Spondyloarthritides (SpA) are a family of interrelated rheumatic disorders with a typical disease onset ranging from childhood to middle age. If left untreated, they lead to a severe decrease in patients' quality of life. A succesfull treatment strategy starts with an accurate diagnosis which is achieved through careful analysis of medical symptoms. Classification criterias are used to this process and are updated on a regular basis. Although there is a lack of definite knowledge on the disease etiology of SpA, several studies have paved the way for understanding plausible risk factors and developing treatment strategies. The significant increase of HLA-B27 positivity in SpA patients makes it a strong candidate as a predisposing factor and several theories have been proposed to explain HLA-B27 driven disease progression. However, the presence of HLA-B27 negative patients underlines the presence of additional risk factors. The current treatment options for SpAs are Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), TNF inhibitors (TNFis), Disease-Modifying Anti-Rheumatic Drugs (DMARDs) and physiotherapy yet there are ongoing clinical trials. Anti IL17 drugs and targeted synthetic DMARDs such as JAK inhibitors are also emerging as treatment alternatives. This review discusses the current diagnosis criteria, treatment options and gives an overview of the previous findings and theories to clarify the possible contributors to SpA pathogenesis with a focus on Ankylosing Spondylitis (AS) and enthesitis-related arthritis (ERA). SpA is a group of immune system related disorders predominantly causing sterile inflammation at sacroiliac joints. In adults, patients often meet the definition of axial spondyloarthritis. In addition to AS, reactive arthritis, psoriatic arthritis, enteropathic arthritis, Reiter's syndrome, Inflammatory Bowel Disease (IBD)-associated arthritis and undifferentiated SpA can be included within this disease subset . The claSpAs affects up to 2% of the population . The preSimilar to other diseases the early diagnosis of SpA is crucial. Delays may result in increased disease activity, irreversible structural damage, low therapy response and limited mobility . PhysiciThorough and distinctive analysis of the symptoms plays a fundamental role in the validity of diagnosis. SpA patients display several common clinical and laboratory findings such as arthritis , psoriasIn recent years MRI has improved the assessment and diagnosis of axial disease. Axial disease was probably underestimated before the widespread use of MRI both in children and adults. Approximately half of the patients are known to be first been diagnosed with sacroiliitis on MRI .Most juvenile SpA are classified as enthesitis-related arthritis or undifferentiated arthritis, depending on whether psoriasis is present in the patient or their family. International League of Associations for Rheumatology (ILAR) criteria are used for the classification of JIA subtypes including ERA  2, 21)., 21.2, 2In adults the diagnosis is based on the Assessment of SpA International Society (ASAS) classification . ASAS crThe ASAS and ILAR criteria set indeed overlap in the defined features and they share several clinical and laboratory findings for classification. However, there are also important differences between the two. Firstly, the ILAR system does not specifically address children who have SpA by fulfilling the criteria for ankylosing spondylitis, or who have coexisting conditions such as inflammatory bowel disease . ReactivIndeed the ILAR criteria has important limitations regarding the classification of patients in the spondyloarthropathy group. Enthesitis-related JIA was considered an undifferentiated SpA in ILAR, whereas all the different forms of adult SpA can be found in children, with the major difference being the higher proportion of undifferentiated forms in children. Thus a new classification criteria have been proposed by researchers from PRINTO, suggesting fundamental changes, in the classification of enthesitis-associated arthritis . This neThe current PRINTO definition has been partly harmonized with the adult one, and an imaging criterion for radiographs  or magneAlthough there is a lack of substantial molecular omics studies, most pediatricians would regard ERA more like a juvenile AS, especially once sacroiliitis is detected. There is a need for more follow-up data on patients with peripheral disease -fulfilling the ERA criteria, to understand whether they constitute a separate pediatric group. Moreover, sophisticated studies are crucial to understand whether axial-ERA is truly an early onset AS or SpA.The lack of knowledge on SpA etiology has been a major concern in diagnosis and disease treatment. The diagnosis is dependent on clinical manifestations which shows heterogeneity between patients whereas the therapeutic interventions were developed based on observational studies. In this section we will discuss the possible risk factors for SpAs and theories related with them.The immune system acts as a safeguard to protect our body from the invasion of harmful intruders. These foreign entities' proteins should be presented as peptides to our immune cells to activate a potent immune response. Peptides loaded on Major histocompatibility complex (MHC) molecules located on cell surface can be recognized by T cells which in turn cause their activation. All nucleated cells have MHC class I molecules that take part in the presentation of intracellular antigens  to CD8+ T lymphocytes and their heavy chains are encoded by genes at HLA-A, HLA-B and HLA-C loci . AntigenThe first report showing the association of the MHC class I molecule HLA-B27 with SpAs was published in 1973 . Since tThe importance of HLA-B27 in SpA etiology was also recapitulated using animal models. Rats having high levels of HLA-B27*05 and human beta 2 microglobulin (B27-Tg) partially phenocopy the human disease with inflammatory bowel condition, inflammatory peripheral arthritis and skin lesions . Interesvia pro-inflammatory stimuli. Of note, this dose dependent effect might also explain why only 2% of HLA-B27 positive patients develop the disease.The HLA-B27 levels seems to be a pivotial factor regulating disease susceptibility. Higher levels of HLA-B27 are typically seen in the peripheral blood mononuclear cells (PBMCs) of patients compared to healthy controls positive for this allele . MoreoveThe positivity of HLA-B27 also has an influence on disease manifestation. In more than 80% of AS patients, symptoms emerge at ≤30 years of age. Interestingly, HLA-B27 positive AS patients show an earlier disease onset compared to negative ones  and haveOverall, it is clear that HLA-B27 somehow plays a role in disease pathogenesis. Its possible contribution to disease progression and related theories are discussed below.via tapasin which is bound to ERp57 to form the peptide loading complex (The mature MHC I molecule is composed of a heavy chain (HC), a β2m light chain and a peptide, 8–10 amino acids in length. Its formation involves a series of protein assembly and disassembly within the complex. First, newly synthesized heavys chains are translocated to the endoplasmic reticulum (ER) and glycosylated. This post-translational modification acts as a signal for incomplete folding which in turn triggers HCs interactions with chaperones calnexin and calreticulin. As HCs gain the correct tertiary structure, they associate with β2m resulting in the dissociation of calnexin , 49. Nex complex . Eventua complex . FinallyThe plausible contribution of APCs in disease pathogenesis has been the center of many studies. The increased abundance of macrophages in sacroiliac  and enthPrevious studies suggested that HLA-B27 binds to a distinctive set of peptides that show similarity to self-peptides . Their pKlebsiella pneumoniae  and CD4+ T lymphocytes in the form of β2m-free homodimers  which isStudies aiming to understand the involvement of dimer formation in disease pathogenesis unearthed that HLA-B27 variants associated with SpA have an increased tendency for dimer formation . The recvia autophagy or Endoplasmic Reticulum Associated Degradation (ERAD) . Proteins with folding deficits can be removed n (ERAD) . Howevern (ERAD) .