VCP gene mutations lead to compartment-specific aberrant accumulation of IRTs. Specifically, we identify >100 IRTs with increased cytoplasmic abundance in ALS samples. Furthermore, these aberrant cytoplasmic IRTs possess sequence-specific attributes and differential predicted binding affinity to RNA binding proteins. Remarkably, TDP-43, SFPQ and FUS—RNA binding proteins known for nuclear-to-cytoplasmic mislocalization in ALS—abundantly and specifically bind to this aberrant cytoplasmic pool of IRTs. Our data are therefore consistent with a novel role for cytoplasmic IRTs in regulating compartment-specific protein abundance. This study provides new molecular insight into potential pathomechanisms underlying ALS and highlights aberrant cytoplasmic IRTs as potential therapeutic targets.We recently described aberrantly increased cytoplasmic SFPQ intron-retaining transcripts (IRTs) and concurrent SFPQ protein mislocalization as new hallmarks of amyotrophic lateral sclerosis (ALS). However, the generalizability and potential roles of cytoplasmic IRTs in health and disease remain unclear. Here, using time-resolved deep sequencing of nuclear and cytoplasmic fractions of human induced pluripotent stem cells undergoing motor neurogenesis, we reveal that ALS-causing  et al. now identify >100 cytoplasmic IRTs and show that they are ‘blueprints’ for ALS-related RNA binding protein mislocalization, as well as potential therapeutic targets.Aberrant cytoplasmic SFPQ intron-retaining transcripts (IRTs) have recently been described in ALS, but their roles remained unclear. Tyzack, Neeves  The molecular pathological hallmark of ALS is a nuclear-to-cytoplasmic mislocalization of key RNA binding proteins (RBPs),VCP gene), which have been fractionated into nuclear and cytoplasmic samples. Indeed, this resource allowed us to make important insights into the nature of aberrant intron retention in a human stem cell model of ALS. Specifically, we provide a taxonomy for aberrant intron retention based on nucleocytoplasmic distribution, cis attributes and predicted intron binding affinities to major RBPs. Remarkably, this revealed >100 IRT species in the cytoplasm of ALS cultures, suggesting that this is a more widespread phenomenon than previously recognized. Furthermore, we confirm direct binding of splicing factor proline and glutamine rich (SFPQ) protein with SFPQ IRT within the cytoplasm of our hiPSC ALS model along with preliminary evidence of a functional interaction between them. In summary, we have uncovered a novel class of cytoplasmic IRTs that exhibits predictive value for the nuclear-to-cytoplasmic mislocalization of key RBPs, a recognized molecular hallmark of ALS.Here, we combine cellular fractionation of hiPSCs undergoing motor neurogenesis with deep RNA-sequencing (RNA-seq) of ∼100 million paired end reads per sample to gain insight into the molecular ‘logic’ governing intron retention programmes in healthy and disease states. This is a rich resource for researchers across the disciplines of basic and applied neuroscience, constituting six time points during motor neurogenesis for four control lines  and three ALS lines .TM (Life Technologies) with Essential 8TM Medium (Life Technologies), and passaged using EDTA . All cell cultures were maintained at 37°C and 5% CO2. Directed differentiation of hiPSCs to motor neurons was performed as previously described.HiPSCs were maintained on GeltrexAll sequence data for this project have been deposited at NCBI GEO database under accession number GSE152983. Additional data supporting the findings of this study are available from the corresponding authors, upon reasonable request.VCP mutations: R155C and R191Q, hereafter termed VCPmu (mu-related ALS); four clones from four different healthy controls and three clones from two VCPmu patients]. Further details of the samples sequenced can be found in GAPDH intronic RNA was negligible in the cytoplasm, suggesting that leakage of RNA from the nucleus to the cytoplasm due to the fractionation protocol was minimal. Importantly, the efficiency of fractionation was comparable between control and VCPmu lines. Singular value decomposition (SVD) analysis of 18 834 reliably expressed genes across the 95 samples revealed that developmental stage and cellular fraction were the largest contributors to transcriptome diversity, explaining 41% and 15% of the variance, respectively. Notably, the VCPmu samples cluster with their age- and fraction-matched control counterparts  RNAseq data derived from nuclear and cytoplasmic fractions of hiPSCs (Day 0), neural precursors , ‘patterned’ precursor motor neurons , post-mitotic but electrophysiologically immature motor neurons , and electrophysiologically active motor neurons . The cellular material was derived from four healthy controls and two ALS patients with ed VCPmu  [95 sampterparts . Unsuperterparts .mu and control samples in at least one time point   , bottom IV = 14) ; most nomucultures: (i) 237 predominantly in the nucleus; (ii) 63 in both the nucleus and the cytoplasm; and (iii) 49 predominantly in the cytoplasm  using RT-PCR, where we not only confirmed the increase in intron retention ratio . We identified three categories of aberrant IRTs in VCPytoplasm . Gene onytoplasm . To addrytoplasm . Next, lytoplasm A. We preon ratio C, but alon ratio . This fi,,,,,Prior studies have shown that retained introns are on average shorter and more G/C rich.SFPQ IRT and the SFPQ protein itself, which are predicted to avidly bind to each other, are exported to the cytoplasm thus providing a potential mechanism for SFPQ protein mislocalization in ALS.,,The finding that cytoplasmic aberrant IRTs abundantly bind to RBPs raises the hypothesis that this interaction drives hallmark RBP mislocalization events in ALS. Indeed, we previously reported that the ,,,,SFPQ IRTs. We first performed RNA immunoprecipitation to demonstrate that the SFPQ protein physically interacts with SFPQ IRT .SFPQ. Furthermore, we specifically demonstrated an increase in the cytoplasmic abundance of the intronic sequences, suggesting aberrant nuclear export and/or cytoplasmic stabilization of a specific pool of IRTs in VCP mutant samples. Importantly, a significant overlap of these aberrant events has been previously recapitulated in RNAseq datasets from terminally differentiated motor neurons carrying mutations in SOD1 or FUS genes.,SFPQ transcript, has been recently reproduced in human post-mortem tissue from sporadic ALS cases, demonstrating the predictive power of our hiPSC model.Recent studies have demonstrated that intron retention is more frequent in mammals than previously recognized.cis features comprising an ‘intron retention code’ that can reliably discriminate retained from constitutively spliced introns.cis attributes of the three categories of IRTs, we found that aberrantly retained introns that are predominantly in the nucleus of VCP mutant cultures display similar lengths and GC content to this ‘intron retention code’.,,,,,To better understand the nature of cytoplasmic IRTs, we categorized the aberrant intron retention events into three classes according to their nucleocytoplasmic distribution . We chose to direct our attention to Day 14 of our motor neurogenesis protocol as this was where the majority of aberrant intron retention events were observed. Retained introns during neurogenesis have previously been shown to exhibit a highly correlated set of SFPQ IRT in the cytoplasm and further show siRNA SFPQ knockdown-related increase in SFPQ intron retention ratio relative to the spliced transcript. These data support a model where nuclear SFPQ binding to its retained intron facilitates splicing under normal circumstances, which is consistent with a recent study showing significant retention of long introns upon SFPQ depletion.SFPQ IRT cytoplasmic abundance may then lead to SFPQ nuclear-to-cytoplasmic mislocalization, SFPQ nuclear loss-of-function and consequently amplify aberrant splicing of SFPQ intron 9, which in turn exacerbates SFPQ nuclear loss-of-function. However, we have not formally excluded the possibility that an increase in SFPQ IRT upon knockdown is caused by differential accessibility or targeting of the SFPQ IRTs by the siRNAs. Therefore, future experiments should further address the specific role of nuclear SFPQ protein in regulating retention of intron 9 within its own transcript. Altogether we propose that a subset of IRTs aberrantly accumulate in the cytoplasm and their intronic sequences serve as ‘blueprints’ for the hallmark protein mislocalization events in ALS by creating a mislocalization-prone environment for their bound (and otherwise predominantly nuclear) RBPs  RBPs . This isThe first systematic characterization of intron retention during neuronal lineage restriction examined time-resolved RNAseq data during the differentiation of cortical glutamatergic neurons from murine embryonic stem cells. This study established that intron retention progressively increases during neuronal differentiation and downregulates non-physiologically relevant transcripts.awab078_Supplementary_DataClick here for additional data file.


