Streptococcus lutetiensis (SL), Streptococcus alactolyticus and three subspecies of the clade Streptococcus gallolyticus, namely gallolyticus (SGG), macedonicus (SGM) and pasteurianus (SGP). The taxonomic assignment of SL as separate species is not fully agreed upon and also referred to as Streptococcus infantarius subsp. coli  was used  or not   to identify and discriminate species of the SBSEC but results in unreliable identification of SGG was found in over 90% of fecal droppings in turkey flocks and reached up to 80% prevalence in pigeon crop and cloaca samples , marsupials, aquatic mammals and game  microbiota  than that of patients without bacteremia (32.7%) based on sodA gene PCR, which increased the detection rate of SGG from 27% and 16%, respectively, based on selective culturing.   and a malate transporter  as well as degrade tannins encoded by tanA, that are otherwise toxic to many bacteria  and peptide metabolism (duplication of oligopeptide transporters) , gastric , intestinal  and venous sites .SB biotype I and II strains was around 2–3 times higher than that of commensal reference strain SB DSM20480T = NCTC8177  adhered highly variable to CMT-93 and Caco-2. Significant adhesion (>0.2–0.3% of the inoculum) to CMT-93 and Caco-2 was rare within this strain pool and at slightly lower levels than the S. Typhimurium reference strain  to intermediate (20–50% of inoculum) for SGG and SGM/SE, respectively  and SGP ATCC43144 (human blood isolate) to human saphenous vein endothelial cell lines  on their cell surface. sLex is normally expressed on the cell surface of leukocytes enabling rolling of leukocytes on the endothelium. This might increase SGG adhesion to endothelial cells  are an important component to facilitate bacterial binding to epithelial and endothelial cell surfaces and thus niche colonization in humans and animals. Collagen thereby plays an important role. Collagen type I is present in organ capsules and scar tissue, such as on damaged heart valves. Collagen type IV is the main constituent of basement membranes and can become exposed at tumor sites  and SL strains derived from sea otters with IE, but also fecal and dairy SBSEC isolates displayed binding to collagen type IV. In contrast, binding to collagen type I was a feature mostly associated with human blood-derived SGG, SGP, SII and SL isolates and partially also with SGM isolates  and to a lesser extent collagen type V (40%)  Table . DiffereSL strains from sea otters adhered to fibronectin, laminin, and hyaluronic acid in all cases  water-soluble glucans, often dextrans, comprised of α-1:6 linked glucose units  Bailey, . GtfA inSII and other SBSEC members were also observed to form biofilms on human teeth featuring various degrees of auto- and co-aggregation with other oral microbes  biotin and ammonium chloride as sole vitamin and N-source, respectively, (ii) calcium, pantothenate, adenine, biotin, thiamine and arginine or glutamic acid, or (iii) xanthine and additional amino acids  displayed increased adherence of SB, other streptococci and staphylococci in contrast to ARH77 cells without syndecan-1 as well as E. faecalis or E. coli and other Gram-negative bacteria  and Gallo_2178 (Pil1B)] represent the typical features of pilin subunits with a pilin motif PK centralized in the protein containing a structural CnaB domain. Only Pil1A harbors a putative collagen-binding domain . Heat inactivation ablated the effect of wall-extracted antigens on IL-8 secretion, suggesting that proteinaceous components of the cell wall are involved in the IL-8 response  showed reduced but not significant lower survival  in contrast to the survival of SGG UCN34Δpil1 suggests that further factors besides pil1 are involved in immune system interactions and the establishment of infection  or capsule (ΔcpsD) and overexpression of pil1 (Δterm). Since kininogen degradation leads to the release of bradykinin, also bradykinin release was significantly reduced in all mutant strains although not completely inhibited  compared to SB biotype II species. The median prevalence of neoplasia in SB infected patients was 60%, and was higher for adenomas (43%) than for carcinomas (18%). SB infection could therefore be predominantly associated with premalignant colonic lesions, which underscores the importance of colon examination for colon pathologies  in 232 patients (24.6%) with a mean follow-up of 41.8 months. In Clostridium septicum bacteremia patients, no new neoplasias were found with a mean follow-up of 37.4 months   and divided by subspecies for SGG in 51.7% (n = 462), SGP in 24.5% (n = 200) and SL in 24.2% (n = 33) , although the association of SGG with adenomas was confirmed in five of seven studies  and SL (25–60%) followed