- Folding rate: HLA-B27 has a slow folding nature which in turn induces homodimer formation and its retention in the ER. These molecules can then activate the UPR. The B pocket which is located at the peptide binding groove of HLA-B27 seems to be crucial for this machinery. Altering residues in this region not only enhances HLA-B27's folding but also alleviates homodimer formation .- ERAD: Another clue showing the misfolded nature of HLA-B27 is its enhanced predisposition to undergo ERAD  in which- Interaction with chaperones: The chaperones help misfolded proteins to gain a proper tertiary structure. The prolonged interaction between HLA-B27 multimers and chaperone Bip indicates the improper folding of HLA-B27 which in turn activates the stress response . In addiMacrophages residing in the peripheral joints of AS patients have increased levels of Bip compared to osteoarthritis patients  are among the gene loci that shows association with AS , 114. ThFor JIA, ERAP1and IL23R are among the disease susceptibility genes for ERA and juvenile psoriatic arthritis respectively . The lowMany rheumatic diseases display gender predominance. Both the incidence of AS  and ERA via downregulating TNF alpha levels  occur concomitantly in up to 10% of SpA positive population . PatientAkkermansia muciniphila species was found to be elevated in B27-Tg rats indicating that SpA related dysbiosis may be involved in impaired gut barrier  were enrolled for an open-label secukinumab trial in the first treatment period. In the second period, responders received secukinumab or placebo. Secukinumab demonstrated a significantly longer time to disease flare and a consistent safety profile similar to adults . MoreoveBridging therapy with systemic glucocorticoids might be used during the initiation or escalation of therapy. Intraarticular glucocorticoid injections of the sacroiliac joints as an adjunct therapy are conditionally recommended . PhysiotAnother important aspect of the treatment is to monitor the side-effects of the drugs. For NSAIDs, gastrointestinal problems may arise thus proton pump inhibitors might also be prescribed. On the other hand, adequate fluid intake is essential to circumvent renal injury . Anti-TNDisease activity has to be followed to evaluate the response to treatment. For JIA patients, Juvenile Arthritis Disease Activity Score (JADAS) and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) are screened whereas BASDAI and Ankylosing Spondylitis Disease Activity (ASDAS) are used for the assessment of therapy success in adult SpAs , 173. ThFor adults, many therapeutics have been used and published. The reader is referred to excellent reviews on the subject. The primary treatment for SpA is NSAIDs and TNFis . Of note, SpA patients have higher levels of TNF-α  and HLA-wIL17 inhibitors (secukinumab and ixekizumab) can also be used for patients. Anti-IL17 is not recommended in patients with IBD or recurrent uveitis. If the patient has tuberculosis or recurrent infections, sulfasalazine is preferred over secukinumab and ixekizumab. Tofacitinib (a JAK inhibitor) is a second-line option for patients with contraindications to TNFi or anti-IL17. Co-treatment with low-dose methotrexate is not generally recommended except with infliximab . BrodaluAs summarized above, studies ongoing for more than 4 decades have led to the discovery of many risk factors for SpA development. Among these factors HLA-B27 seems to be the spearhead helping us to better understand the etiology of the disease. HLA-B27 driven mechanisms are thought to involve UPR activation and switching on the IL23/IL17 axis.The fact that only a part of HLA-B27 positive people develop SpA indicates that there are additional factors contributing to disease pathogenesis. Although the threshold effect for HLA-B27 might be a possible explanation for this observation, it is most likely that further investigation of factors other than HLA-B27 is required that will also pave the way for the development of alternative therapies. The current treatment regimen involves NSAIDs, TNF inhibitors and possibly DMARDs. However, only some patients respond to the treatment which in turn causes a significant decrease in the non-responders' quality of life. Therapies targeting UPR and IL23/IL17 axis have recently gained attention but clinical trials are needed for further validation.


Introduction: Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in CCR5 CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos.Methods: DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis.Results: Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. De novo mutations were also identified at predicted CRISPR-Cas9 off-target sites.Discussion: This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations. CCR5 gene (CCR5-Δ32). CCR5 serves as an HIV co-receptor  are superior for modeling human diseases as they share similar aspects of immune, neuro-, and reproductive physiology and are ideal for transplant and neurodevelopmental disorder research. The interest in creating NHP models of human disease has been augmented by the need to better define the etiology of a disease and for the development of treatments and therapeutics . For exareceptor . Transplll cases . GeneratHIV cure .Genome editing approaches to create gene disruption in NHP embryos have been successful, yet evidence of CRISPR-Cas9-induced chromosomal anomalies in mammalian cells and embryos warrants further investigation of embryonic editing outcomes in primate embryos. CRISPR-Cas9 editing has been shown to result in large scale deletions (up to 6 kb) and whole chromosome loss leading to genomic instability in mouse embryonic stem cells  and embrCCR5 in Mauritian cynomolgus macaque  embryos using PCR-based methods to confirm successful targeting of the locus . Embryos were cultured individually in a microwell of a CultureCoin MIRI-TL dish  containing 25 μL of Global medium overlaid with mineral oil and the culture dish was placed in a MIRI TL Time-Lapse incubator  to monitor embryo development. Individual blastomeres from two embryos arrested at the 6-cell and 9-cell stage were isolated. The zona pellucida was removed by treatment with 1 mg/ml of activated pronase E  and the embryo was washed in calcium- and magnesium-free PBS with .2% EDTA and 1 mg/ml human albumin . Individual blastomeres were then dissociated by gently pipetting the embryo. DNA was amplified from individual blastomeres using a REPLI-G single cell kit .escribed . BrieflyCCR5 region  CCR5 PCR amplicon size of 613 bp or a biallelic mutation producing a product of 415 bp. To evaluate large-scale deletions near the on-target site, a long-range CCR5 PCR was performed. PCR primer sequences and expected amplicon sizes are listed in PCR amplifications were performed as previously described to assess the targeted 5 region  using prBlood draws from both the oocyte and semen donors of the in vitro-produced embryos were performed to obtain parental DNA from peripheral blood mononuclear cells. Genomic DNA was isolated from blood cells using a Quick-DNA Miniprep kit .DNA quality was assessed at the University of Wisconsin Biotechnology Center’s by the NexGen DNA Sequencing Core using an Agilent Femto Pulse system  to confirm a uniform yield of DNA product with the average product length of greater than 9.4 kb.Macaca fascicularis reference genome, M_fascicularis_5.0, using an Illumina Dynamic Read Analysis for GENomics (DRAGEN) Bio-IT platform version 3.7. Small variant and calling was performed using DRAGEN. Variants from control  samples were used to filter and identify de novo mutations. Variant annotation was performed using SNEPeff tool that will predict synonymous or non-synonymous amino acid changes, gains or losses of start/stop codons, and frame shifts due to insertions or deletions. Structural analysis was performed using Parliament2  was used to view the WGS data.Whole genome sequencing was performed by the University of Wisconsin-Madison Biotechnology Center using the Illumina short-read platform and a NovaSeq 6000 instrument. Reads were trimmed to remove sequencing adapters and low quality base calls using the software skewer  and thenliament2  and onlyhttp://www.rgenome.net/cas-offinder/) and allowing for three mismatches. Regions of interest were then evaluated in the WGS dataset to see if de novo mutations were present in individual blastomeres compared to the parental DNA. The presence of de novo mutations in three predicted off-target genes were assessed by Sanger sequencing of PCR amplicons containing the region of interest for individual blastomeres. DNA obtained from a wild-type cynomolgus macaque iPSC line was sequenced in parallel. PCR reactions were performed using the Q5 Hot start High-Fidelity DNA polymerase kit following manufacturer recommendations and the reactions were cleaned up using a Gel extraction and PCR clean up kit . PCR primer sequences are listed in https://nucleobytes.com/4peaks/index.html) application.Potential CRISPR-Cas9 off-target regions were identified using the Cas-OFFinder tool   and determined the genotype for one blastomere in which the d by PCR . Sequenc methods . If ther methods . Regardldetected . PCR usidetected . MoreoveDe novo structural variants that were not present in the parental DNA but were within individual blastomeres of both embryos 4 and 5 were identified by WGS  within the on-target region was evaluated. Single nucleotide variants (SNVs) were identified and considered to be in silico Cas-OFFinder tool to identify off-target sites based on sequence homology to the gRNA sequences and the total number of off-target sites when allowing for up to 9 mismatches between the gRNA and off-target sequence is provided in De novo mutations within individual blastomeres were identified by WGS in 16 predicted off-targeted sites of which 7 were located within genes and nine were located in intergenic regions  and the two PINK1 mutants were triplets that died days after birth. These studies demonstrated that implantation and pregnancies can be achieved despite the presence of large-scale editing errors. Furthermore, on-target deletions have now been observed across studies in both NHP embryos and tissues where different genes were targeted by wild-type CRISPR-Cas9, which necessitates refinement of genome editing tools for creating precise disease-associated mutations.Undesired editing events at the on-target site, including large-scale deletions, have been observed in human and mouse embryos targeted with wild-type Cas9 nuclease . In the s monkey , a ∼7.2  monkeys , and 920s monkey . The SHACCR5 targeting site). Mosaicism in blastomere genotypes suggests that editing was delayed and did not occur at the one-cell stage as the deletion was not detected in all cells of the embryo. Editing mosaicism has been observed in tissues of edited NHPs produced from embryo transfer of CRISPR-Cas9 targeted embryos  and an incomplete reference genome, a large number of false positives might be called due to errors incurred during WGA or due to differences in the reference assembly.A goal of the present study was to assess the feasibility of using WGS to assess off-target effects in individual blastomeres. An smatches . Moreovesmatches . In vitrde novo mutations that the authors thought were not introduced by CRISPR-Cas9 and rather could be attributed to natural spontaneous generational mutations or that were due to technical noise. In these previous NHP studies, WGS analysis was performed on DNA obtained from cells or tissues of live offspring or miscarried fetuses whereas here we reported WGS on individual blastomeres of NHP embryos. The higher incidence of off-target editing in this study could be explained by the editing efficiency of the RNP or it is possible that such significant off-target errors could have been embryonic lethal and therefore not present in offspring that survived to or near term. Editing has shown to be more rapid and efficient when targeting with an RNP versus Cas9 mRNA in NHP embryos  use  embryos , or 3) u embryos .Off-target INDELs and segmental chromosome errors introduced by CRISPR-Cas9 have been observed in human embryos . The forin vitro fertilized cleavage stage rhesus macaque embryos, and when analyzing individual blastomeres from 50 embryos, 73.5% showed whole and/or partial chromosomal losses or gains , be reproducible and have a lower error rate  comparedOverall, utilizing a WGS approach to determine CRISPR-Cas9 editing outcomes allows for the identification of edits not identified by PCR. In this study, WGS revealed the incidence of on-target large-scale deletions and INDEL formation at off-target sites. Imprecise editing could hinder the development of an NHP disease model that both genocopies and phenocopies the disease. The consequences of undesired editing events on gene expression of off-target and/or neighboring genes was not evaluated in this study, but should be considered in future studies. Based on evidence from human and mouse embryonic targeting by CRISPR-Cas9, it is likely that chromosomal damage incurred early in embryo development could negatively impact embryo viability. It remains unclear whether a reduced concentration or volume of the RNP delivered to the one-cell embryo would have a dose-dependent impact on on-target errors. Additional studies are needed to optimize embryonic editing by wild-type Cas9 and/or to use alternative next-generation Cas9 nucleases that do not create a double-stranded DNA break . Regardl


In particular, we emphasize strategies for manufacturing tissue engineering hydrogels in which both macroporous scaffold fabrication and cell loading can be conducted in a single manufacturing step – electrospinning, 3D printing, and in situ tissue engineering. We suggest that combining such integrated fabrication approaches with the lessons learned from previously successful translational experiences with other hydrogels represents a promising strategy to accelerate the implementation of hydrogels for tissue engineering in the clinic.While the soft mechanics and tunable cell interactions facilitated by hydrogels have attracted significant interest in the development of functional hydrogel-based tissue engineering scaffolds, translating the many positive results observed in the lab into the clinic remains a slow process. In this review, we address the key design criteria in terms of the materials, crosslinkers, and fabrication techniques useful for fabricating translationally-relevant tissue engineering hydrogels, with particular attention to three emerging fabrication techniques that enable simultaneous scaffold fabrication and cell loading: 3D printing,   Every year, a high number of deaths or disabilities results from the loss