Mucosal surfaces are the first site of infection for most infectious diseases and oral vaccination can provide protection as the first line of defense. Unlike systemic administration, oral immunization can stimulate cellular and humoral immune responses at both systemic and mucosal levels to induce broad-spectrum and long-lasting immunity. Therefore, to design a successful vaccine, it is essential to stimulate the mucosal as well as systemic immune responses. Successful oral vaccines need to overcome the harsh gastrointestinal environment such as the extremely low pH, proteolytic enzymes, bile salts as well as low permeability and the low immunogenicity of vaccines. In recent years, several delivery systems and adjuvants have been developed for improving oral vaccine delivery and immunogenicity. Formulation of vaccines with nanoparticles and microparticles have been shown to improve antigen stability, availability and adjuvanticity as well as immunostimulatory capacity, target delivery and specific release. This review discusses how nanoparticles (NPs) and microparticles (MPs) as oral carriers with adjuvant characteristics can be beneficial in oral vaccine development. Mucosal immunization has numerous advantages over parenteral (needle-based) administrations such as socio-economic benefits, relatively improved safety by a lower risk of needle injury infection/inflammation, self-delivery, capacity for mass immunizations, no special training required, patient compliances and ability to elicit mucosal immune responses . MucosalIntranasal and oral routes are both considered as the main options for mucosal immunization. As intranasal immunization could have some harmful effects on people with asthma and other chronic pulmonary or cardiovascular disorders, the oral route appears to be the safest, more applicable and most preferred mode of vaccination for the development of new generation vaccines.2 of the mucosal surface containing immune inductive tissues such as Peyer’s patches , lymphoid follicles (large intestine) and intraepithelial lymphocytes which are important for antigen presentation and appropriate functioning of the immune system The gastrointestinal (GI) tract of humans has over 260–300 mHelicobacter pylori infection  or whole inactivated pathogens  . The comnfection .On the other hand, oral vaccine  delivery has always been a significant challenge for pharmaceutical technology and vaccine development due to its very poor bioavailability through the GI tract. The possible explanation for the low oral absorption of the vaccines might include enzymatic degradation, poor membrane penetration, hepatic metabolism and the unique physicochemical characteristics of the GI mucosa .As the GI tract is continuously exposed to a broad range of pathogens, successful oral vaccines need to induce appropriate strong signals to be recognized by the immune system, otherwise, the host immune system would consider the vaccines as non-immunogenic and resulting in immune tolerance instead of conferring broad protection. Therefore, it is important to design an effective vaccine carrier including safe effective adjuvants to sufficiently stimulate the mucosal immune system . An ideaMost of the soluble antigens are unable to be efficiently endocytosed by the antigen-presenting cells (APCs) and cannot induce any protective immunity. Conjugation or encapsulation of soluble antigens with nanocarriers could improve their immunogenicity and facilitate recognition and uptake of antigens by APCs. Therefore, the immunogenicity of soluble antigens could be improved by conjugation or encapsulation with nanocarriers that could facilitate the recognition and uptake by APCs . SeveralThe surface of the GI tract contains a chemical and physical barrier formed by an impermeable layer of epithelial cells. The M cells are mainly located within the epithelium of Peyer's patches in the ileum and have some noticeable features for the uptake of particles. However, the lower percentage of M cells in the GI tract  causes significant problems in humans towards oral vaccine development. The efficacy of oral vaccination is mostly impaired due to the low populations of M cells in the intestines. To increase the efficiency of the delivery of oral vaccines, it is necessary to target the vaccine complex to M cells. Various absorption parameters that play an important role in the uptake of NPs/MPs via M cells include particle size, charge, hydrophobicity/hydrophilicity balance and existence of a targeting molecule at the surface of the particles. Generally, M cells could uptake particles ranging from below 1 µ to above 5 µ in size. Particles smaller than 1 µ are passed into the basal medium, while particles above 5 µ are delivered to Peyer’s patches. The optimum size of NPs for transcytosis by M cells is proposed to be smaller than 200 nm. The formulation of negatively charged and hydrophobic particles is favorable because of the optimal absorption by M cells .The intestinal immune system is regulated by gut-associated lymphoid tissues (GALT) which contain inductive and effector sites. Inductive tissues include the Peyer’s patches, lymphoid follicles (within lymph nodes), and APCs. Meanwhile, effector sites comprise the lamina propria and the surface epithelium. After oral administration with particulate vaccines, antigens could migrate through the GI tract.After entering the small intestine, specialized M cells in the Peyer’s patches sample and transport the antigens across to APCs. The antigens are processed into small fragments by DCs that present antigenic fragments on their surface.These antigen-loaded DCs provide costimulatory signals to activate naive CD4 T cells. The primed helper T cells further interact with antigen-specific B-cells that undergo class-switching to become immunoglobulin-secreting cells.Upon maturation, IgA B cells leave the Peyer’s patches through afferent lymphatics to the regional mesenteric lymph node before reaching the systemic blood circulation  Brayden. FinallyAdjuvants originated from the Latin word “adjuvare”, which means “aid or to help” and was first explained by Ramon in 1924 as “substances used in combination with a specific antigen to enhance the immunological responses” .Generally, adjuvants are non-specific immunopotentiators which together with the antigen(s), could boost the body's immune responses as well as changing the type of the immune response .Aluminum salts, developed in the United States in the 1920s and for more than 7 years, was the only adjuvant available in the United States until MF59 (incorporated in influenza vaccine) was approved in the 1990s .Haemophilus, Influenzae Type b (Hib), tetanus, and meningococcal vaccines (Alum is still an important component of most licensed human vaccines like human papillomavirus (HPV), Hepatitis A virus (HAV), diphtheria, Hepatitis B virus (HBV), vaccines .Alum as a potential adjuvant have been tested in the formulations of a few under exploratory and pre-clinical coronavirus vaccine investigations. Furthermore, virus-like particle (VLP) and inactivated vaccines containing E, M, and N proteins were formulated with Alum and showed enhanced IgG1 and neutralizing antibody titers and prolonged durability. Studies also demonstrated that alum adjuvant plays an essential role in the dose-sparing of CoV vaccines . MoreoveAluminum-based adjuvant might be sufficient for eliciting humoral immune responses with acceptable safety and efficiency, but it is a poor immunostimulator of cellular immune responses and has limited application as an adjuvant for vaccination against intracellular pathogens. Aluminum is not effective at triggering the molecular events that support IgA class switching, recombination or homing of activated T and B cells in mucosal tissues . TherefoAdjuvant selection and formulation can be based on several parameters including the type of disease, route of vaccination, vaccine platform, physical and chemical natures of antigen, type of required immune response and age of the target population. Moreover, the selection of the wrong adjuvant could reduce vaccine efficiency. Thus, the selection of vaccine antigens must take into account the selection of adjuvant to enhance the potential effectiveness of vaccine candidates .Many different types of compounds have been evaluated as adjuvants for human and animal applications and these include; mineral salts, microbial products, emulsions, saponins, cytokines, polymers, NPs, MPs and liposomes. To date, only a few adjuvants have been used in licensed human vaccines: including Alum , MF59 c, squalenEven though adjuvants derived from bacteria or plant components and synthetic materials are immunostimulatory, many of them exhibited toxicity, leading to undesirable adverse reactions . These ain vivo. These data suggested that larger NPs prefer interacting with tissue-resident APCs, while smaller NPs (<200 nm) could circulate through the vein and lymphatic drainage, thus providing better antigen presentation   while MPentation . Moreoveentation . The NPsentation .Liposome technology was established by Bangham and Horne in the 1960s as a system for the diffusion of ions across biological membranes and later in the 1970s, there was an interest in drug delivery applications . LiposomThe cationic liposomes have been extensively considered as an effective oral vaccine carrier for diverse antigens and adjuvanticity applications due to their unique properties such as biodegradability, biocompatibility, surface charge variability, and membrane fluidity . EncapsuLiposomes significantly protect the delivery of DNA vaccines in the GI against DNases and promote absorption at the cellular level. The fusion of liposomes to host cells could facilitate the internalization of encapsulated DNA vaccine into the cytoplasm and adjuvanticity of liposomes could enhance the expression of the recombinant plasmid .Mycobacterium antigen 85A (Ag85A) encapsulated in liposomes. After three oral vaccinations in mice, the Ag85A protein antigen was detected in the epithelium, M cells, dendritic cells (DCs) and Peyer’s patches of the small intestine and antigen-specific mucosal, systemic humoral and cellular immunity against tuberculosis were generated  protein from eriditis . Oral imeriditis . In anoteriditis .H. pylori urease B subunit and cholera toxin B were expressed and purified in E.coli as a recombinant fusion peptide. The recombinant fusion peptide was encapsulated in the phospholipid bilayer vesicle liposome with encapsulation efficiency of 71.4%. Liposome as the oral carrier could protect fusion peptide in the GI tract from protease, acids and other harmful components. Oral vaccinations of mice with liposome-encapsulated recombinant fusion peptide administered at four weekly intervals were able to increase the specific IgG and IgA antibodies as well as providing prophylactic and therapeutic effects against H. pylori infection  is a type of lipid NPs containing a hydrophobic solid and liquid lipid core covered by the surfactant monolayer . Comparein vivo studies. However, both studies delivered the NLC-rvRNA and HIV p24 peptide vaccines through intramuscular (IM) and intraperitoneal injections (IP), respectively. Thus, further studies to evaluate the potential or even to improve the formulation of the NLC-mRNA or NLC-peptide vaccines as oral vaccines can be considered.NLC has been recognized as a nano-delivery solution to increase the oral bioavailability of poorly soluble drugs . InteresIn addition to traditional liposomes, bilosomes are non-ionic lipid-based vesicles (niosomes) containing biodegradable and biocompatible bile salts (sodium deoxycholate). They have been extensively used for oral vaccine delivery due to their adjuvanting properties, encapsulation of antigens, flexible size formation, and rapid uptake by M cells . RecentlAs most of the hepatitis B vaccines were administered via the parenteral route and they failed to provide mucosal immune responses, oral vaccination could be considered as an alternative to overcome this failure. Oral immunization of mice with bilosomes containing the recombinant hepatitis B surface antigen was found to increase uptake of bilosomes entrapped in gut-associated lymphoid tissues and produced both systemic as well as mucosal