by SGG (2–35%) and rarely for SII, SGM and S. alactolyticus  than non-encapsulated strains (range 28.9–56.1%). This suggested antigenicity of capsule components and therefore a potential contribution of capsule and cell wall to the antigenicity of SB. In fact, colonization and antibody response of pigeons immunized with an SGP strain containing a thin irregular capsule (PDH 827) did not protect against a high-virulent strain SGG strain with a thick capsule (STR357). These findings indicate that supernatant proteins, glycans, fimbriae and the capsule may be involved in the induction of protective immunity against SGG infections but likely not across SBSEC species   that were only recognized on low virulent SGG strains  was tested in single plex and multiplex assays. The IgG response to Pil1B was the best predictor for tumor presence, but did not result in the same response in all IE infected patients. CRC patients generally had response to only one of the four antigens with a maximum sensitivity of 20–43% by combination of four antigens  (Butt et al., SGG with CRC and adenomas of the colon.The IgG response to four SGG retained most genetic and functional properties for a large variety of niches, SGP, SL, SII and particularly SGM have adapted more specifically to certain niches by gene gain and loss. SBSEC colonization as part of the rumen and gut microbiota is not ubiquitous and confers only a small sub-population of the overall microbiota during symbiosis. However, their ability to proliferate and colonize in dysbiosis, e.g., ruminal acidosis or in the tumor microenvironment is significantly enhanced and defines their pathogenicity.SBSEC pathobionts inhabit the GIT of animals and humans as well as food products featuring significant niche adaptation. While SGG may shape their microenvironment further and support the association with neoplastic sites. Increased immune responses in CRC patients toward SGG suggests a strong association with these sites. Considering the bacterial driver-passenger model, it seems that particularly SGG could be considered as a neoplastic site hijacker. SGG only excels when colonization factors and growth conditions in the environment are favorable. SGG is then able to further shape the microenvironment to its benefit.Although SBSEC are not often found in association with tumors in microbiome studies, they likely have increased colonization levels at tumor sites. A potential tumor promoting ability of SGG, are only able to colonize neoplastic tissues selectively. Subsequently, some strains have the capacity to translocate across the epithelial barrier into the blood system. Human blood-derived SGG possess the full pil1, pil2, and pil3 loci, which enable adhesion to endothelial cells, exposed collagen and fibrinogen on heart valves and activate the human contact system. Via pili phase variation and expression of a capsular polysaccharide they can survive phagocytosis and minimize activation of the humoral immune response. SGG strains of animal origin and other SBSEC members without full pilus loci or different pilus proteins likely do not possess the same degree of infection ability and might depend on other factors to establish infection. Thus, animal and food-derived strains often adhere best to collagen type IV, whereas human-derived strains, particularly those of bacteremia or IE origin, predominantly show highest adhesion toward collagen type I. However, data is so far limited to presumed commensal animal strains whereas animal pathogens are not characterized leaving open question regarding virulence factors, host specificity but also zoonosis. In the light of SBSEC zoonotic potential, association with rural areas and prevalence in food, it will be relevant to determine the factors that enable colonization and establishment in animal and human hosts alike and provide comparison to food-derived strains. This will greatly contribute to a better understanding of transmission routes and development of intervention strategies to mitigate health risks associated with individual SBSEC members.This could also explain controversies between the relation of SBSEC with CRC in IE matched and unmatched patients. As SBSEC members are not strong ubiquitous gut colonizers and nonobligatory passengers on CRC, their prevalence among the general CRC population is low in contrast to patients with SBSEC IE. This also supports the proposed route of infection in humans and animals alike: SBSEC members, mostly CJ and AB equally shared all aspects of topic development, analysis, interpretation, manuscript writing and revision. Both authors have read and approved the submitted version of the manuscript. Both authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.