All participants received NSPT, whereas the patients in the EG also received one-on-one 30 min lessons from a CHW over 4 weeks. The EG exhibited greater improvement in the probing pocket depth  and clinical attachment level  at 1-month follow-up than the CG did. The ES increased over the 1-, 3- and 6-month follow-ups, indicating an increase in OHQoL  in the EG. The patients in the EG were more likely to change their oral self-care behaviors than patients in the CG were. The NSPT with CHW strategy had a positive effect on 1-month periodontal treatment outcomes, long-term OHQoL and oral self-care behaviors in patients with T2DM.Interventions engaging community health workers (CHW) for diabetes management aim to improve diabetes care and self-management behaviors among patients. We evaluated the effects of nonsurgical periodontal treatment (NSPT) with the CHW strategy on oral self-care behaviors, periodontal status and oral health-related quality of life (OHQoL) in patients with type 2 diabetes mellitus (T2DM). The participants were randomly assigned to experimental (EG;  Diabetes has become one of the most pressing health issues worldwide. In Taiwan, the prevalence of diabetes among patients aged 20–79 years was approximately 10.1% in 2014, and it has increased annually ,4. For pUnlike health educators, known as health education specialists, community health workers (CHWs) are lay people who have in-depth knowledge of the communities they serve, and they usually provide a link between the community and healthcare professionals. The advantages of the CHW strategy include its ability to make up for the shortage of health care professionals in rural communities, and increase the acceptance of patients toward health care or health education. CHWs have a similar culture, lifestyle and language to those of residents; therefore, a wide range of work may be implemented by CHWs ,15. The The American Diabetes Association recommend that patients with diabetes should take good care of their teeth and gums and should go for regular checkups every 6 months . A studyA randomized experimental design was used. This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (reference numbers: KMU-IRB-20140291). We obtained informed consent from all participants. The study was also registered on the ISRCTN Registry (reference number: ISRCTN17756516). We recruited participants from the endocrinology and metabolism divisions of Kaohsiung Medical University Hospital, Kaohsiung Municipal Ta-Tung Hospital, and Kaohsiung Municipal Hsiao-Kang Hospital, Taiwan, from 2015 to 2018. Patients aged 35–65, having severe periodontal status  with at f = 0.25  ohen’s f ). The toohen’s f , was 30.We drew random numbers from a paper bag to divide the participants into the experimental group  and control group . A double-blind experimental design was used; participants did not know which group they were assigned to, and the dentists providing NSPT were also blinded to the participant grouping.W was 0.929. Glycated hemoglobin (HbA1c) was collected by research staff from the electronic medical records system. A questionnaire survey was conducted to measure OHQoL, oral self-care behaviors, oral health-related knowledge and attitude toward periodontal health. The questionnaire was modified from that used in our previous study [Periodontal data was collected using the hospitals’ routine periodontal chart with an additional chart designed for the study. We used Kendall’s coefficient of concordance to test the interrater reliability of periodontal examination among three dentists prior to the study, and Kendall’s us study  and was Five periodontal parameters were measured, namely probing pocket depth (PPD), clinical attachment level (CAL), gingival index (GI) , plaque HbA1c reflects average glycaemia trends over the previous 8 to 12 weeks . We collOHQoL was measured using the Taiwanese version of the 14-item Oral Health Impact Profile-14 (OHIP-14T). The OHIP-14T comprises seven domains: functional limitation, physical disability, physical pain, psychological discomfort, psychological disability, social disability and handicap. Participants respond on the following scale: very often = 4, often = 3, occasionally = 2, rarely = 1 and never = 0. Total scores range from 0 to 56; lower scores indicate a better OHQoL. The evaluation period of OHQoL was the year before treatment (baseline) and each follow-up after treatment.Patients were asked questions regarding their tooth brushing method and interdental brush use. Brushing method was coded as 0 (other methods) or 1 (modified Bass brushing technique), and interdental brush use was coded as 0 (no) or 1 (yes).Thirteen statements were used to measure oral health-related knowledge, including “People with diabetes are more likely to have gum disease” and “It’s better to choose the bigger size interdental brush”. Possible responses included true (1), false (0) or unknown (0), with possible scores ranging from 0 to 13; higher scores indicated a greater degree of oral health-related knowledge. The Kuder–Richardson 20 coefficient was 0.74 for this scale.Seven statements were used to measure attitude toward periodontal health, including “The condition of our teeth can influence our dietary intake” and “I think periodontal disease is serious”. Each statement was evaluated using a 5-point Likert scale from 1 (strongly disagree) to 5 (strongly agree), with possible scores ranging from 7 to 35; higher scores reflected more positive attitudes toward periodontal health. Cronbach’s α was 0.72 for this scale.Demographic characteristics encompassing five variables, namely gender, age, duration of diabetes, body mass index (BMI) and education level, were assessed at baseline for each participant in this study.Four CHWs were recruited from the community by using flyers and social media. We conducted a 4 h training course containing a curriculum on periodontal disease and care, teaching and communication skills. The CHWs received a training manual reviewed by a panel of experts. The training manual described the goals and contents of each lesson in detail. All four candidates passed the test and became certified CHWs. The research staff maintained contact with CHWs to address any difficulties in reading the training manual during the month prior to the intervention.The T2DM patients in the EG received the NSPT and periodontal care curriculum taught by