antibody responses .Glucomannosylated bilosomes containing BSA were found to induce mucosal, systemic as well as cell mediated immune responses . Oral vaThe optimized bilosome formulation was assessed as an oral vaccine delivery system with respect to its biodistribution and vaccine efficacy. The larger bilosome vesicles (∼6 vs. 2 µm in diameter) were demonstrated to have increased uptake within the Peyer’s patches. Oral immunization of ferrets with recombinant hemagglutinin (HA) incorporated into bilosome was able to reduce fever, suppressed lung inflammation and reduced viral load in the influenza challenge model. Thus, the bilosome has shown promising results as an oral vaccine carrier and could induce higher immune responses .Oral immunizations of mice using HA entrapped in bilosomes with particle sizes between 400–2000 nm elicited an immune response that was significantly biased towards Th1 as measured by serum antigen-specific IgG2a and splenocyte IFN-γ production rather than vaccination using bilosomes with size 10–100 nm . These rOral immunization of bilosomes was able to induce both mucosal and systemic immune responses. Surface modification of bilosomes with anchoring ligands demonstrated their potential to target specific immune cells. Availability and low cost of bile salts and acids could thus transform such chiral carriers into attractive building blocks for targeting novel drug carrier systems. Moreover, bilosomes could enhance the bioavailability of drugs, increase the efficacy of drugs and the ability to entrap proteins, peptides and antigens .ISCOMs were first described by Morein et al. (1984) as a vaccine delivery vehicle . ISCOMs 323–339 peptide epitope linked to CTA1-DD  were highly immunogenic when administered by the subcutaneous (s.c.), oral, or nasal routes and induced a wide range of T cell-dependent immune responses as well as systemic immune responses (IgG2a and IgG1 isotypes). Their results demonstrated that ISCOMs could induce a broad range of cellular and humoral immunity  antigens  were able to induce high levels of IgA and IgG  as well as conferring sufficient protection against heterologous lethal dose of HSV-2 in mice . In anotimmunity . Oral imimmunity . Most ofGold (Au) has been used extensively in nano-medicine (in the form of NPs) due to its therapeutic effects on several diseases. Gold could also play an important role in the vaccine development field as a carrier and an adjuvant, enhancing the immunogenicity of antigens, reducing toxicity, and providing stability  Carabin.Saponaria extract (QS) as an adjuvant. Oral immunization of mice with CsAuNPs-TT-QS induced up to 28-fold immune responses (TT-specific IgG and IgA) compared to TT and TT-QS controls. Thus, combination of adjuvants with NPs can play an important role in the efficacy and stability of mucosal vaccines  were used as a carrier for tetanus toxoid (TT) as an antigen model along with the immunostimulant of Quillaja vaccines .Asparagus racemosus extract (ARE) in oral delivery of TT using CsAuNPs as a carrier was evaluated in the murine model. A significant local and systemic increase in TT-specific IgG and IgA were observed when TT-CsAuNPs were formulated and delivered with ARE. As an immunomodulatory adjuvant for mucosal delivery of vaccines, ARE showed no effect on charge, size and loading properties of CsAuNPs. Additionally, ARE and CsAuNPs were observed to have no effects on antigenicity and the secondary structure of TT  and polyglycolic acid . PLGA NPPLGA vaccine/adjuvant encapsulation could provide slow release of antigens and adjuvants which could stimulate innate immune responses and potentially induced both mucosal and systemic immune responses as well as improving humoral and long-term memory CD8 T cell . BesidesM. tuberculosis lipoprotein MPT83 together with the adjuvant trehalose-dibehenate (TDB) or Monophosphoryl lipid A (MPL). Mucosal immunization of mice with PLGA-encapsulated protein-based subunit vaccine was found to stimulate strong anti-MPT83 antibody or Th17 responses and PLGA could be considered as a potential carrier for vaccines against extracellular pathogens . The results showed oral administration of encapsulated OmpW using PLGA could provide dose-dependent protection against A. hydrophila infection in fish (The outer membrane protein W (OmpW) of Aeromonas hydrophila was cloned, purified, and encapsulated in PLGA NPs for oral vaccination of rohu  overexpressed tissue factor (TF) which was targeted by the fusion protein EGFP-EGF1 nanoparticles loaded with TF siRNA as a potential treatment. EGFP-EGF1 conjugated PLGA NPs (ENP) were used as a new targeted carrier for TF-specific siRNA which could be effectively delivered to atherosclerotic plaques on of TF .Ma et al. (2014) developed ulex europaeus agglutinin-1 (UEA-1) conjugated PLGA-lipid NPs containing a Toll-like receptor (TLR)-agonist monophosphoryl lipid A (MPL) as an oral vaccine delivery system. Oral immunization of mice with UEA-MPL/PLGA lipid NPs could protect and released the entrapped OVA through the GI tract. The entrapped ovalbumin (OVA) was protected from exposure to the GI tract and the OVA was released in a controlled manner. These results suggested that designed OVA-UEA-MPL/lipid NPs could be effectively transported by M cells, captured by mucosal dendritic cells (DCs) and induced mucosal IgA and serum IgG antibodies .Bacillus subtilis. Due to the unique characteristics of γ-PGA like low toxicity, biodegradability and biocompatibility with tissues and cells as well as non-immunogenic properties, it has been used extensively for vaccine development and pharmaceutical applications. γ-PGA NPs could induce cellular and humoral immune responses as well as having great potential as an antigen-delivery system and vaccine adjuvant . Orally administered γ-PGA enhanced levels of various chemokines in intestines, accumulation of CD8 DC subsets in mesenteric lymph nodes and activation of DCs in Peyer’s patch. Oral inoculation of mixed γ-PGA with OVA was shown induce activation of OVA-specific T cells as well as levels of both IgA  and IgG (sera). In the study, OVA was used as the model protein to develop a protein-based vaccine .Chitosan is a linear polysaccharide predominantly composed of β-(1–4)-linked D-glucosamine and N-acetyl-D-glucosamine. The N-acetyl-D-glucosamine moiety of chitosan is recognized by the mannose receptors on the DCs . The chiChitosan has attracted considerable attention as a biodegradable, non-toxic and biocompatible polymer to encapsulate a range of vaccines such as DNA, RNA, proteins, peptides as well as drugs as a novel delivery vehicle for both oral and intranasal administration. Various studies have shown chitosan as an oral vaccine delivery system by activation of macrophages, dendritic cells (DC) and lymphocytes which induce higher immune responses. Moreover, an ionic crosslinking method was used to encapsulate Newcastle disease virus (NDV) with chitosan NPs to enhance the efficacy of a lentogenic live-virus vaccine against ND. Single oral immunization of chickens with chitosan-live NDV provided full protection after challenge with a highly virulent NDV strain F48E9 .Salmonella. Oral immunization of layer chickens with OMP-F-CS NPs led to localization of the nanovaccine in ileal Peyer’s patches and induced significantly higher OMP-specific mucosal IgA production as well as lymphocyte proliferation response and reduced salmonellosis in poultry  as well as improved surface expression of costimulatory molecules on macrophages. y system .In another study, bovine serum albumin (BSA) was used as a protein-based vaccine model and was loaded into the mannosylated chitosan NPs (MCS) by ionic gelation method with tripolyphosphate (TPP), followed by coating with Eudragit L100 (Eud) and electrostatic interaction. MCS NPs were accumulated more specifically into PPs after Eudragit L100 was dissolved in intestinal juices. Oral immunization of rats by using BSA-loaded Eudragit L100-coated MCS NPs elicited strong mucosal IgA and systemic IgG antibody responses. These results suggested that enteric-coated MCS NPs could serve as a promising carrier for oral protein-based vaccine delivery .Streptococcus iniae and Lactococcus garvieae was examined in rainbow trout. Oral vaccination of fish with chitosan-alginate coated microparticle vaccine increased immunity and improved the survival rate by the expressions of IL-6 and IgM  as a bivalent streptococcus-lactococcus vaccine against  and IgM .Vibrio anguillarum showed elevated specific antibodies and higher concentrations of lysozyme and complement activities in fish serum than ECPs. CMCS/CS-NPs loaded with ECPs could improve both innate and adaptive immune responses and suggested that it could serve as a potential oral antigen delivery system in fish  against V. anguillarum infection  loaded with extracellular products (ECPs) of  in fish . The appnfection .Alginate is a hydrophilic anionic polysaccharide obtained from the brown seaweed. Alginate can be orally administered or injected and has been extensively investigated in drug and vaccine delivery due to its low toxicity, biocompatibility, biodegradability  and was Pasteurella multocida and OVA antigens could produce higher serum IgG, IgA, and sIgA responses than those immunized with unencapsulated antigens  of around 50% and this failed to warrant the efficacy of the vaccine formulation as a primary vaccination method. In another study, the efficacy of alginate-encapsulated killed vaccine as a booster immunization was evaluated 3 months after primary intraperitoneal immunization of fish with aqueous-based bacteria. The relative percent survival could reach up to 87%. These results highlighted the value of alginate encapsulation which led to an increase in the duration of protection of the rainbow trout against lactoccocosis  is the only approved vaccine against Tuberculosis. This vaccine is currently administered intradermally and has shown variable effectiveness of between 0 and 80% in various clinical trials . As mycoOral administration of encapsulated OVA (as a model antigen) in alginate microparticles to calves demonstrated that encapsulation of OVA in alginate microparticles could successfully induce pulmonary immunity and increased antigen-specific IgAs in bronchoalveolar lavage fluids . These sIn order to develop an effective oral vaccine carrier, immunological adjuvant, antigen protection and M cell antigen uptake, hydrophobic carbon NPs were synthesized by taking silica as a template and sucrose as a carbon source. Carbon NPs provided large mesopores and macropores which could encapsulate a large amount of antigens and could be considered as a potential antigen delivery system. Bovine serum albumin (BSA) was used as an antigen and loaded into the pores of carbon NPs. Oral immunization of mice with BSA/carbon NPs induced IgG in serum and mucosal IgA in salivary, intestinal and vaginal secretions as well as both T helper 1 and helper 2 mediated immune responses .Oral vaccine development is complex and is considered the most challenging vaccination method due to the route of administration. Current licensed oral vaccines target mainly enteric pathogens and viruses that invade via the intestinal mucosa. In the last few years, material science applications in the field of vaccine development are growing rapidly and showed some promising results. Although the development of human oral vaccines have not been so successful, biocompatible and biodegradable NPs/MPs as vaccine carriers in animal models offer promising and novel vaccination methods that might act synergistically both as a delivery vehicle and an adjuvant. Engineered NPs/MPs have demonstrated their potentials to ensure the induction of both cellular and humoral immune responses as well as offering much greater advances for the future development of oral vaccines for humans. The progress of developing oral vaccines required careful consideration of multiple physicochemical and biological barriers in the GI tract as well as delivery systems and adjuvants. However, with increased understanding of intestinal biology, mucosal immunity and the next generation of NPs/MPs as vaccine carriers and adjuvants, there is great hope to address the limitations and develop more novel oral vaccines.