or damage of tissues and organs from injuries or diseases , the numHydrogels, networks of water-soluble polymers physically or chemically crosslinked to form a gel, have been widely used as vehicles to deliver cells to a designated location in the body , scaffol®, AlloDerm®, and Juvéderm®) have been approved for use in the clinic  of the targeted tissues . The ECM3 g/mol)  .Finally, the compositional and morphological diversity  of a given hydrogel in relation to its target application should be considered in choosing the correct hydrogel material. The exceptional control that modern polymer chemistry techniques can impart on both the chemistry and the molecular structure of a synthetic polymer can enable precise tailoring of the physiochemical and mechanical properties of synthetic hydrogels in a way that is challenging to reproduce with naturally-derived polymers that typically have broad molecular weight distributions and complex variable compositions . SynthetBalancing these competing considerations makes the selection of the type of polymer(s)  and the specific polymer(s) within one of those two groups to design a hydrogel-based tissue engineering scaffold complex . In the in vivo by naturally occurring collagenase  as induced by cytomodulin-2 (CM) that was also covalently linked to HA  is a linear polysaccharide that is found throughout the body but particularly in connective tissues . HA playreaction . The in ed to HA . HoweverChitosan is a positively charged polysaccharide formed through the deacylation of chitin, the main structural component of crustacean exoskeletons . Althougin vivo  is a non-toxic, degradable, and thermoresponsive polymer derived from cellulose . Given tadhesion . The lowadhesion . Howeveradhesion , while tadhesion .block-poly (ε-caprolactone), PEG-PCL) or thermoresponsive block copolymers (PEG-block-poly (propylene oxide), PEG-PPO); the latter two approaches also enhance the mechanics of the hydrogel via a dual covalent bonding/hydrophobic interaction crosslinking mechanism)  with GelMA to prepare a printable hydrogel ink for fabricating vasculature-like structures (Poly (ethylene glycol) (PEG) is a synthetic hydrophilic polymer used for a variety of biomedical applications due to its low cytotoxicity, non-immunogenicity and non-specific protein adhesion properties . PEG-baschanism) . The chochanism) . Howeverchanism)  due to echanism) . PEO-basreatment . The in neration . Howeverructures . The micructures . The surructures  that mayPoly  (PVA) has attracted attention for its high-water retention and superior mechanical strength to PEG, enabling its use in applications requiring stiffer scaffolds such as the replacement of articular cartilage . PVA hydN-isopropylacrylamide) (PNIPAAm) is an amphiphilic temperature-responsive smart polymer typically fabricated via free radical polymerization. PNIPAM has a lower critical solution temperature (LCST) of ∼32°C and therefore can form a hydrogel at physiological temperatures by thermally-driven self-assembly  to mainassembly , althougassembly . The maiassembly , which rnologies . PNIPAM hallenge .n = 2–3 ethylene oxide side chains) to being highly protein-repellent (n > 5–6 ethylene oxide repeat units)  methyl ether methacrylate (M(EO)2MA) with longer chain (n = 7–8 ethylene oxide repeat units) OEGMA monomers, with the transition temperature varying linearly with the mole percentage of each monomer (in vivo (Poly (oligoethylene glycol methacrylate) (POEGMA) has a methacrylate-based backbone  and PEG-based side chains. By tuning the length of the PEG side chains, the polymer properties can be switched from being temperature-responsive (t units) ; couplint units) , POEGMA backbone . POEGMA backbone . In situ monomer  while pr monomer . In addi monomer . For exa(in vivo .Poly (2-hydroxyethyl methacrylate) (PHEMA) hydrogels were first introduced in the 1960s for use in contact lenses and have since been widely explored in tissue engineering given that they have many of the same advantages as PVA and PEG previously described . HoweverIn situ gelling in which the hydrogel gels upon injection in vivo via either physical crosslinking  or chemical crosslinking  offers particular promise for preparing injectable hydrogels for cell delivery in that such hydrogels avoid the need for surgical implantation, facilitating their practical clinical use  tend to be more specific to particular polymer types.Once the backbone polymer is chosen, the method of crosslinking the polymer chains together must be judiciously selected. Physical crosslinking and chemical crosslinking strategies are available, each with their own advantages and drawbacks in the context of tissue engineering. Compared to physical crosslinking, chemical crosslinking strategies typically result in linkages with more controllable degradation profiles and better mechanical properties; however, chemical crosslinking typically requires some kind of chemical derivatization of the backbone polymer and/or additional synthetic steps that may not be fully compatible with cells, unlike most physical crosslinking processes . In situical use . Herein,ical use . Note th2+, Mg2+, Fe3+) have been particularly widely leveraged to prepare hydrogels for tissue engineering given the high cell compatibility of the divalent cation crosslinkers, although as previously noted ion exchange with the ions in the physiological environment can make it challenging to control the degradation of the hydrogels  is used to initiate the polymerization of unsaturated vinyl, acrylate, methacrylate or allylic groups conjugated to the gel precursor polymers, typically aided by water-soluble photoinitiators  to promote free radical generation under lower intensities of light than would be required to form a gel via only hydrogen abstraction from the precursor polymers . While pications .Click chemistry, a range of reactions that can occur rapidly and spontaneously under physiological conditions without producing toxic by-products  has also attracted significant increasing attention given that no additional crosslinkers/initiators/catalysts are required to prepare the hydrogels, no post-treatment is necessary, and (if delivered using a double barrel syringe and/or a static mixer) hydrogels can be administered directly into the body by simple injection to avoid the need for surgical implantation . A rangeA range of other chemical crosslinking methods based on di/multi-functional small molecule crosslinkers has been reported to prepare hydrogels . Howeverin situ gelling hydrogels under physiological conditions in the presence of cells (2O2) and horseradish peroxidase (HRP) in which the stiffness of hydrogel was directly tunable by adjusting the concentrations of H2O2 and Gtn-HPA to optimize the proliferation of chondrocytes  to create hydrogels with low inherent immunogenicity . Enzymatof cells . For exadrocytes . Howeverdrocytes .As another alternative, instead of using physical, chemical, or enzymatic processes to crosslink individual polymers together that were not previously networked together, ECM-mimetic hydrogel scaffolds can also be created by decellularizing native tissues. Decellularization refers to the removal of cells and other potential components that may introduce an immune/inflammatory response  from native tissues using methods that do not disrupt the strong physical and/or chemical interactions in the native ECM , methodsin vivo cell delivery; control over the pore size and shape is also very challenging with these methods, creating a situation in which cells in different parts of the scaffold receive different cues from their microenvironment. Photopatterning  bioprinting is an additive manufacturing process that creates highly-complex tissue constructs using the layer-by-layer deposition of biomaterials onto a computer-controlled build platform . 