CHWs. The curriculum included four 30 min, one-on-one lessons taught by CHWs during NSPT. The teaching dates and locations were determined according to the participants’ convenience. To encourage their effort and participation, the CHWs received USD 70 upon completing the curriculum with each patient. The primary goal of the lessons was to provide information in the selection of tooth-brushing tools, correct toothbrushing methods based on the Bass and modified Bass technique and periodontal care related to diabetes mellitus . Standardized lecture slides were prepared based on the curriculum and presented on a tablet computer. The tablet computers featured the ability to enlarge the slides to help patients understand the course content and a built-in camera function to help patients check their cleaning condition after using a plaque disclosing agent. A set of toothbrushing tools comprising a soft-bristled toothbrush, fluoride toothpaste, dental floss, an interdental brush, a mouthwash cup and fluoride mouthwash was offered to participants to help them learn how to select and use toothbrushing tools. A working log was designed for CHWs to record the implementation of the intervention and the degree of participants’ cooperation. Using the working log, the CHWs and researchers could evaluate the patients’ understanding of the intervention process and the effectiveness of teaching in each course to ensure that the participants achieved the course goals.The patients with T2DM in the control group (CG) received only NSPT. A simple leaflet presenting information on the relationship between periodontal disease and diabetes was provided to patients in the CG at the end of the study. The NSPT included full-mouth scaling, root planning and oral hygiene instructions (OHI). The course of treatment was completed in 1 to 2 months, depending on patients’ periodontal status. Routine OHI based on personal oral conditions were provided by a dental hygienist, including tooth brushing technique, flossing skills and interdental aids.Data were collected by trained and certified study personnel at four time points, namely at baseline and at the 1-, 3- and 6-month follow-ups. At baseline, our staff assisted participants in completing the self-administered questionnaire, and patients were then appointed to receive oral examination by dentists to obtain the periodontal parameters. The participants completed an identical questionnaire and oral examination at the 1-, 3- and 6-month follow-up appointments. The HbA1c data were collected at baseline and at the 3- and 6-month follow-ups by staff using an electronic medical records system under the authority of the endocrinology and metabolism doctor.t-test were used to compare the demographic variables of the EG and CG. A logistic regression model using generalized estimating equations (GEEs) analyzed the change in oral self-care behaviors between baseline and the follow-up appointments. Comparisons of periodontal parameters, OHQoL, HbA1c, knowledge, and attitude between the EG and CG were analyzed with a linear regression model using GEEs. The intervention effects were adjusted for age, gender, educational level, BMI and T2DM duration. The ES (Cohen’s d) of continuous variables were calculated from the mean difference between baseline and the follow-ups within the EG and CG. An effect of 0.20 is small, 0.50 is medium and 0.80 is large [This study explored the relationships among the variables by using STATA version 13.0 . A chi-squared test and two-sample is large .The distribution of sociodemographic characteristics between the EG and CG are shown in The changes in oral health-related knowledge, attitude toward periodontal health, and oral self-care behaviors at different stages are presented in We converted the 5-point scale of the satisfaction survey into a 100-point scale, as shown in This is the first study employing the CHW strategy to improve daily oral self-care behaviors in patients with T2DM, with the aim of improving treatment outcomes.We observed a greater improvement in 1-month short-term clinical outcomes with respect to PPD and CAL in the EG than in the CG. Using the CHW strategy in the EG improved patients’ toothbrushing technique and interdental brush use in home care at the 1-month follow-up, which may contribute to improved clinical outcomes after periodontal treatment. Moreover, the positive changes in oral health-related knowledge and attitude toward periodontal health at all follow-ups were higher in the EG than in the CG.In the present study, no differences were found between EG and CG in OHQoL at 3-month and 6-month follow-ups. However, both EG and CG exhibited improvement in OHQoL at 3-month and 6-month follow-ups. An increase in ES in OHQoL from 1-month to 6-month follow-ups was observed in the EG and the ES was medium at 3-month and 6-month follow-ups. This positive effect of using CHWs is consistent with the findings of previous studies focused on different health issues ,18,19. TA greater improvement in PPD and CAL was observed in the EG than in the CG at the 1-month follow-up. Relatively large ESs in PPD, CAL and BOP were also noted in the EG than in the CG. This observation may have occurred because the health education provided by diabetes educators to patients does not include adequate content related to periodontal care; one previous study even indicated that most education curricula from diabetes educators do not include an oral health module . AlthougIn this study, the patients in the EG demonstrated good effects on changes in knowledge, attitude and behaviors. This finding is consistent with previous studies using CHWs for other health issues ,17,18. PIn our study, the average HbA1c level of neither group showed clinical improvement at follow-up after patients received NSPT; this finding is consistent with that of a previous study . AccordiThe present study has several limitations. First, the attrition rate of the entire study was 15.8%  from baseline to 6-month follow-up; however, a dropout analysis using baseline data indicated no difference in demographic characteristics between dropouts and participants. Second, maturation bias may have occurred because the teaching skill of the CHWs may have improved over time. Patients who received curriculum later may receive more proficient teaching. However, this bias was potentially limited by the standardized design of our