Human adipose-derived stromal/stem cells (hASC) are widely used for in vitro modeling of physiologically relevant human adipose tissue. These models are useful for the development of tissue constructs for soft tissue regeneration and 3-dimensional (3D) microphysiological systems (MPS) for drug discovery. In this systematic review, we report on the current state of hASC culture and assessment methods for adipose tissue engineering using 3D MPS. Our search efforts resulted in the identification of 184 independent records, of which 27 were determined to be most relevant to the goals of the present review. Our results demonstrate a lack of consensus on methods for hASC culture and assessment for the production of physiologically relevant in vitro models of human adipose tissue. Few studies have assessed the impact of different 3D culture conditions on hASC adipogenesis. Additionally, there has been a limited use of assays for characterizing the functionality of adipose tissue in vitro. Results from this study suggest the need for more standardized culture methods and further analysis on in vitro tissue functionality. These will be necessary to validate the utility of 3D MPS as an in vitro model to reduce, refine, and replace in vivo experiments in the drug discovery regulatory process. Stem cells are widely used for in vitro tissue engineering due to their ability to self-renew and differentiate into multiple cell lineages . In the https://www.cdc.gov/obesity/data, accessed on 10 February 2021). Obesity is associated with several comorbidities including type 2 diabetes, cardiovascular disease, and metabolic syndrome; therefore, an understanding of the mechanisms driving obesity and discovery of prevention methods are of great interest [Adipose tissue recently emerged as a research area of great interest due to its endocrine activity and role in disease conditions such as obesity ,4. AccorIn vitro models of adipose tissue can be constructed using hASC cultured in adipogenic inductive medium to study adipose tissue biology and pathophysiology. Many in vitro studies involve culturing cells in a 2-dimensional (2D) format; however, this technique fails to recapitulate the native 3-dimensional (3D) microenvironment. Therefore, 3D cell culture methods have been developed to better model the structure and function of human tissue as compared to their 2D counterparts. 3D cell culture mechanisms involve seeding cells into a scaffold or allowing them to self-assemble into cell aggregations called spheroids in a scaffold-free environment.Scaffold and scaffold-free culture conditions can be employed to engineer constructs for implantation and soft tissue regeneration or to develop adipose tissue microphysiological systems (MPS). MPS are defined as 3D, multicellular, tissue-engineered organ constructs developed using human cells . MPS havThe purpose of this review is to evaluate emerging methods of culturing and assessing hASC in vitro for the development of engineered adipose tissue constructs. The identification of the most physiologically relevant culture and characterization methods for engineering constructs that recapitulate native human adipose tissue will support efforts in soft tissue regeneration and in vitro modeling for drug discovery.We conducted a systematic review by searching PubMed using 7 subsets of keywords as defined in Duplicate articles identified using different subsets of keywords were removed from the original search results. Review articles were excluded from analysis as were articles that did not utilize human adipose-derived stromal/stem cells (hASC) or stromal vascular fraction (SVF) cells. As a note, SVF cells represent all other cell subtypes besides the mature adipocytes in adipose tissue, and contain endothelial progenitors, fibroblasts, lymphocytes, monocyte/macrophages, pericytes, pre-adipocytes, and stromal/stem cells, among others . This coOur database search and independent acquisition of relevant articles resulted in the identification of 184 records. After 30 duplicates were removed as well as one article for which the full text could not be obtained, 153 independent articles were screened further based on the abovementioned exclusion criteria. Thirty-one articles were excluded in this screening process, leaving 122 records to be analyzed in the systematic review . ResultsHuman adipose-derived stromal/stem cells are capable of differentiation along multiple lineage pathways. As such, this database search identified studies on a variety of differentiation pathways. The most common hASC differentiation pathways studied were adipogenic 36.9%) [% 9,10,1,48,49,50Articles that assessed adipogenesis were divided further according to the flow chart in Out of the 27 relevant articles, 26 articles used adipogenic medium to differentiate hASC into adipocytes in vitro. The studies reviewed used either commercial (40%) or custom-made medium (60%). In vivo, mature adipocytes often have a complex three-dimensional structure that is characterized by a single large (unilocular) lipid droplet along with other cellular components contained within a thin cytoplasm. Immature adipocytes are described as multilocular as they contain multiple small lipid droplets. Throughout maturation, these small lipid droplets aggregate together to form the aforementioned unilocular droplet . This maIt can be challenging to image adipocytes using light microscopy because of their high lipid content which causes autofluorescence that is enhanced by light diffraction. Confocal imaging, used in combination with the introduction of fluorescently-tagged lipophilic dyes, is a useful alternative as it operates in a narrow plane of focus allowing for the removal of autofluorescence. Additionally, a z-stack of images can be combined to recreate an image of the complex three-dimensional structure of adipocytes . Of artiOil red O (ORO) is a staining solution that accumulates in cellular compartments that are lipid rich; therefore, it can be used to detect the neutral lipid amounts after hASC adipogenesis. ORO can be observed using bright field and fluorescent microscopy . Of artiAlternatively, BODIPY is a commercially available fluorescent dye that labels lipid droplets and is detected as bright green fluorescence . Of artiAnalyses associated with a specified single time point or “snapshot” assay are used to quantitatively assess hASC biomarker expression. Snapshot assays confirm the status of cells at a given time point but lack substantial information about their temporal functionality in vitro.Flow cytometry is a widely used assay for assessing the immunophenotype of mesenchymal stromal/stem cells (MSCs). According to the International Federation for Adipose Therapeutics (IFATS) and Science and the International Society for Cellular Therapy (ISCT), hASC in culture maintain markers similar to other MSCs as they are positive for CD90, CD73, CD105, and CD44 and negative for CD45 and CD31 . Flow cyQuantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a quantitative method to assess gene expression pre and post-hASC adipogenesis. Of articles evaluated in this review, 40.7% employed qRT-PCR to assess adipogenesis-related gene expression. The most commonly assessed genes were peroxisome proliferator activated receptor γ (PPAR-γ) (84.6% of articles using qRT-PCR), lipoprotein lipase (LPL) (46.2%), fatty acid binding protein 4 (FABP4) (46.2%), and adiponectin (ADIPOQ) (30.8%). hASC adipogenesis is driven by PPAR-γ expression and characterized by the upregulation of LPL, FABP4, and ADIPOQ .Functional biochemical assays quantify the dynamic functionality of cellular enzymatic activities based on biomarkers at the carbohydrate, lipid, or protein levels. Four functional biochemical assays were routinely employed monitoring triglyceride, lipolysis, glucose uptake, and adipokine secretion. The ASCs’ accumulation of neutral triglycerides with the formation of intracellular lipid droplets was assessed quantitatively based on biochemical detection of triglyceride levels. Following triglyceride lysis into free fatty acids and glycerol, glycerol levels are evaluated based on spectrophotometric detection. Alternatively, the ASC intracellular lipolytic enzyme activity can be monitored following stimulation with exogenous isoproterenol based on release of glycerol within the cellular lysates . Both asGlucose uptake following activation of the GLUT4 transporter in response to insulin can be monitored based on colorimetric or fluorescent detection of 2-deoxyglucose uptake by ASC. Only a single report in this systematic review (3.7%) reported this outcome. Finally, adipokine assays using antibody-based methods (ELISA or bead-based assays) are used to quantify ASC cytokine secretion activity. While 25.9% of the reports evaluated adipokine secretion, none of the articles characterized the cellular inflammatory response in the context of adipogenesis.Three-dimensional stromal/stem cell culture techniques have been developed for the purpose of engineering in vivo-like adipose tissue constructs. Three-dimensional culture mechanisms can be subdivided into either scaffold or scaffold-free culture systems. Likewise, culture conditions can also be described as either static or dynamic. In static culture, cells are placed in non-circulating media that is replaced every few days throughout the culture period. In dynamic culture, media is continuously perfused through the culture construct to model the circulating in vivo environment.One critical aspect of 3D stem cell culture is developing an in vitro environment that closely mimics native in vivo conditions ,21,25. ANewman et al. developed collagen-elastin-like polypeptide scaffolds for culturing hASC and found that more stiff, dense, and crosslinked scaffolds led to a spheroid morphology whereas less stiff, non-crosslinked scaffolds lead to a spread morphology. The spheroid morphology is more representative of native adipose tissue cell morphology, which may explain why cells seeded on stiff, crosslinked scaffolds experienced enhanced adipogenesis compared to their less-stiff, non-crosslinked counterparts .Clevenger et al. developed three different hydrogels with various RGD peptides  to assess impact on hASC adhesion, proliferation, and differentiation. Their results suggested that the cyclic and vitronectin-derived RGD sequences allowed for robust initial attachment of undifferentiated hASC. Vitronectin-derived RGD maintained cells in a more rounded morphology. This rounded morphology supported increased adipogenesis in hydrogels containing vitronectin-derived RGD compared to other RGD peptides as determined by percentage of cells containing lipid vacuoles as well as lipid vacuole size ,31.Despite the work by Newman et al. and Clevenger et al., studies on the interaction between ECM-derived scaffold biomaterials and hASC throughout proliferation, differentiation, and maturation are limited. Notably, only two publications utilized decellularized adipose tissue (DAT) as a scaffold material for the culture of hASC ,31. DAT Cheung et al. found that incorporation of DAT into methacrylated glycol chitosan (MGC) and methacrylated chondroitin sulphate (MCS) hydrogels enhanced long-term viability and hASC adipogenesis based on GPDH enzyme activity, adipogenic gene expression, and intracellular lipid accumulation. Cells seeded on DAT hydrogel scaffolds exhibited better cell retention than culture on a pure DAT scaffold. Additionally, hydrogel choice significantly impacted hASC viability and adipogenesis . These rMohiuddin et al. analyzed the effect of seeding hASC on