3D biopIn extrusion bioprinting, a hydrogel or hydrogel precursor bioink is extruded out of a narrow print-tip such as a needle or conical nozzle that is moving through space to build a 3D structure layer by layer. The flow of the hydrogel can be driven by pneumatics  or mechain vitro by mixing alginate with HA to allow for rapid ionic crosslinking immediately after printing while maintaining a HA-rich hydrogel phase   or microIn digital light processing (DLP) bioprinting, a photosensitive hydrogel is crosslinked by patterns of light in a layer-by-layer approach. Typically, the build platform is lowered into a hydrogel precursor solution placed on top of a transparent substrate and a light source. A thin layer of the hydrogel is exposed to a light pattern, the build platform is raised a small distance, and the process is repeated until the print is complete. The light pattern for each layer can be produced by a photomask , projectSoft hydrogels provide a favorable environment for cells to grow, proliferate and differentiate ; howeverin situ application of polymeric nanofibers , poly (lactic acid) (PLA) and poly (vinylidene fluoride) (PVDF)) in the clinic for wound healing and other applications  and subsequently exposed to 10 min of UV irradiation (enabling secondary GelMA photo-crosslinking). High cell viability (>90%) could be maintained coupled with an 8-fold increase of cell number in human iPSC-derived ventricular cardiomyocytes in 3D culture over 2 weeks of observation  a secondary crosslinking step toward producing a multi-crosslinked hydrogel network . For exaervation .In vivo work showed that implantation of the GelMA scaffolds in rats enabled decreased glial scar tissue formation, increased vascularization, and increased neuronal development compared to electrospun gelatin fibers crosslinked with glutaraldehyde. The degree of crosslinking can also be tuned by adjusting the UV-light exposure time post-fabrication. in vitro) is recommended.For soft tissues, photocrosslinking can be used directly to form the hydrogels. For example, GelMA dissolved in the solvent hexafluoroisopropanol (HFIP) was reported to fabricate aligned electrospun nanofibers that were subsequently submerged in anhydrous alcohol containing Irgacure 2959 and crosslinked under UV light for 60 min to form hydrogel nanofibers . In vivoElectrospun hydrogels can also be formed by chemically crosslinking the polymer components via covalent bond forming chemistries. Common chemical crosslinking agents include N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride , genipin (for crosslinking aminated polymers), glutaraldehyde , and glyoxal (for crosslinking hydroxylated polymers) . Such crChemical crosslinking with GTA has also been explored in comparison to the physical crosslinking method of dehydrothermal treatment (DHT) for fabricating an electrospun collagen scaffold . The tril-arginine to form hydrogel fibers with the PVA component chemically crosslinked through the addition of anhydrous citric acid prior to electrospinning. CNC addition serves to increase the mechanical strength of the fibers while l-arginine promotes ECM collagen synthesis, angiogenesis, and epithelialization, promoting improved cell viabilities when cells were seeded on the scaffold. The scaffold also had anti-microbial properties related to l-arginine release and faster wound closure times compared to controls. However, the inclusion of multiple components in the electrospinning solution that may cross-interact in multiple ways can pose challenges with controlling the precursor solution viscosity and thus the uniformity of the electrospun hydrogel.Covalent chemical crosslinking can also enable more efficient entrapment of additives into the electrospun hydrogels without significantly compromising the crosslinking and mechanical strength of the scaffold. For example, 2O2  scaffold electrospun in hexafluoroisopropanol (HFIP) was crosslinked via enzymatic oxidation of the HPA moieties with the addition of horseradish peroxidase and H2O2 . Enzymatetration ; howeverin situ-gelling polymer pairs are co-delivered through a double-barrel syringe directly into the electrospinning process. For example, While the use of organic solvents can be a useful tool in the formation of electrospun nanofibers due to the ease of solvent evaporation between the nozzle and the collector, emerging studies have focused on electrospinning polymer solutions in water to remove the need for solvent removal before cell seeding and do not risk exposure of the seeded cells to cytotoxic crosslinking chemicals or photoinitiators. Altered experimental setups and polymer chemistries have both been explored as methods to reduce the need for electrospinning with organic solvents. First, the use of sacrificial sheaths can be used. in situ around the cells during electrospinning  in terms of structuring different cell types within a single fabricated scaffold. The reactive electrospinning technique  offers pspinning , 2020.A few other examples cell electrospinning have been reported using post-crosslinked nanofiber scaffolds, although successful cell electrospinning remains somewhat uncommon. In one such example, C2C12 cells were suspended in a mixture of fibrinogen and the electrospinning aid PEO and electrospun into a collection bath containing thrombin to crosslink the fibers into fibrin  Guo et . Cell vi2)  . Howeverin situ tissue engineering involves the injection of hydrogels  to the defect site to direct regeneration using a minimally-invasive technique  is most common given that in situ gelation facilitates local injection of the hydrogel without the need for transplantation  to maintain the viability and function of encapsulated cells; 2) to reproduce the target tissue morphology, including its mechanical profile and adhesion to surrounding tissues; and 3) to tune the degradation rate to ensure that complete tissue replacement can occur on a clinically-relevant timescale  printing  uses a near-infrared pulsed laser beam coupled to a scanning mirror and focusing system to enable high cell printing resolution and precision   . The inhin situ tissue engineering approaches to create skin substitutes, though only a few studies have been reported. Hakimi et al. developed a portable handheld extrusion-based skin printer that allowed for the co-printing of both ionically crosslinked biomaterials  and enzymatically crosslinked protein  with dermal and epidermal cells to form cell-laden sheets of consistent thickness, width, and composition  may further improve the efficacy of this therapeutic option.Skin is the largest and most accessible organ and has thus become an ideal platform to validate position   (in situ via ultraviolet (UV) photocrosslinking in the chondral defect of a sheep knee joint   is curedee joint . The in slinking .