curriculum. Third, social desirability may influence results, especially in the EG. Patients might provide responses that conform to social expectations rather than responses that accurately reflect their condition. Fourth, the patients were not placed under any special restrictions regarding daily diet, drug use or treatment methods related to their diabetes during the research period; these factors should be carefully controlled in future research. Finally, the participants were patients in a hospital; therefore, the generalization of findings to other T2DM patients should be carefully discussed.This study reported that NSPT with the CHW strategy improved short-term clinical treatment outcomes  and exerted a positive effect on long-term OHQoL in T2DM patients. The CHW strategy also had a positive effect on oral health-related knowledge, attitude toward periodontal care and oral self-care behaviors. Our study further suggests that in areas where health care professionals are scarce, CHWs should be employed to improve preventive oral  self-care behavior as part of diabetes management.


Modern vaccine design has sought a minimalization approach, moving to the isolation of antigens from pathogens that invoke a strong neutralizing immune response. This approach has created safer vaccines but may limit vaccine efficacy due to poor immunogenicity. To combat global diseases such as COVID-19, malaria, and AIDS there is a clear urgency for more effective next-generation vaccines. One approach to improve the immunogenicity of vaccines is the use of nanoparticle platforms that present a repetitive array of antigen on its surface. This technology has been shown to improve antigen presenting cell uptake, lymph node trafficking, and B-cell activation through increased avidity and particle size. With a focus on design, we summarize natural platforms, methods of antigen attachment, and advancements in generating self-assembly that have led to new engineered platforms. We further examine critical parameters that will direct the usage and development of more effective platforms. Particles in this size range can significantly influence biological systems, driving the expansion of nanoparticle research in biology and medicine. In vaccinology, nanoparticles serve three major roles: as adjuvants, carriers, or presentation platforms, determined by how the vaccine antigen interacts with the nanoparticle  classifies a nanoparticle as a material or substance that has been deliberately manipulated to have dimensions between 1 to 100 nm or up to 1000 nm if it exhibits physical, chemical, or biological effects dependent on its size14. In contrast, protein-based platforms are highly biocompatible, can assemble homogenously, and can be effectively tailored to suit any antigen. Protein nanoparticles injected intravenously have been shown to freely travel through circulatory and lymph vessels with rapid accumulation in the thyroid and spleen, advantageous for establishing humoral immunity15. Furthermore, protein-based platforms enable antigen attachment through genetic fusion or affinity tags complexes, which allows for a homogenous decoration of antigens on the platform. The first protein platforms utilized virus capsid proteins isolated from their infectious components and exploited their naturally oligomeric nature to form stable nanoparticles called virus-like-particles or VLPs5. Subsequently, many naturally oligomeric proteins such as ferritin, lumazine synthase, and C4b-binding protein (C4bp) orthologs have been developed for platform design. These platforms vary in size, ranging from as little as 4 nm with C4-binding protein-like particles to as large as 120 nm with Influenza M1 VLPs6  followed by the activation of B-cells by antigen and a specialized T helper cell, the T Follicular helper (TFH)17. APCs phagocytize, process, and present antigens to T-cells, which if recognized, become activated to differentiate into T helper cells. Naive B-cells, which require two signals to mature into high-affinity IgG plasma cells, receive the first signal from the crosslinking of multiple B-cell receptors (BCR) with an antigen that has directly entered the lymph node or was presented by APCs, followed by endocytosis of the receptor and antigen, and then presentation of the processed antigen on the surface. The second signal is delivered upon recognition of the B-cell-presented antigen with a TFH cell, which activates the B-cell to mature into an IgG-producing plasma cell.The effectiveness of a prophylactic vaccine is determined by the generation of a long-lasting T-cell-dependent IgG antibody response18. This success has been attributed in part to the ability of the antigen, the major capsid protein, to self-assemble into highly oligomeric spherical VLPs. However, many antigens, such as those used in influenza subunit vaccines, do not self-assemble into nanoparticles. In these cases, self-assembly can be generated by attachment of these antigens to an oligomeric protein platform. Studies have shown that scaffolded antigen induce stronger and longer lasting neutralizing antibody titers, as well as greater protection24.Many successful vaccines in the past consisted of attenuated vaccines that were highly effective but were potentially infectious. This deficiency prompted the creation of inactivated components or subunit vaccines, for example those used in modern influenza vaccines. Subunit vaccines typically have excellent safety profiles by consisting of isolated antigens necessary to establish an immune response, and as a result may also be less immunogenic. However, vaccination with certain subunit vaccines such as those against Human papillomavirus (HPV), Cervarix and Gardasil, has seen remarkable success in protection26 , elongating retention in lymph follicles, and enhancing antigen presentation to B-cells27. Particles displaying numerous antigens can then facilitate B-cell activation through efficient crosslinking with multiple BCRs28 , suggesting an effect outside the total amount of antigen. In addition to enhanced B-cell activation caused by repetitive antigen surfaces, the immune system appears to be trained to recognize these surfaces, such as viral capsids and bacterial pili, as a non-self indicator30. Because of this effect, protein platforms have enabled self-antigen, that are normally dispersed, to break self-tolerance and mount an immune response31. The