DAT hydrogels in terms of hASC proliferation and differentiation as well as hydrogel microstructure. Their results suggested that the DAT hydrogel could support the attachment, proliferation, and adipogenic differentiation of hASC. Adipogenic differentiation was analyzed by adipogenic gene expression and BODIPY staining. Additionally, DAT hydrogels cultured with hASC in adipogenic media had a lower mean pore size and a higher number of fiber intersections compared to unseeded hydrogels. However, this change in hydrogel structure was less significant than that of constructs cultured in osteogenic media . These fLouis et al. created vascularized adipose tissue constructs by bioprinting a mixture of mature human adipocytes, hASC, human umbilical endothelial cells (HUVEC), collagen microfibers, and fibrinogen into a supporting bath. The constructs remained viable and demonstrated vascularization with capillary structures surrounding mature adipocytes after seven days of culture in vitro .Three-dimensional scaffold culture is typically done in static conditions wherein media is replaced every few days of culture ,24. In aScaffold design for adipose tissue engineering can be challenging due to the significant increase in adipocyte size throughout maturation. Many scaffolds are unable to adapt their pore size, which can restrict adipocyte volume expansion. Therefore, scaffold-free culture approaches leading to the formation of 3D spheroids have been studied extensively. There are four main scaffold-free culture mechanisms: low-attachment culture, hanging drop, magnetic levitation, and dynamic culture conditions . Low-attKapur et al. found that hASC spheroids formed using a hanging drop method followed by adherent or ultra-low attachment culture spontaneously generate an extracellular matrix as evident by positive staining for type I and type III collagen. hASC survived for up to six months in ultra-low attachment culture and were able to produce new, replication-competent cells when moved to adherent culture. Additionally, spheroids demonstrated adipogenic differentiation potential while cultured in ultra-low attachment culture and exposed to adipogenic induction media .Fitzgerald et al. compared three different culture methods: ultra-low attachment static culture, ultra-low attachment dynamic culture, and elastin-like polypeptide–polyethyleneimine (ELP–PEI) coated surfaces. They assessed spheroid size and number of spheroids through time-lapse microscopy and adipogenic differentiation by measuring triglyceride content. They found that ultra-low attachment static and dynamic culture led to a reduction in number of spheroids due to spheroid merging and ultimately the formation of larger spheroids. PEI molecular weight was shown to impact cell retention and spheroid size as lower molecular weight PEI (800 g/mol) exhibited better retention and larger spheroid size than higher molecular weight PEI . Additionally, triglyceride accumulation on ELP–PEI coatings was equal to or higher than that in ultra-low attachment static and suspension culture .Zhang et al. assessed the effects of hASC spheroid formation in a microgravity bioreactor on stemness properties and differentiation potential. Spheroids exhibited increased stemness properties, including increased proliferation and colony forming efficiency, compared to monolayer culture. Additionally, spheroids exhibited increased adipogenic differentiation capabilities as demonstrated by an upregulation of adipogenic marker genes (PPAR-γ and LPL) and ORO staining .Labusca et al. used proprietary magnetic nanoparticles and magnetic field levitation to form hASC spheroids. Spheroids exhibited improved viability and proliferation compared to non-levitated spheroids and 2D culture. Additionally, hASC experienced increased adipogenesis when cultured in spheroids compared to 2D; however, magnetic field levitation was associated with a smaller increase in adipogenesis compared to non-levitated spheroids .Results from these studies indicate the potential use of scaffold-free hASC culture methods for adipose tissue engineering applications. While 3D hASC spheroid culture has demonstrated increased adipogenic differentiation potential and improved maintenance of stemness properties, more research needs to be conducted to determine the best scaffold-free culture methods for engineering adipose tissue. Results from this systematic review indicate that studies on 3D culture methods in the field primarily use “snapshot” assays  rather than functional assays  to assess hASC adipogenesis in vitro. Further research is needed to evaluate the impact of 3D culture methods on hASC functionality to engineer adipose tissue constructs that are more physiologically relevant.Scaffold and scaffold-free 3D culture techniques have been employed to develop microphysiological models of adipose tissue. Adipose tissue MPS aim to recapitulate the functionality of adipose tissue as an organ system in vitro with the ultimate goal to reduce, refine, and replace animal models during the drug discovery process for human diseases. Out of the relevant articles included in this review, six articles (22.2%) self-reported the use of an adipose tissue MPS in their research. One article reported the use of an adipose tissue organoid. The remaining five articles reported the use of an adipose tissue “organ-on-a-chip” .Tseng et al. published a methods paper about their successful development of an “adiposphere” via magnetic levitation of human SVF, preadipocytes and endothelial cells, and hASC . This isPaek et al. created a vascularized adipose tissue microphysiological system by coculturing hASC and human adipose microvascular endothelial cells (hAMECs) in a fibrin hydrogel scaffold. The hAMECs underwent vasculogenesis and formed a network of endothelial tubes throughout the scaffold. The presence of the vasculature increased the rate of adipogenesis allowing for the formation of twice as many lipid droplets in 28 days compared to a non-vascularized model. Furthermore, cells were cultured in the MPS for 40 days leading to the robust maturation and growth of adipocytes to form a densely packed tissue construct .Yang et al. created a microfluidic device to study the effects of flow rate on hASC adipogenic differentiation. The device is composed of five round, 3D culture chambers filled with a hASC-laden fibrin hydrogel scaffold interconnected by two fluidic channels. By administering media at a volumetric flow rate of 1, 4, and 10 µL/hr, average cellular shear stress was estimated to be 0.79 mPa, 3.13 mPa, and 7.86 mPa, respectively. Despite the fact that all three average shear stresses are within physiologic limits (less than 10 mPa), several adipogenic markers were downregulated in response even the smallest amount of shear stress. This suggests that even physiological levels of interstitial shear stress can have inhibitory effects on hASC adipogenesis in vitro [Bender et al. used human SVF and a human blood product-derived biological scaffold (ObaGel™) to create a “fat-on-a-chip” construct. These 3D structures demonstrated increased adipogenesis based on number and size of lipid vacuoles. Additionally, SVF cultured in ObaGel demonstrated increased glucose uptake, leptin secretion, and lipolysis in response to isoproterenol, metformin, and compound C compared to 2D culture ,48.Several reports identified in this systematic review discuss the use of an adipose tissue MPS. Despite significant findings that hASC culture in 3D cellular aggregates (spheroids) experience increased adipogenesis likely due to the replication of in vivo adipocyte morphology, there is a lack of research on the development of self-assembled adipose tissue organoids . AdditioArticles that self-identified as using an MPS tended to use more functional assays for characterizing their in vitro adipose tissue constructs compared to articles that did not identify as using an MPS. This is likely due to the focus of MPS development for drug discovery applications which necessitates the formation and, therefore, validation of physiologically relevant adipose tissue. Even so, there is a general lack of focus on functional assays within the published literature. Additionally, only one article in this review performed drug testing on a construct . TherefoDespite the systematic approach utilized in the current meta-analysis, there are several limitations to the analysis in this systematic review. The keywords used in the database search did not capture all relevant articles, as evident by the inclusion of several related articles identified only through independent search efforts. It is possible that this was amplified through the use of only a single literature database (PubMed). Additionally, we limited our search to articles that used either adipose-derived stem cells or stromal vascular fraction from human sources. This potentially limited the methods that we identified by not including studies that used animal-derived sources. Additionally, with a narrow focus on hASC and SVF culture methods, we were not able to capture related studies that may have used different cell types such as mature adipocytes or explanted adipose tissue constructs.Significant advances have been made in developing physiologically relevant MPS models of adipose tissue using hASC and SVF cells. However, there are several gaps in the published literature that were identified in this systematic review.First, there is no standardized cocktail of adipogenic factors that are used to routinely induce either beige/brown or white adipose differentiation. In the absence of such a fundamental standardized growth medium, there is likely to be considerable variability in the adipogenic process across laboratories and studies. Despite the widespread use of ECM-derived biomaterial scaffolds for culturing hASC, there are few reports on cell-material interactions throughout proliferation, adipogenesis, and maturation. Among articles studying scaffold-free culture methods, there are few reports on the impact of culture type on adipogenic potential. Additionally, no articles identified in this review used functional assays to assess the impact of 3D culture methods on the creation of a physiologically functional in vitro tissue construct. Therefore, there is a need for more studies to assess the impact of culture environment on the development of physiologically relevant adipose tissue constructs.Similarly, very few articles reported the use of perfusion culture to mimic interstitial flow in adipose tissue. Articles using perfusion culture reported decreased adipogenic potential compared to static culture ,26,45. TDespite the fact that white adipose tissue is dependent on vascularization for the transport of nutrients, oxygen, and other soluble factors to maintain homeostasis, only three articles identified in this review focused on engineering vascularized adipose tissue constructs . NotablyAccording to the FDA, predictive tools such as engineered tissue constructs are critical to the goal of reducing, refining, and/or replacing animal testing. Standardization of variables such as reagents, culture methods, and analysis methods (summarized in In summary, there remains a need for an increased focus on the assessment of in vitro functionality within the field of adipose tissue engineering for both regenerative medicine and MPS models. Advancements in 3D culture methods for the generation of physiologically relevant adipose tissue constructs will synergistically support the discovery of novel methods for soft tissue regeneration and models for drug discovery.