® is a bovine collagen-I hydrogel scaffold loaded with human neonatal fibroblasts that is covered on one side by a stratified layer of neonatal keratinocytes  but are generally very expensive given the extensive purification steps required to ensure sufficiently consistent batch-to-batch manufacturing of non-inflammatory decellularized matrix. A cell-loaded alginate-based matrix is the only currently non-ECM derived approved tissue regeneration hydrogel (AlgiMatrix®) but to-date is only used for 3D tissue models for in vitro drug screening. As such, despite the massive developmental efforts invested to-date in the design and fabrication of tissue engineering-based hydrogels, the translation of such hydrogels remains slow.To be appropriate for clinical translation, a hydrogel must be biocompatible and have suitable mechanics and an appropriate degradation profile while addressing key biological challenges  and logistical challenges  associated with the targeted application. The majority of clinically approved injectable or implantable hydrogels are targeted for skin or joint regeneration or drug delivery applications . Table 3inocytes . This tiinocytes . The hydinocytes . A studyraftment ; howeverf action . A cliniol group , suggest® and Bulkamid®.Clinically approved hydrogel therapies that do not include cells fall into four broad categories: natural hydrogels for cosmetic applications , natural® is a 1,4-butanediol diglycidyl ether (BDDE)-crosslinked hyaluronic acid (HA) gel clinically approved in 2006 in the United States for aesthetic treatment of dermal wrinkles and folds (Juvédermnd folds . Pre-clind folds  and the nd folds  are suitnd folds . Slight nd folds . Clinicand folds  as well ® is an injectable synthetic polyacrylamide hydrogel approved by the FDA for use in the United States in 2020 to treat stress urinary incontinence (SUI) by functioning as a bulking agent to add a total volume of up to 2 ml to the walls of the urethra  are key parts of the functional product. These barriers, and the regulatory challenges in dealing with regenerative tissue scaffolds, have been recognized by the FDA, which in 2017 acknowledged that as we enter a “new era of 3D printing of medical products regulatory issues related to the bioprinting of biological, cellular and tissue-based products” will have to adapt as the applications and practical implementations of tissue engineering continue to drift from the definitions of traditional therapies . This emWhile the majority of discussion on translation has focused on the materials design, the cell sourcing and production of large number of functional cells for practical translation is perhaps an even more challenging problem given the limited potential of many cell types to be expanded in a lab and the potential for non-host transplants to induce potential immune response for patients. The ability of tissue engineering scaffolds to fully recapitulate the full complexity of the microenvironment some more sensitive cells need to function is also to-date weakly addressed in the area, at least using scalable methodologies for scaffold manufacturing. While not the main focus of this review, it is critical that methods to use the patient’s own cells  and/or to expand immune-compatible cell lines are developed and scaled to ensure consistency in the cell responses and thus reliable tissue regeneration responses.Hydrogels as tissue engineering scaffolds offer enormous potential for clinical translation given their clear advantages in maintaining a hydrated environment for cells, mimicking the mechanics of soft tissues, and facilitating the presentation of appropriate interfaces and cell cues to promote tissue regeneration. However, the slow translation of these materials to the clinic goes beyond simple regulatory barriers. For natural polymer-based hydrogels, the effective scale up and management of batch-to-batch differences in natural polymers remains a huge challenge, as is addressing the often weaker mechanical properties of natural polymer-based hydrogels for promoting proper signaling to regrow stiffer tissues. For synthetic polymer-based hydrogels, challenges around degradability can be limiting . Combining natural and synthetic polymers into a single scaffold may offer benefits to leverage the benefits (and dilute the drawbacks) of each individual polymer type, although the inclusion of multiple materials is also likely to further complicate the regulatory process., cell electrospinning, and in situ tissue engineering) in our view offer the most practical ways forward to commercialize cell-loaded tissue engineering hydrogels given that they all streamline multiple fabrication steps, make maintaining sterility much easier, and could in principle be applied directly in the operating room. However, each method still offers challenges, particularly in terms of broadening the types of materials that can be used for scaffold fabrication. For bioprinting, a relatively small number of bioink options is currently available due to the requirements of printing and the need to maintain precise crosslinking kinetics to fix the printed shape in place, although the emergence of embedded printing approaches  can help to offset some of these challenges. The still relatively low feature resolution printable with high-throughput printing strategies like extrusion also offers a limitation of current bioprinting strategies for reproducing the nanofibrous structure of native ECM. Cell electrospinning can provide similar feature sizes to native ECM  essential to provide cues for cell adhesion and proliferation but involves the exposure of cells to a strong electric field and  induces at least some degree of cell dehydration as the electrospun scaffolds are collected, both of which apply mechanical and/or osmotic stresses on cells that may compromise the viability of particularly more sensitive primary cell lines. Electrospinning directly on tissues  or into cell media instead of on dry collectors may address some of these challenges, while minimization of the applied voltage can ensure as high as possible cell membrane viability during the electrospinning process. In situ tissue engineering approaches offer a mechanism to directly administer cells and hydrogels into the body via injection but are more difficult to fabricate into controlled internal microstructures and morphologies optimized for cell growth. Methods to perform in situ gas foaming using biologically-safe foaming agents, incorporate sacrificial pore-forming materials that can be dissolved or otherwise in situ degraded over time to introduce tunable porosity, and/or provide alignment cues  all off


Background:Modeling of single cell RNA-sequencing (scRNA-seq) data remains challenging due to a high percentage of zeros and data heterogeneity, so improved modeling has strong potential to benefit many downstream data analyses. The existing zero-inflated or over-dispersed models are based on aggregations at either the gene or the cell level. However, they typically lose accuracy due to a too crude aggregation at those two levels.Results:We avoid the crude approximations entailed by such aggregation through proposing an Independent Poisson Distribution (IPD) particularly at each individual entry in the scRNA-seq data matrix. This approach naturally and intuitively models the large number of zeros as matrix entries with a very small Poisson parameter. The critical challenge of cell clustering is approached via a novel data representation as Departures from a simple homogeneous IPD (DIPD) to capture the per-gene-per-cell intrinsic heterogeneity generated by cell clusters. Our experiments using real data and crafted experiments show that using DIPD as a data representation for scRNA-seq data can uncover novel cell subtypes that are missed or can only be found by careful parameter tuning using conventional methods.Conclusions:This new method has multiple advantages, including (1) no needfor prior feature selection or manual optimization of hyperparameters; (2) flexibility to combine with and improve upon other methods, such as Seurat. Another novel contribution is the use of crafted experiments as part of the validation of our newly developed DIPD-based clustering pipeline. This new clustering pipeline is implemented in the R (CRAN) packagescpoisson.