ability to break self-tolerance has facilitated the use of protein platforms in cancer vaccine development through the presentation of tumor-associated antigens (TAAs), which are self-antigens overexpressed in cancerous tissue but rarely observed in normal tissue31.Two characteristics of nanoparticle platforms contribute to generating the B-cell IgG response: (1) the attachment of the antigen to a larger scaffold, which improves APC uptake and retention in lymph follicles and 2) the repetitive array of antigens, which enables efficient binding and activation of multiple B-cell receptors , which contributes to the infection of HIV and Dengue, occurs when non-neutralizing antibodies bind and encourage infection of immune cells32. To counter this possibility a technique called epitope focusing has been used to design antigens that direct an antibody response to neutralizing epitopes. This technique that involves isolating neutralizing epitopes from antigens are oftentimes poorly immunogenic in isolation. However, when attached to a nanoparticle platform, the increased presentation allows these antigens to yield a strong yet targeted humoral response33. In addition to epitope focusing, T-cell epitopes introduced in the interior of the platform can be used to activate a cell-mediated immune response35. Incorporation of universal CD4 + T-cell epitopes can help establish a humoral response against the antigen through recruitment of helper-T cells while CD8 + T-cell epitopes can generate killer T-cells against a specific pathogen. The combination of these techniques may enable successful vaccination against difficult pathogens.Protein nanoparticles can help in the development of vaccines for immuno-evasive pathogens such as human immunodeficiency virus (HIV), influenza, and malariaThree different methods are used to attach antigens for presentation on nanoparticles: chemical conjugation, genetic fusion, and tag coupling Fig. . These m36  vaccines37, which conjugates polysaccharides of Hib to an immunogenic protein platform such as Tetanus toxoid. The established nature of this method has led researchers to explore vaccines that use conjugation to attach protein-based antigen. Decoration of Pseudomonas aeruginosa exoprotein A (EPA), an inherently immunogenic protein, with a malaria antigen has led to a 75- to 110-fold increase in specific antibody generation versus immunization without EPA40. For this case the antigen was lysine-treated and conjugated to the cysteine of EPA41. Unlike other methods, conjugation can be used to decorate monomeric proteins with numerous antigens. However, the uneven decoration of this method may lead to poor display of antigen and inefficient B-cell activation against neutralizing epitopes42. Nevertheless, chemical conjugation has been proven to significantly improve immune responses despite these supposed limitations.In a typical chemical conjugation used for antigen attachment to VLPs, the protein nanoparticle undergoes chemical treatment to crosslink surface-exposed lysines to engineered or surface-exposed cysteines of a target antigen36 Fig. . This me36.Genetic fusion and tag coupling can often provide greater precision through site-specific antigen attachment. Genetic fusion of the antigen to the nanoparticle platform is the most direct method Fig.  if the a44. This system uses Streptococcus pyogenes fibronectin-binding protein FbaB, which has been split in two components, SpyCatcher (113 aa) and SpyTag (13 aa). Nanoparticle platforms that are fused to a SpyCatcher form an irreversible peptide bond with the SpyTag fused to the antigen or vice versa and can be fused to either the N- or C-terminal. Attachment of two different antigens can be accomplished by combining SpyTag/SpyCatcher with another tag-coupling system such as SnoopTag/SnoopCatcher45. Tag coupling can result in a lower numbers of antigen per particle than genetic fusion, due to inefficient coupling46. In addition, the genetic fusion of a large protein  to the nanoparticle may interfere with the attachment of antigen. Ultimately, tag-coupling systems allow for rapid modular attachment of different antigens to a nanoparticle platform, at the cost of introducing more components.SpyTag/SpyCatcher has had the most extensive use in antigen presentation platforms2. VLPs were the first nanoparticles to be used in vaccines due to the relative ease of design that exploited naturally high stability and self-assembly. VLPs have seen extensive use in clinical trials and commercially approved vaccines as platforms and demonstrate a reliable proof-of-principle for protein platform technology.Virus-like particles (VLPs) are composed of self-assembling viral envelope or capsid proteins, ranging from 20 nm to 800 nm in diameter Table , which a47. RTS,S uses the hepatitis B virus surface antigen small-envelope protein (HbsAg) to self-assemble into a spherical nanoparticle48. HbsAg, which is the main component in currently approved hepatitis B vaccines49, forms the lipid viral envelope that encloses the capsid and is able to form noninfectious particles 22 nm in diameter50. Further structural characterizations suggests that an HbsAg VLP consists of 24 tetramers, with a rhombicuboctahedron-like shape, although exact composition may vary due to lipid nature51. Antigen attachment to the envelope in RTS,S is accomplished by genetic fusion of a malaria antigen to the N-terminus of HbsAg48. The fused HbsAg is then coexpressed with unmodified HbsAg to allow for nanoparticle formation, although nanoparticles consisting solely of fused HbsAg have also been created52. Vaccination with RTS,S confers moderate protection against malaria, and attachment of the antigen to the VLP has been credited with significantly increasing its efficacy53.The world’s first licensed and approved malaria vaccine, RTS,S, is based on VLPs54. This platform forms large spheroid particles around 120 nm in diameter55. To assemble into VLPs M1 must interact with proteins containing a cytoplasmic tail region such as HA (hemagglutinin) and NA (neuraminidase)56. M1 VLPs have been made with different combinations of influenza surface glycoproteins or envelope proteins from other viruses and has been expressed in mammalian and insect systems58. M1 VLPs were used in a SARS-CoV vaccine, consisting of M1 envelope protein and chimeric spike protein19. Replacement of the spike protein transmembrane and C-terminus regions with those of HA allowed the antigen to insert itself into the lipid membrane formed by the envelope protein. Vaccination of mice with the chimeric spike VLP protected mice against SARS-CoV. Comparatively, vaccination with spike alone required the addition of adjuvant for protection.Influenza Matrix 1 (M1) protein-based VLP is a second enveloped VLP platform, which has seen usage in clinical trials for an influenza vaccine59. In contrast to enveloped VLPs that incorporate a lipid membrane, capsid-based VLPs are completely composed of protein. 