About 10% of reproductive-aged couples suffer from infertility. However, the genetic causes of human infertility cases are largely unknown. Meiosis produces haploid gametes for fertilization and errors in meiosis are associated with human infertility in both males and females. Successful meiosis relies on the assembly of the synaptonemal complex (SC) between paired homologous chromosomes during the meiotic prophase. The SC is ultrastructurally and functionally conserved, promoting inter-homologous recombination and crossover formation, thus critical for accurate meiotic chromosome segregation. With whole-genome/exome sequencing and mouse models, a list of mutations in SC coding genes has been linked to human infertility. Here we summarize those findings. We also analyzed SC gene variants present in the general population and presented complex interaction networks associated with SC components. Whether a combination of genetic variations and environmental factors causes human infertility demands further investigations. It is estimated that about 8% to 12% of reproductive-aged couples worldwide suffer from infertility Meiotic cell division is required for all sexually reproductive organisms to generate haploid gametes, i.e., sperm and eggs. Defects or failure in meiosis can cause NOA or POI in humans Studies from various organisms have revealed the critical roles of the SC in meiotic progression. A list of SC gene mutations has been identified in infertile patients, which show NOA or POF/POI phenotypes. In this review, we focus on the role of the SC in meiosis and summarize how mutations in SC coding genes are linked to human infertility.The chromosomes are organized as loop-axis structures during the meiotic prophase. The formation of chromosome axis provides the framework for SC assembly, and the axis is then referred to as lateral elements (LEs). The fully assembled SC consists of two parallel LEs, a central element (CE) in the middle and the bridging transverse filaments (TFs) Figure . The CE The formation of the SC lateral elements likely requires the self-assembly property of SYCP proteins in mammals. Structural analyses showed that human SYCP3 forms tetramers, which can further assemble into filamentous structures and have DNA binding properties Human SC central region proteins also exhibit self-assembly properties. TF protein SYCP1 serves as the core architecture of the SC. X-ray crystallographic and biophysical studies showed that SYCP1 has an obligate tetrameric structure, and through two self-assembly sites, assembles into a zipper-like lattice, in which the N-terminal sites assemble in the midline, and the C-terminal tails face towards the chromosome axis Caenorhabditis elegans also exhibit dynamic properties The highly ordered SC structure and the self-assembly properties of SC components have promoted the hypothesis that SC structure is static. However, studies from model organisms suggest that the SC is dynamic C. elegans, and Sycp2 in zebrafish, are required for recombination initiation C. elegansC. elegans and yeast C. elegans, SC central region promotes the recruitment of pro-CO factors, and the intact SC is required for wild-type level of CO interference Arabidopsis, SC TFs are required to regulate CO rates and probably interference The tripartite proteinaceous ultrastructure of the SC provides the spatial scaffold for homologous recombination and CO formation. Studies from multiple organisms showed that SC components play important roles in various steps of meiotic recombination, including recombination initiation, feedback control of DSB formation, choice of DSB repair pathways, and the control of CO number and distribution In addition to controlling homologous recombination and CO formation during early meiotic prophase, SC proteins may also have additional roles during meiosis. Studies from various organisms suggest that SC components do not dissociate from the chromosomes simultaneously upon pachytene exit. Instead, some components are retained until late meiotic prophase or metaphase I While synapsis and meiotic recombination are essential for establishing chiasmata and preventing aneuploidy, synapsis defects during early meiotic prophase can cause meiotic arrest and apoptosis, which are more directly associated with infertility in mammals. Meiotic sex chromosome inactivation (MSCI) is an essential process in mammalian male germline development, and the failure to initiate MSCI causes the complete arrest and elimination of germ cells during the mid pachytene stage So far, mutant mouse models are available for all known SC components, and their phenotypes are summarized in Table SYCP2, SYCP3, SYCE1, and C14ORF39  in a cohort of 627 male patients with diverse infertility phenotypes, three heterozygous SYCP2 frameshift mutations were identified in three azoospermia patients. Such frequency is significantly higher than that in the general population, suggesting the tight association between SYCP2 mutations and male infertility SYCP3 also shows a high mutation frequency in infertile patients. In a mixed ethnic origin of 23 couples with recurrent miscarriages, a heterozygous change c.548T>C was detected in SYCP3, which caused a missense mutation in the coiled-coil domain of SYCP3 SYCP3, i.e., c.553-16_19del and c.657T>C, both of which disrupt normal splicing and possibly result in C-terminally truncated proteins SYCP3 that results in a premature stop codon and truncation of its C-terminal coiled-coil region was identified in two patients SYCP2 and SYCP3 are the core axis proteins in mammals and have a direct interaction C14ORF39 and SYCE1. Interestingly, protein interaction analysis suggests their protein products form heterodimers in vivo. So far, six mutations in C14ORF39 were identified in infertile males and females by whole-exome sequencing Mutations in two SC central element genes have also been linked to human infertility, i.e., SYCE1. Two splicing mutations of SYCE1 were identified in two independent autosomal recessive NOA families SYCE1 mutation c.613C>T was identified in a consanguineous Israeli Arab family with POI A growing list of pathogenic mutations has also been identified in In addition to mutations within SC genes, copy number variants and translocation-mediated gene dysregulation may also be linked to human infertility. For example, multiple groups have revealed the associations of POI with deletions or duplications of chromosome regions encompassing SYCE1 SYCP2, SYCP3, C14ORF39, and SYCE1). However, given the critical roles of the other SC components in meiotic progression, it would not be surprising to identify infertility-related mutations in the other SC genes as the number of studied cases increases. Moreover, although most infertility-related SC mutations were reported only for a specific gender, mutations within the same SC gene were usually found in both male and female infertile patients. Those reported infertility-related SC mutations may cause infertility in both genders.Interestingly, although mutations in both LE and CE coding genes can cause infertility, most identified mutations in LE genes have a dominant-negative effect, while mutations in CE genes are generally homozygous recessive mutations https://gnomad.broadinstitute.org), which includes WES data of 123,136 individuals and WGS data of 15,496 individuals of different ethnic origins SC genes only express in the germline during meiosis, and they are unlikely to be required for normal human development SYCE1, C14ORF39, SYCP2, and SYCP3, corresponding to the four reported genes with infertility-related mutations. The ratio of observed/expected (OE) number of variants measures how tolerant a gene is to a certain class of variation, and low OE values indicate strong intolerance. Surprisingly, SC genes likely have distinct LoF intolerance levels .SC genes have significant differences in exon counts and protein product sizes, resulting in the differences in their expected numbers of loss-of-function (LoF) or missense variants Figure A, which SYCP2 and C14ORF39 have slightly higher OE values than the other SC genes. Interestingly, proteins encoded by these two genes contain the longest intrinsically disordered regions (IDRs), and the majority of the missense variants locate in their IDRs. SC IDRs are likely more tolerant than helix regions to missense mutations. This is consistent with the observation that helix regions of the SC proteins usually mediate stable protein-protein interactions and are thus critical for SC functions. However, recent findings have also highlighted the important roles of IDRs in SC regulation in C. elegansCompared to LoF, missense OE values show less variation among the SC genes Figure C. SYCP2 Cellular activities are mediated by complex interactome networks of physical, biochemical, and functional interactions of the proteins The study in humans and model organisms has provided insights into how internal interactions between SC components mediate SC formation. However, it remains to be explored how SC assembly and functions are regulated by non-structural proteins, and what biological processes are regulated by SC components. Available protein interaction databases may provide clues about how SC proteins interact with the rest of the proteome. To this end, we extracted an SC binary interaction network from the human reference interactome, which covers about 90% of the protein-coding genome in vivo and whether abnormal interactions between them may contribute to human infertility.Within the SC interactome, several proteins are potentially involved in meiotic cell division or gametogenesis, including the spindle and kinetochore-associated protein 1(SKA1) It is well established that SYCP2 and SYCP3 form the LE core, and the C-terminus of SYCP1 faces towards LEs. Thus, the C-terminus of SYCP1 was expected to interact with LEs. However, direct interactions between SYCP1 and LEs have not been able to be detected. This raises the possibility that SC central region proteins may interact with the LEs through multivalent weak interactions, which may not be detected by conventional methods. Remarkably, SC central region proteins are evolutionarily divergent. The application of multivalent weak interactions to mediate synapsis may underlie the rapid evolution of SC components Infertility remains a severe problem affecting couples wishing to have children. It is ranked third in major diseases after tumors and cardiovascular diseases. The cause of infertility is very complicated. For about one-fifth of the cases, the causes are still unknown. Genome or exome sequencing for infertile patients combined with mouse models will still be a powerful strategy to identify the genetic causes of at least some types of infertility. The SC plays an