Motion sickness is characterized by nausea and vomiting among a constellation of symptoms. Symptom severity is dynamic and distressing. Most validated motion sickness scales are time-intensive and effortful, with alternative scales having uncertain performance or non-specific measures. A validated instrument allowing for facile, rapid assessment of core motion sickness symptom severity would therefore be valuable. We assessed the performance of the Motion Sickness Severity Scale (MSSS), a six-item questionnaire designed to measure real-time motion sickness symptoms.MSSS construct validity was assessed as a secondary analysis of data from 63 healthy participants without antiemetic treatment in a clinical trial (Unique Identifier = NCT03772340) conducted to evaluate the safety and efficacy of Tradipitant—a novel neurokinin-1 receptor antagonist—in the treatment of motion sickness. Clinical outcome assessments included the MSSS, the Patient Global Impression of Severity (PGI-S), and the Motion Sickness Assessment Questionnaire (MSAQ). The performance of the MSSS through Pearson correlation coefficients, within-group analysis of variance, empirical cumulative distribution functions, and Kolmogorov-Smirnov tests.The MSSS correlated very highly with the PGI-S  and highly with the MSAQ . Mean MSSS scores between increasing PGI-S severity levels increased significantly in all four increments . There were statistically significant differences in MSSS score distributions stratified by PGI-S severity level, with higher MSSS scores associated with higher PGI-S severity levels and lower MSSS scores associated with lower PGI-S severity levels.The MSSS is a valid instrument for the assessment of the core motion sickness symptoms and is reflective of global disease severity. Implementation of the MSSS and comparable simplified, short questionnaires in motion sickness research will provide rapid and accurate measures of disease severity. These measures will enable further elucidation of motion sickness as an illness and inform the development and evaluation of motion sickness therapies. Several questionnaires have been developed to assess motion sickness , which aThe Motion Sickness Severity Scale (MSSS), previously used in an efficacy study of Hyoscine (Scopolamine) , would met al. published a study that included assessment of the MISC [Similarly, the 11-point Motion Illness Symptoms Classification (MISC) scale, which was also developed to assess motion sickness symptomatology during stimulus presentation, has been employed prior to its performance evaluation . Encourathe MISC . Using dTherefore, in this secondary analysis of data from a clinical efficacy study (Motion Sifnos) for the development of Tradipitant (a novel neurokinin-1 antagonist) for the treatment of motion sickness , we sougThis manuscript reports finding from a secondary analysis of data collected in the Motion Sifnos Study . ConstruA total of 126 participants  were recruited from the greater Los Angeles, California, USA area through radio and online streaming advertisements. Participants completed the Motion Sickness Eligibility Questionnaire (MSEQ), a phone interview, and an in-person medical screening. Screening for participation criteria included medical and psychiatric history, physical examination, electrocardiography, serum chemistry, hematology, urinalysis, and urine toxicology. Participants who met the inclusion criteria had a significant history of motion sickness, were otherwise in good health, and had no history of any other nausea-inducing disorders. The institutional review board at Advarra  reviewed and approved the study protocol and provided ethical oversight for the study. All participants provided written informed consent prior to enrollment, and all methods were carried out in accordance with relevant guidelines and regulations.Motion Sickness Severity Scale (MSSS)–The MSSS is a self-reported clinical outcome assessment that evaluates the severity of motion sickness symptoms, including the core symptoms nausea and vomiting. In 30-minute intervals throughout the duration of the boat trip, participants rated the severity of their motion sickness symptoms on a 7-point scale: no symptoms, stomach awareness or discomfort, mild nausea, moderate nausea, severe nausea, retching, or vomiting .Patient Global Impression of Severity (PGI-S)–The PGI-S is a self-reported clinical outcome assessment that evaluates the global severity of a sickness or disease. Approximately 60 minutes following the boat trip, participants rated the severity of their motion sickness on a 5-point scale: none, mild, moderate, severe, or very severe .Motion Sickness Assessment Questionnaire (MSAQ)–The MSAQ is a self-reported clinical outcome assessment that evaluates the severity of motion sickness with specificity for individual symptoms that may be experienced. Approximately 60 minutes following boat travel, participants rated their degree of agreement with each of 16 items (statements) about different motion sickness symptoms on a 9-point scale from “Not at all” to “Severely” . The MSAEligible participants were randomized to take Tradipitant 170mg or placebo approximately 60 minutes prior to the initiation of boat travel. Groups of between 10 and 26 participants each participated in one of seven boat trips lasting between 237 and 250 minutes, except where extreme sea conditions limited one trip (Boat 5) to 148 minutes. Boat travel occurred in the Pacific Ocean near Los Angeles between January and May 2019. Participants rated their motion sickness symptom severity using the MSSS in 30-minute intervals throughout the trip. Participants also completed the PGI-S and MSAQ approximately 60 minutes after the conclusion of the trip. Additional information about the study procedure is available in the study efficacy report [To eliminate treatment effects of neurokinin-1 receptor antagonism (which has demonstrated anti-nauseogenic and antiemetic properties) as a potential confounder of validity testing, the Tradipitant group was excluded from the primary evaluation of the validity of the MSSS. The decision to conduct the primary evaluation using data from healthy participants without antiemetic treatment is supported by a demonstrated treatment effect, including significantly reduced vomiting observed in the Tradipitant group compared to placebo  . As an eStatistical analyses performed included Pearson correlations, one-way analysis of variance (ANOVA), empirical cumulative distribution functions (ECDF), and two-sample Kolmogorov–Smirnov tests (K-S test). To evaluate the relationship between mean scores across questionnaires, analyses were anchored to the global PGI-S and compared to the motion-sickness-specific questionnaires (MSSS and MSAQ).There were four core elements of the validation study. Within the analytic sample , first, Pearson correlation coefficients were calculated to estimate the linear association between MSSS scores and each of PGI-S and MSAQ scores. Second, in subpopulations stratified by the five PGI-S severity levels , within-group differences in mean MSSS and MSAQ scores were evaluated using a one-way ANOVA . Third, There were two additional exploratory analyses of the validation study. First, we evaluated the Pearson correlation between the MSSS and subscales of the MSAQ, including items conceptually directly related to MSSS scores  and the group of gastrointestinal MSAQ items. Second, we calculated Pearson’s correlation of MSSS and PGI-S and MSAQ scores on the overall Motion Sifnos Study population . The purpose of this analysis was to evaluate whether correlations differed with the expanded study population that included a group on an investigational antiemetic therapeutic.The significance level of all tests was set at α = 0.05 given that the statistical tests were performed for different purposes and nonduplicative. Multiple statistical measures were used without thresholds to characterize the performance of the MSSS relative to established measures.Pearson’s correlation coefficient is a measurement of association between two variables. The strength of association between variables is described by -1.0≤r≤+1.0 where -1.0 represents a perfect negative correlation and +1.0 represents a perfect positive correlation. Pearson’s correlation was chosen rather than Spearman’s correlation to assess the relationship between continuous rather than ranked scale scores. Using guidelines established for appropriate use of correlation for medical research, a threshold of r ≥│0.70│was used to interpret correlations as high (│0.70│≤r ≤│0.90│) or very high (r>│0.90│) .A one-way analysis of variance (ANOVA) was used to compare MSSS (or MSAQ) in subgroups corresponding to consecutive and ascending PGI-S severity levels. Statistically