180 monomers of the coat protein oligomerize to form a capsid that resembles a truncated icosahedron with 20 hexameric facets and 12 pentameric vertices  antigen from P. falciparum erythrocyte membrane protein 1 (PfEMP1) was attached to AP205 with the Spytag-Spycatcher coupling system20. AP205 coupled with CIDR produced significantly higher antibody titers after immunization in mice versus non-coupled AP205 & antigen or antigen alone. As a cancer vaccine, AP205 elicited an auto-antibody response against human epidermal growth factor receptor-2 through genetic fusion to the AP205 VLP61.Capsid VLPs based on bacteriophage AP205 coat protein have shown relative ease of antigen attachmentces Fig. 60. The c62. VLPs assembled in vivo can encapsulate host DNA and other host proteins, potentially due to their nature, which encourages the packaging of genomic information. Removal of such contaminants involves complex purification or in vitro disassembly and reassembly steps62. VLPs also suffer from stability issues, possibly due to a lack of viral genome63. Additionally, enveloped VLPs require eukaryotic host expression systems in order to acquire their lipid membrane. VLPs are well-established, however, manufacturing complications hinder their adoption as platforms.VLPs have demonstrated remarkable improvements in improving the humoral immune response when used as a platform for antigens. However, VLPs can be difficult to produce due to low expression yields and the presence of host cell contaminants from expression systemsAs an alternative to VLPs, non-viral proteins that are highly oligomeric have been used as platforms. These platforms are often enzymes or homeostasis proteins that can be easily produced. A hallmark of self-associating proteins is a single protein component that readily assemble into stable highly oligomeric structures that complement icosahedral VLPs.64. The complex consists of 8 trimers with octahedral symmetry and resembles a rhombic dodecahedron: a polyhedron with both threefold and fourfold symmetry64 , which was truncated and stabilized with a coiled-coil linker to the C-terminus of LS from Aquifex aeolicus significantly improved B-cell activation compared to antigen alone21. Similar to ferritin, LS has also successfully presented SARS-CoV-2 trimers on LS with a similar construct design, glycosylation modifications, and expression system used for ferritin46. LS is typically expressed in a mammalian cell secretion system, although E. coli expression has also been successfully utilized for LS70.Lumazine synthase (LS), an enzyme involved in riboflavin synthesis, is an icosahedral non-VLP platform that has significantly more subunits than ferritin. Sixty monomers of pentameric protein naturally assemble into particles resembling a dodecahedron71. The E2p nanoparticle is slightly larger than LS  is inaccessible.An alternate icosahedral non-VLP platform is dihydrolipoyl acetyltransferase (E2p), an enzyme that assembles into a 60-mer hollow dodecahedron from 20 trimers and forms the core of the pyruvate dehydrogenase complex72. Nsp10 consists of 12 monomers that assemble into a spherical particle resembling a dodecamer with twelve faces. Both N- and C-terminals are surface-exposed and are located on separate threefold axes, which allows for the attachment of antigen, especially trimeric, to both terminals. Genetic fusion of nsp10 with a γ-herpesvirus antigen, gD, elicited binding antibody titers in rabbits72.The nonstructural protein 10 (nsp10) has recently gained interest as a nanoparticle due to its presence within the coronavirus genus, including the SARS-CoV-2 virusGallus gallus ortholog of C4bp, forms a heptameric ring-like protein73. The malaria antigen Pfs25 was fused to the N-terminal of IMX313, expressed in yeast, with vaccination resulting in significantly increased antibody titers compared to vaccination with the antigen alone73. A dual component, dual antigen IMX313 was also successfully developed through genetic fusion of SpyCatcher at the N-terminus and SnoopCatcher at the C-terminus, which was expressed in E. coli with refolding. The genetic fusions enabled attachments of two distinct malarial antigens Pfs25 and Pfs28 to IMX313 through coupling with SpyTag and Snooptag, respectively45. The multimeric antigens produced a significantly stronger antibody response than monomeric antigens.Smaller non-spherical particles have also been utilized. IMX313, a Many highly oligomeric proteins have been successfully used as platforms. These proteins have distinct arrangements than their capsid VLP counterparts enabling unique nanoparticle presentation and design. Platforms such as Ferritin, LS, and E2p also contain a trimeric threefold favorable for the attachment of trimeric viral antigens such as the Spike protein of SARS-CoV-2. However, most of these self-assembling platforms are smaller in size than VLPs and may have only one terminus available for antigen attachment, which can limit the valency and compatibility for antigens.Through rational or computational design, dimeric, trimeric, tetrameric, or pentameric proteins have been engineered to assemble into larger, highly oligomeric complexes that offer greater control over antigen stoichiometry, spacing, and particle size. These engineered particles present additional platforms for vaccines beyond the limited number of natural platforms. In order to develop novel platforms several methods and techniques of generating self-assembly have been developed. There are several design parameters for designing self-assembling proteins for use as a platform. First, the geometric symmetry and shape of the desired nanoparticle are determined by type of building blocks used. Second, self-assembly must be promoted by either fusing two different building blocks or engineering an interface between building blocks. Lastly, at