essential role during meiosis to produce the haploid gametes for fertilization. Understanding how alterations in SC genes are linked to infertility will improve the diagnosis, prevention, and treatment of this disease. Addressing the following outstanding questions will help us to make a better connection between the SC and human fertility.Have all human SC components been identified? Functional redundancy may hinder the discovery of new SC components through phenotype analysis. Given the conservation of SC components in mammals, proteomic analysis in mice or primates will help to identify new SC components or its binding proteins.How is the SC assembled into the functional tripartite structure? Although structural and biochemical studies have provided insights into the self-assembly properties of SC subcomplexes, how the entire SC is assembled remains not fully understood. For example, how are the SC central region proteins organized when all components are present? How do the SC central region proteins interact with the lateral elements? What are the roles of the IDRs in SC assembly? Characterizing the precise interaction network in the SC will help identify the critical sequence elements essential for SC formation.Does the human SC have dynamic properties? While the dynamic properties of the SC have been observed in multiple organisms, whether the SC in humans has the same properties remain to be determined. Addressing this will help to understand how the SC performs its conserved functions in crossover regulation.Can combined gene variants produce synergistic effects? Attention should be paid when multiple genetic variants are observed in physically or functionally associated components, although the single variant might not look harmful. Importantly, with a growing set of individual genomes available, humans are likely emerging as a direct biological model to study rare genetic variations and combinations, which are actually more difficult to explore in other systems.Can SC gene variants combine with internal or external factors to cause infertility? Some SC gene mutations have been shown to cause severe meiotic defects only when grown at suboptimal temperatures in worms


Shoulder abduction/adduction, elbow flexion/extension, wrist extension/flexion, respective range of motion (ROM), and joint patterns were obtained using inertial measurement unit (IMU) sensors. The results showed clear differences in upper limb kinematics between the able-bodied and wheelchair players, especially in the elbow and wrist. For the para-players, noticeable variations in techniques were also observed among the different disability classes. In conclusion, wheelchair players likely adopted distinct movement strategies compared to AB to compensate for their physical impairments and functional limitations. Hence, traditional table tennis programs targeting skills and techniques for able-bodied players are unsuitable for para-players. Future work can investigate how best to customize training programs and to optimize movement strategies for para-players with varied types and degrees of impairment.To better understand the biomechanics of para-table tennis players, this study compared the shoulder, elbow, and wrist joint kinematics among able-bodied (AB) and wheelchair players in different classifications. Nineteen participants (AB,  Para-table tennis is a popular sport in the Summer Paralympic Games, consisting of players with both physical and intellectual impairments. Para-athletes can compete in 11 classifications (Classes) ,3. GenerTraditionally, table tennis techniques and players’ movements are analyzed using video analysis , high-spTo the best of authors’ knowledge, research on para-table tennis are very limited and all previous studies have focused on non-technical skills of para-table tennis players. For example, when comparing the psychomotor performance between elite and non-elite para-table tennis players, elite players demonstrated superior cognitive flexibility, attention control, information processing, and eye-hand coordination . Lim andSingapore is one of the most successful countries for para-table tennis, consistently achieving podium results at international competitions. For example, Singaporean Classes 1 and 2 players clinched silver and bronze medals at the 2015 ASEAN Para Games (APG) and gold medals at the 2017 APG. Research on the technical skills of para-players will enable coaches and sport practitioners to better understand their performance because the game characteristics , technicThis study was carried out following the Declaration of Helsinki, and the methods were approved by the Nanyang Technological University Institutional Review Board (Protocol Number: IRB-2017-08-044). All participants provided written informed consent to volunteer for the study. Parental consent was also sought for minor participants who were under 21 years old.A total of 19 male table tennis athletes were recruited , includi® long sleeve compression shirt and leggings  mounted with 17 inertial 3D orientation sensors inserted into fixed pockets over specific anatomical locations. Three sensors  from the playing limb were interested in this study  was adopted in the present study to capture the shoulder, elbow, and wrist joint motions of all 19 participants. This system is consisted of a Men’s Under Armor HeatGearis study a. Each 3One researcher (JWY) assisted all participants to wear the 3DSuit for good consistency in fitting the IMU sensors on the athletes. After checking the position of each sensor, which should be on the appropriate anatomical landmark, a Velcro strap was tightened to secure the sensors. The IMU sensors were connected with the long sleeve compression shirt and leggings via the Sensor Bus Splitter (SB-Splitter). The SB-Splitter supported joining upper and lower body chains of sensors into a single data acquisition and transmission unit. The SB-Splitter was then connected to the Sensor Bus Universal Serial Bus Control Unit (SB-CU-USB), a data acquisition system that provided power, received, and transmitted data to a computer simultaneously via a standard mini-universal serial bus (USB) cable b.Before commencing all trials, participants were required to face south in an initialization posture to calibrate the system. This prerequisite posture entailed them standing in the anatomical position with face directed forward, arms at the side with palms facing forward, and feet shoulder-width apart to reset sensors 13–15 to 0°. Subsequently, participants moved to their ready position before executing the table tennis techniques. Next, data recording was initiated and terminated using the AnimaDemo program (version 11.6) on a computer. Finally, participants’ motions were reconstructed into three-dimensional real-time movements in the AnimaDemo program at a sampling rate of 60 Hz.An International Table Tennis Federation (ITTF) approved Donic Stress table tennis net set  was mounted onto the ITTF approved Delhi 25 table tennis table . The standard D40+ 3 stars table tennis balls  were loaded into the Newgy Robo-Pong 2050  to project balls to 2 oscillator positions (positions 15 and 5) for forehand and backhand topspin drives . Valid rAll participants were given 2 min to warm up the forehand and backhand topspin drives by rallying 30 consecutive forehand topspin drives followed by another 30 consecutive backhand topspin drives. This sequence, which replicated a table tennis player warming up in competitions prior to the match, allowed participants to familiarize themselves with the height, speed, and spin of the balls projected by the robot when they were wearing the 3DSuit. The tests started after concluding the familiarization period. The order of executing forehand or backhand topspin drive first was randomly assigned for each participant. They performed a total of 30 trials (3 sets of 10 consecutive drives) for forehand topspin drives and 30 trials (3 sets of 10 consecutive drives) for backhand topspin drives. Only the trials with projected balls landed diagonally on the lower half of the table within the targeted zone (0.80 m × 0.76 m) were deemed valid b. All inJoint angle data were recorded using the AnimaDemo program. The raw data were exported as .bvh files and then low-pass filtered at 8 Hz using the 4th order zero-lag Butterworth filter in MATLAB  to smooth and remove random errors. Subsequently, each participant’s joint angle data were time normalized (0 to 100%) to obtain an average angle-time history of the 10 trials for the shoulder joint in the frontal plane, elbow joint in the sagittal plane, and wrist joint in the frontal plane .Minimum angle—the minimum angle for shoulder, elbow, and wrist joints is maximum shoulder abduction, elbow flexion, and wrist extension, respectively;Final angle—the angle at the end of the follow-through phase of each drive;Range of motion (ROM)—t he excursion from minimum to final angles of each drive.Each table tennis stroke can be split into different phases. Positions 1 to 2 refer to the preparation phase, positions 2 to 3 refer to the execution phase, and positions 3 to 4 refer to the follow-through phase . For eacThe average values of 10 trials were calculated per participant in each type of drives. Group mean and standard deviation for the AB and para-players in different classifications were also determined.Descriptive data and graphical comparison of angle-time histories are presented to illustrate the different in movement patterns among AB and wheelchair players in varies classes . No infeAB demonstrated the greatest ROM for all joints of the forehand topspin drives . All parThe backhand shoulder angles differed between AB and wheelchair players, while the profiles were similar among the different wheelchair classification groups . For theThis study applied IMU sensors to profile the kinematics of the playing limb in table tennis players with and without physical disabilities. Shoulder, elbow, and wrist joint angles were compared among AB and wheelchair players in different classifications while performing the forehand and backhand topspin drives. To the best of the authors’ knowledge, the present study is the first to comprehensively report the upper limb kinematics of wheelchair table tennis players while executing table tennis strokes. The results showed clear differences between the AB and wheelchair players, especially in the elbow and wrist. Among the wheelchair table tennis players, noticeable variations in techniques were also observed in different disability classes.Using portable IMU sensors, the AB’s shoulder and elbow joint angles observed in this study were similar to those reported by Xia and colleagues  who usedAs with other wearable devices and compression garments, there will be some degrees of