significant increases in mean scores accompanying ascending PGI-S levels would be indicative of an association between the MSSS (or MSAQ) and the validated questionnaires.x, the ECDF value at x is the percentage of observations that are less or equal to x. ECDFs of MSSS scores were generated for subpopulations corresponding to each PGI-S severity level. When comparing ECDFs, increasingly right ECDFs correspond to stochastically greater values. Non-parametric kernel smoothing was applied to ECDF curves.An empirical cumulative distribution function (ECDF) of a sample is an ascending function ranging from 0 to 1 over all possible values. At each point The Kolmogorov-Smirnov test (K-S test) serves as a nonparametric test to compare two samples. K-S tests were applied to MSSS scores corresponding to increasing PGI-S severity levels. The underlying hypothesis of the K-S test is that the two ECDFs were generated from the same population-level Cumulative Distribution Function (CDF). Statistically significant K-S test results between MSSS score ECDFs at increasing PGI-S severity levels would indicate that MSSS scores between PGI-S severity levels were likely sampled from different distributions and that MSSS scores are differentiated by PGI-S severity level.Included in the analysis were 62 healthy participants without antiemetic treatment who completed the PGI-S, MSAQ, and MSSS per the study protocol. Paired questionnaire scores of the MSSS and the PGI-S, MSAQ, and MSAQ-GI from individual participants are presented in scatterplots as panels A, C, and D of MSSS scores correlated very highly with scores on the PGI-S  , and higMSSS scores increased significantly with increasing PGI-S severity levels across all four increments . For exaIn the exploratory analysis of the overall Motion Sifnos Study population (treatment and placebo groups), the correlation remained high between the MSSS and the PGI-S  , as wellEvaluation of the construct validity of the MSSS was motivated by the desire for a reliable diagnostic instrument to assess the severity of motion sickness with the following elements: measurement of core symptoms, brevity, clarity of scale items for undemanding completion, potential for repeated and rapid assessment of acute motion sickness severity during stimulus presentation (including when the respondent is distressed), and scalability. To evaluate the MSSS with attention to these elements, the scale was compared to two validated assessments: a brief questionnaire (PGI-S) used as an index of global illness severity, and a comprehensive questionnaire (MSAQ) used as an assessment of the multiple dimensions of motion sickness. This analysis of the construct validity of the MSSS provides evidence that the MSSS correlates strongly with validated and widely used questionnaires and is therefore a valid instrument for the assessment of motion sickness.Scores on the MSSS correlated very highly with scores on the PGI-S, and sufficient differentiation is supported by the within-group ANOVA demonstrating significant increases in mean MSSS scores with increasing PGI-S severity level. The order and shape of the PGI-S ECDFs reveal that grouped MSSS score distributions indicate that higher PGI-S severity levels are associated with higher MSSS scores, and lower PGI-S severity levels are associated with lower MSSS scores. This analysis suggests that the MSSS accurately measures motion sickness severity progression from no symptoms to stomach awareness and eventually to severe nausea and vomiting , 10. TheSimilarly, scores on the MSSS correlated highly with scores on the MSAQ. The high degree of association of scores between the questionnaires supports the construct validity of the MSSS based on the MSAQ, which was designed to capture the highly variable symptom profiles of motion sickness and validated against two widely used motion sickness severity assessment instruments: the Pensacola Diagnostic Index and Nausea Profile . Scores et al. in evaluating the 11-item MISC suggest that with simplified and shorter questionnaires than the MSAQ, the MSSS and MISC assess motion sickness severity and reflect global disease state by focusing on core symptoms. This distinction highlights differences in purpose for questionnaires. The MSAQ can capture the multiple dimensions of motion sickness symptoms that may be experienced. The MSSS can rapidly and acutely measure severity of the core symptoms of motion sickness in a single-item questionnaire. The MISC provides yet another viable scale to assess motion sickness symptom severity, with enhanced resolution during pre-nauseated states [Together, the findings of this paper in evaluating the 7-item MSSS and those of Reuten d states . Future Of note, the MSAQ was validated based on motion sickness ratings from 21 students following exposure to a rotating optokinetic drum . While tThis evaluation of the validity of the MSSS was performed on data collected in a single study in which participants with a history of motion sickness were exposed to different sea conditions and randomized to take Tradipitant or placebo as part of the Motion Sifnos Study, with the primary analysis focused on participants without antiemetic treatment. While these environmental and interventional variables may increase the variability in questionnaire scoring as potential confounders, results from the exploratory analysis further support the MSSS for assessing motion sickness. The robustness of scores with the inclusion of these variables indicates that the utility of the MSSS may extend across populations, interventions, and stimuli.The findings reported in this study establish the MSSS as a highly performing instrument for the assessment of motion sickness designed for use during stimulus presentation, presenting motion sickness researchers with a valuable symptom-screening instrument comparable to the recently evaluated MISC. Further research using the MSSS for repeated assessment of motion sickness severity in larger populations with exposure to variable provocative stimuli will further inform its utility. Going forward, the MSSS can serve as a valuable instrument—in both clinical and research settings—that can be administered to assess motion sickness severity and reflect global state of disease.


HIV-1 controllers comprise a heterogeneous group of people living with HIV-1 (PLWH) with the ability to maintain low viral loads and high CD4+ T cell counts for years in the absence of antiretroviral therapy (ART). Within these is a special group of individuals termed “elite controllers” (ECs) who durably maintain undetectable plasma HIV-1 RNA levels without medication. Immune profiles in these rare individuals have been proposed as a model to guide the development of a functional HIV-1 cure. Several protective immune signatures have been linked to better HIV-1 control but host factors described thus far only partly explain effective HIV-1 control . In parthi phenotype  cells, macrophages and natural killer T cells, the secretion of soluble cytokines and signaling pathways are emphasized and deemed critical to the natural control of HIV-1 infection. The involvement of both arms of the immune response described above can be demonstrated by the contribution of DCs to the enhancement of the overall HIV-1 specific T cell immunity observed in ECs  activity earlier in infection. Reports suggest that ECs can maintain high levels of potent gp120 specific antibodies with ADCC or antibody dependent NK cell activation (ADNKA) function in the absence of detectable virus  that triggers an epigenetic and metabolic reprograming, allowing them to provide an enhanced response to a subsequent exposure to the same or another unrelated agent . HIV-1-itrollers , 19. TheOverall, the articles presented here provide an important basis and mechanistic view of how innate immune responses may partner with adaptive immune responses to contain the virus in HIV-1 controllers. Some questions remain regarding the contribution of the innate immune response to the high levels of chronic inflammation and the risk of non-AIDS-related conditions in ECs. It is possible that high levels of immune activation from low-level viral replication trigger sustained immune activation and a chronic inflammatory state that would be beneficial in the early stages of HIV-1 infection for disease containment but detrimental in the chronic state. Therefore, initiation of ART could be a possible solution to avert effects of chronic immune activation in specific populations of ECs. Given that it would be challenging to identify EC populations at high risk of comorbidities, further investigations are required to determine whether treatment should be offered to all EC groups to alleviate the risks.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.