least one terminal should be exposed and accessible for antigen attachment. Described below are the design and evolution of novel self-assembling proteins that have led to engineered platforms.34, and the utilization of a trimeric coiled-coil that binds to a dimeric coiled-coil74. SAPNs are limited by the relatively small surface area of the monomeric building blocks. It is difficult to fuse large proteins to the monomers without them interfering with assembly, thus limiting the use of SAPNs to display small peptides or epitopes.Self-assembling peptide nanoparticles (SAPNs) are large protein assemblies composed of numerous coiled-coil domains. SAPNs are able to oligomerize into large icosahedral complexes because of interactions between individual coiled-coil domains. Formation of these complexes has been accomplished through the genetic fusion of a trimeric coiled-coil to a pentameric coiled-coil75 and optimized in 201276. Producing a tetrahedral platform requires the oligomerization of four trimeric building blocks  was also successfully fused to T33-21 using an α-helical linker86 resulting in the presentation of 12 copies on the nanoparticle and enabling the small protein to be visualized by Cryo-EM. The I3-01 nanoparticle has been fused to malarial antigens using SpyCatcher-Spytag43 in comparison with the AP205 VLP platform. Although I3-01 presented less antigen, 60 compared to 180 in AP205, they both induced similar antibody levels. The I3-01 design has also been genetically fused to a stabilized trimeric HIV envelope antigen gp14023, with a ten amino acid length linker to account for the relatively large spacing of the N-termini. An alternative linker, which incorporated a T-cell epitope was also successfully incorporated. The trimeric antigen presented on I3-01 generated significantly higher levels of antibodies compared to the trimeric antigen alone. Similarly, I53-50 has been developed as an HIV vaccine, utilizing a prefusion trimer stabilized envelope antigen called SOSIP fused to the N-terminus of the trimeric secondary building block24. Presentation of ConM genotype SOSIP trimers on I53-50 demonstrated increased neutralizing antibody titers compared to SOSIP trimers alone. However, presentation of BG505 genotype SOSIP on I53-50 elicited lower antibody titers and poorer neutralizing antibody response than BG505 SOSIP alone. This occurrence was attributed to less accessibility to the neutralizing epitopes of BG505, which were located at the base of the spike, due to attachment with the platform. However, further analysis demonstrated no change in antibody titers against other base-located epitopes with I53-50 fused and non-fused SOSIP. Ultimately, this result suggests that it may be beneficial to orient antigen with neutralizing epitopes apically exposed. I53-50 design has been utilized in a vaccine for respiratory syncytial virus (RSV)87. A prefusion-stabilized variant of the RSV F glycoprotein trimer was fused to a trimeric foldon domain located at the N-terminus of the trimeric secondary building block. Neutralizing antibody titers induced by the antigen fused to I53 were ten times greater than those that were induced by the trimeric antigen alone. I53-50 has recently been utilized as a vaccine for SARS-CoV-2 with the Spike glycoprotein receptor-binding domain (RBD) genetically fused to the N-terminus of the trimeric building block with varying flexible linker lengths88. In mice, immunizations with the RBD fused with I53 designs generated a strong antibody response, while monomeric RBD failed to generate a detectable response, and stabilized prefusion Spike trimer (S-2P) elicited a weaker response. Similar results were seen in neutralizing antibody productions. RBD fused to I53 was able to confer protection against SARS-CoV-2 replication while monomeric RBD and S-2P did not.Engineered interfaces follow similar geometric symmetry principles to the fusion strategy, but instead of fusing two oligomeric stoichiometries together it introduces another oligomeric binding site. The specificity of the engineered interface limits undesired assemblies, promoting the use of this strategy for platform development. GFP was successfully fused to the trimeric building block of the In conclusion, advancements in protein design have allowed for engineered platforms that have been successfully used in prototype vaccines. With comparable immunogenicity to natural platforms, these platforms surpass natural non-VLP platforms in size Table  while haSeveral factors such as size, antigen density, and surface properties are critical for the successful implementation of nanoparticles in vaccines. These parameters are described below in an attempt to guide the usage and development of platforms for improved immunogenicity. Although discrepancies may arise due to differences in nanoparticle composition the findings highlight mechanisms such as cellular uptake, opsonization, trafficking, and B-cell activation, that can be used to evaluate the performance of a platform.25. Testing of polystyrene particles ranging from 40 to 1500 nm in diameter determined that particles below 500 nm had the highest uptake by dendritic cells89. Specifically, polystyrene particles of 40, 100, and 500 nm all had relatively similar levels of uptake. Mathematical models have predicted the optimal size for cellular uptake to be between 20 and 30 nm90. Spherical gold particles, conjugated to albumin as an antigen, had a lymph follicle retention time of 5 weeks with particles 50 and 100 nm in diameter compared to 48 h with particles 5 and 15 nm in diameter91. Furthermore, the larger particles were more likely to be presented on the FDC surface, allowing greater delivery of antigen to B-cells, and higher levels of B-cell maturation. Conversely, the smaller particles were able to enter the lymph node more rapidly than the larger particles. A similar result was seen with polypropylene sulfide nanoparticles, where particles 25 nm in diameter entered the lymph nodes 10 times more efficiently than 100 nm particles92. Thus, the optimal platform may be around 20–50 nm, large enough for efficient uptake and retention in lymph follicles, small enough for entry into the lymph node (Fig. Efficient cellular uptake, lymph follicle retention, and entry into lymph nodes are three critical factors that lead to improved immunogenicity and protection that is influenced by the size