movement impediment resulting from wearing the 3DSuit. While we cannot quantify how the 3DSuit may have affected natural table tennis movements, we expect a small effect because the compression shirt and leggings are generally comfortable and flexible. In addition, the sensors were light and hence would not add much weight to the players’ limbs. Thus, we believe that the table tennis kinematic data measured using the gyroscopic 3DSuit inertial motion capture system are a good representation of the players’ usual techniques.Findings from the present study provide empirical evidence to confirm the previous speculation that para-table tennis players would use alternative motion patterns to compensate for their physical impairments . This isNo previous studies have examined wheelchair table tennis biomechanics while executing dynamic sport-specific strokes. This present study reports novel results that the playing limb kinematics differed not only between AB and para-athletes but also among different wheelchair disability classes. Since, our wheelchair participants were elite athletes competing at the national and international levels, it is believed that they had adapted optimal movement strategies to generate speed and power while maintaining balance to compensate for their respective impairments. Making direct references or replicating AB athletes’ movement patterns can lead to detrimental outcomes such as poor performance and injury. Given the movement strategies of para-athletes likely to be different from their AB counterparts, they should adapt and modify the movement patterns to suit their nature of physical impairment and sporting requirements for successful performance. The differences between AB and para-athletes have also been acknowledged in other sports. For instance, para-swimmers favored the parabolic fast start strategy in 400-m freestyle swimming, while the AB swimmers favored only the fast start strategy . Thus, iConsidering the marked differences in playing techniques observed in the present study, coaches should not ask para-athletes to simply replicate the skills and techniques used by AB table tennis players. Countless movement strategies exist, and some are more favorable depending on the interacting constraints of an individual’s abilities, task requirements, and environmental conditions. The ideal technique for para and AB players will very likely be distinctive even under the same condition such as returning a ball at a certain speed towards a certain direction. Traditional table tennis training for the AB players has greatly emphasized footwork training on how to move around the table efficiently . HoweverThere are a few limitations to the current study. Firstly, at a starting point on wheelchair table tennis, kinematic data were only obtained from the upper extremities of the playing limb. Future studies could investigate the trunk movement of the para-players as well as the manipulation of the wheelchair. Secondly, the sample size of each disability class was rather small due to the limited number of para-players available in Singapore. In the future, studies are recommended to include more para-players as well as those from the standing classes  who may adopt different techniques compared to wheelchair players. Finally, all para-table tennis players recruited in our study had a disability due to an accident later in adulthood. They picked up table tennis much later than able-bodied players who started training at a young age. The disparity in the number of years of experience between able-bodied and para-players may have affected the maturity of their table tennis technique.The study revealed that wheelchair table tennis players exhibited distinctly different upper limb kinematics compared with AB participants while executing the forehand and backhand topspin drives. For the wheelchair table tennis players, noticeable variations in techniques were also observed among different disability classes. The differences were more pronounced in the elbow and wrist joints than the shoulder joint. The marked differences in techniques could be due to functional limitations associated with the wheelchair players’ physical impairment. Thus, coaches should be aware that traditional table tennis training programs targeting skills and techniques for AB players are unsuitable for para-players. Communication among para-players, coaches, and medical professionals are critical to better understand the players’ physical abilities and functional limitations. Future research can further investigate how to customize training programs and to optimize para-players’ movement strategies for successful performance.


Cancer is a multifactorial disease and a leading cause of deaths worldwide. An estimated 1.89 million new cancer cases and about 600,000 cancer related deaths were reported only in United Stated in the year 2021 . A systeLately, plant bioactives are gaining increased attention for their therapeutic activity against many cancers (nearly 2000 publications per year) as they possess versatile biological properties and target multiple pathways in cancer. Plant-derived compounds have historically led to some of our most useful cancer drugs . These chemopreventives can block initiation, reverse promotion and/or halt the progression of precancerous cells into malignant cells. Since advanced, recurrent and metastatic tumors are practically lethal and cannot be cured by any therapy, cancer chemoprevention of earlier lesions should be the definitive goal if we are to eradicate this deadly disease. To fulfil the unmet needs of cancer treatments, a scrupulous repurposing of plant-based phytochemicals such as chemical modification of the pharmacophores, development of target-based delivery strategies including nano formulations, and their uses in adjuvant settings need to be considered. In this regard, we launched the current special issue to highlight the current development in the anticancer drug research.Al-Odat et al. discuss the role of Mcl-1 inhibition in the management of multiple myeloma. They further highlight that the most prominent BH3 mimetic and semi-BH3 mimetic selectively inhibit Mcl-1 and could be used by itself or in combination with existing therapies for the management of multiple myeloma. In another article, Cui et al. showed that the tubulin Inhibitor VERU-111 in combination with vemurafenib, an FDA-approved BRAF inhibitor, provides an effective treatment for vemurafenib-resistant A375 melanoma.Most of the chemotherapy treatment failures occur due to drug resistance subsequently limiting its use and available treatment options. Several factors like tumor microenvironment, size, heterogeneity, involvement of immune system and undruggable genes necessitate in evoking the drug resistance . For insWet et al. successfully demonstrated using in vitro and in vivo assays that curdione isolated from Curcuma zedoary suppressed uterine leiomyosarcoma cell proliferation by inducing G2/M phase arrest, apoptosis, and autophagy via targeting IDO1. In the similar line, Macleayins A and phenethyl isothiocyanate were shown to inhibit the growth of cervical cancer cells .Besides repurposing the existing drugs, discovery of novel drug molecules is the most explored alternate option to overcome chemotherapy related drug constraints in cancer therapy. In this context, new plant bioactives fill the void of limited chemotherapy options. Uterine leiomyosarcoma is a rare gynecological malignancy with no standard approved drug at present. Peng et al. concluded that ginsenoside Rg 3 isolated from Korean Panax ginseng in combination with first line chemotherapeutic drugs demonstrate enhanced efficacy towards advanced non-small cell lung cancer (NSCLC) patients. Ginsenoside Rg 3 also reduced the chemotherapy induced toxicity. Similarly, phyto-chemicals from banana also showed great potential for future development of drugs for cancer prevention and therapy .In a systematic review and meta-analysis of randomized clinical trials, Wagh et al., provides an up-to-date summary on the effect of celastrol and its nano formulations in cancer prevention. This review discusses a multitude of celastrol nanoformulations that have been developed and tested for various therapeutic applications. Outside the scope of this review, exosomal formulations using milk derived exosomes have been demonstrated against lung cancer using in vitro and in vivo models -induced head and neck cancer (HNC) and concluded that the therapies to distinguish HPV-positive HNC from HPV-negative HNC could help to achieve better outcome. This HPV-associated HNC can be treated with well-established phytochemicals targeting the HPV-mediated carcinogenic mechanisms.Despite potential anticancer abilities, many novel anticancer drugs suffer from poor bioavailability to achieve therapeutic drug level. The review by o models . In anotAlbini et al. demonstrate that the polyphenol-rich extract of olive mill waste water enhances the anticancer potentiality of chemotherapeutic drugs cisplatin and 5-fluorouracil while mitigating their dose related cardiotoxicity. In another study, Oxyresveratrol was shown to have the ability to alter the cancer-causing genes and subsequently inducing apoptosis and cell cycle arrest in breast cancer MCF-7 cells .Plant polyphenols are known for their pharmacological activities and therapeutic benefits. A study conducted by Otaibi et al. shed the light on this aspect in their study and show chemically synthesized biologically active α-amino amide analogs alone and in combination with T cells exhibit pronounced anticancer activity against leukemia cancer cell lines (HL-60 and K562). In another study, Rampogu et al. used chemically modified butein derivatives to inhibit breast cancer progression through modulation of PI3K/AKT signaling pathway and other molecular markers.Growing body of evidence suggests that approaches like chemical synthesis, analogue modification and development of nano formulations might help to achieve better anticancer therapies. in vitro and in vivo data for cancer prevention, the review articles shed light on preclinical and clinical studies in cancer prevention and therapy.In summary, this research topic comprises eight informative research and five authoritative review articles written by expert scientists and expansively discusses various aspects of cancer prevention and control. This special issue highlights strategies for cancer prevention and therapy by plant therapeutics alone and in combination with already in use therapeutics drugs, repurposed molecules for other diseases, and various nanotechnology-based techniques to enhance efficacy, reduce dose related toxicity and to better manage resistant and metastatic cancers. While articles in this special issue show