of a nanoparticle platform Fig. . Particlode Fig. .Fig. 5Cr93. The 8-mer and 60-mer antigens demonstrated enhanced B-cell activation, B-cell proliferation and B-cell localization upon immunization of mice and this effect was not observed with lower valency antigens. Vaccination with the 60-mer also showed improved T-cell recruitment, IgG titers, and was able to rescue a low-affinity version of the antigen to induce B-cell differentiation. In contrast, examination of B-cells induced by 4-mer and 60-mer vaccinations revealed that the lower valency 4-mer had higher titers of B-cells binding to antigen probes, suggesting that lower valency antigens generated higher affinity B-cells. However, differences in the angle and orientation with which the antigen was presented or other differences due to protein composition may have impacted the display of accessible epitopes. Additionally, differences between the protein platforms such as size, flexibility, and composition may also introduce confounding factors. In order to eliminate these factors a recent study used DNA origami platforms that could present antigens with defined valency and organization94. The study compared valency using icosahedron shaped DNA platforms that presented 1, 2, 3, 5, 10, 30, or 60 copies of eOD-GT, with the benefits of increased valency on B-cell activation plateauing at 5. However, this result may have been hindered by inadequate antigen spacing. The study measured effects of antigen spacing between two antigens on a rod-shaped DNA platform with distances from 7 to 80 nm. B-cell activation increased as distance increased, plateaued at a distance of 28 nm and remained similarly elevated at 80 nm. The findings from this study suggest that steric constraints hinder BCR recruitment to antigen less than 30 nm apart (Fig. Optimal B-cell activation is dependent on the antigen valency and spacing Fig. . One stuart Fig. .95. On the other hand, hydrophobic particles have been shown to promote DC uptake, possibly through enhanced cell membrane interaction96 or complement recruitment26. However, the attachment of antigens can significantly change these surface properties, and thus it may be more appropriate to evaluate these properties in context of compatibility with the antigen. Further research is needed to comprehensively analyze how the surface charge and hydrophobicity of the platform affects trafficking and immune response.Beyond size and structure other factors such as surface charge and hydrophobicity can influence the trafficking of particles and thus affect immunogenicity. Positively charged particles has been shown to promote uptake by macrophages and DCs due to electrostatic attraction to negatively charge membranes97, which may potentially divert the humoral immune response from the antigen. Continued research and testing of more platforms are needed to determine the effects of this implication, as well as evaluate parameters for the development of more optimized platforms.These findings suggest the parameters for the ideal platform may be 20–50 nm in diameter with antigen spaced 30 nm apart. However, a more comprehensive study containing more particles with smaller intervals in size is needed for testing of uptake, opsonization, and lymph node trafficking. Furthermore, differences in particle composition may affect the results of size as-well. A conclusive answer to the impact of valency will require testing of valencies with optimally spaced antigen. Antibody responses against the platform has been demonstrated in vaccinesProtein-based nanoparticles have demonstrated the ability to revolutionize vaccine development for various diseases. The design of optimal nanoparticles that effectively present antigens to produce the desired immune response is an active area of investigation. Computational design of nanoparticles has matured and allows for the expansion of available platforms for study. There are countless other possible building block arrangements that have yet to be explored as platforms. Additionally, all the engineered platforms share a common trait—a reliance on creating a dimeric interaction between the building blocks for self-assembly; more complicated symmetries may be investigated in the future. These approaches, along with more standardized methodology, are expected to contribute significantly to improving vaccines and alleviating the burden of global diseases.


Hunter-gatherer lifestyles dominated the southern African landscape up to ~ 2000 years ago, when herding and farming groups started to arrive in the area. First, herding and livestock, likely of East African origin, appeared in southern Africa, preceding the arrival of the large-scale Bantu-speaking agro-pastoralist expansion that introduced West African-related genetic ancestry into the area. Present-day Khoekhoe-speaking Namaqua (or Nama in short) pastoralists show high proportions of East African admixture, linking the East African ancestry with Khoekhoe herders. Most other historical Khoekhoe populations have, however, disappeared over the last few centuries and their contribution to the genetic structure of present-day populations is not well understood. In our study, we analyzed genome-wide autosomal and full mitochondrial data from a population who trace their ancestry to the Khoekhoe-speaking Hessequa herders from the southern Cape region of what is now South Africa.We generated genome-wide data from 162 individuals and mitochondrial DNA data of a subset of 87 individuals, sampled in the Western Cape Province, South Africa, where the Hessequa population once lived. Using available comparative data from Khoe-speaking and related groups, we aligned genetic date estimates and admixture proportions to the archaeological proposed dates and routes for the arrival of the East African pastoralists in southern Africa. We identified several Afro-Asiatic-speaking pastoralist groups from Ethiopia and Tanzania who share high affinities with the East African ancestry present in southern Africa. We also found that the East African pastoralist expansion was heavily male-biased, akin to a pastoralist migration previously observed on the genetic level in ancient Europe, by which Pontic-Caspian Steppe pastoralist groups represented by the Yamnaya culture spread across the Eurasian continent during the late Neolithic/Bronze Age.We propose that pastoralism in southern Africa arrived through male-biased migration of an East African Afro-Asiatic-related group(s) who introduced new subsistence and livestock practices to local southern African hunter-gatherers. Our results add to the understanding of historical human migration and mobility in Africa, connected to the spread of food-producing and livestock practices.The online version contains supplementary material available at 10.1186/s12915-021-01193-z. Hunting and gathering was the only lifeway practiced in southern Africa until approximately 2000 years ago. Previous studies have suggested that at that time, a herding group of East African origin introduced herding practices and livestock into southern Africa and admixed with local hunter-gatherer groups to form what became known as the Khoekhoe populations –6. This In contrast to Kx’a and Tuu that are spoken exclusively by hunter-gatherers, Khoe-Kwadi was historically spoken by hunter-gatherers and pastoralists. The Khoe-Kwadi language family can be structured in three sub-branches: Kalahari Khoe, Khoekhoe and Kwadi (extinct)   allowing the digestion of milk in adulthood [Ancient DNA studies on human remains demonstrate that all extant Khoe-San groups have admixture with a mixed group containing East African and Eurasian ancestry , 4. Thisdulthood . In contdulthood .The present-day Western and Eastern Cape provinces in South Africa were once home to many, now extinct, Khoekhoe languages, linguistically grouped as Cape Khoe , East African (brown), West African (gray), European (red) and Asian (dark-red). We report ADMIXTURE results for K = 5 in detail since it is the K with the lowest cross-validation error , with a San ancestry of 37.7% and 19.1%, respectively [Z-score = 2.424).We started by investigating the genetic structure of Hessequa descendants in comparison to African (and worldwide) datasets Fig. . We estiectively . Howeverectively  under a The East African ancestry is visible in several Khoe-San groups from southern Africa  information to infer time estimates . We initWe further estimated the arrival of the East African ancestry across southern Africa by dating admixture times in other Khoe-San groups  ratio from the admixture fractions in Khoe-San populations with an average East Africa ancestry in autosomes and X chromosome higher than 2% : the Hessequa descendants, Nama from Windhoek, Khwe, ǂKhomani and Coloured population of Wellington. Most populations have a X/A ratio above 1 for San ancestry, indicating a higher female-to-male ratio for the San ancestry , the single uniparental loci, mtDNA and Y chromosomes, should, to some extent, reflect the same patterns observed in the X/A ratio. Since haplotype inference from SNP-chip data is unreliable , we geneTo compare with our mtDNA results, we use the Y chromosome markers present in the H3Africa SNP array and classified their Y chromosome haplogroups using SNAPPY   of the Khoe-San groups. Additional inter- and intra-continental genetic ancestries were introduced into the Khoe-San gene pool during the colonial period (1600s onward). This complex history hinders inferences about preceding (and perhaps more subtle) demographic events. Therefore, we applied an ancestry-specific haplotype-based approach to obtain time estimates for East African admixture into the autochthone Southern African gene pool. Khwe speakers had the oldest East African admixture date in our dataset, i.e. 2142 years ago (CI 2081–2977)  but livestock remains found at the Blombos site in the Western Cape started to appear ~ 2000 years ago in the archaeological record . SimilarIt is worth mentioning that Nama speakers lived exclusively in the present-day Northern Cape province region of South Africa until recently . The datPrevious studies suggested that the East African component from modern Khoe-San individuals show high affinities with present-day Afro-Asiatic speakers from Ethiopia, such as the Amhara . Based oThe East African-Eurasian mixed ancestry in Khoe-San groups has been reported before , 4–6, 39The colonization of southern Africa by Europeans from the 1600s onwards, further impacted the genetic landscape of the indigenous communities of southern Africa, particularly in the area where Cape Khoe speakers once lived. Previous studies on Western Cape Coloured populations have reported similar ancestry fractions to the ones observed on Hessequa descendants , 91–93).The genomes of Hessequa descendants trace 58.1 to 70.1% of their genetic ancestry to groups that immigrated into southern Africa during the colonial period. Apart from European admixture from settlers and mariners, they also received genetic contributions from Bantu-speakers and slaves of Southeast Asian ancestry. The West African-related component in the Hessequa descendants has the closest match to Bantu-speaking groups of Mozambique and northeast South Africa  but not with neighboring Xhosa speakers. These results could possibly be explained by the fact that Bantu-speaking groups from Mozambique and northeast South Africa have little to no admixture with local Khoe-San speakers , 94, 95.Our results support previous findings that East African-Eurasian ancestry arrived in southern Africa around 2000 years ago, possibly introducing pastoralism and livestock into the area. Although the complete history of the introduction of pastoralism to southern Africa remains to be uncovered, we detect that the East African component in southern African genomes is genetically most similar to several pastoral Afro-Asiatic speaking groups from Ethiopia and Tanzania. Furthermore, we also inferred that the East African pastoralist expansion was significantly male-driven. This result is supported by different sections of the genome: autosomes vs. X chromosome ancestry ratios, mtDNA and NRY markers. Although based on very few archaeological and genetic data points, we see some similarities and trends between archaeological proposed routes for the spread of pastoralism across the southern Africa region and genetic admixture dates of the East African component into an autochthonous southern African hunter-gatherer background. Apart from the earliest contact between autochthonous hunter-gatherers and the East African-origin group(s), our haplotype-based admixture time estimates tentatively indicate that this East African ancestry might have spread at a slower pace than livestock remains—support is lent to this hypothesis by the archaeological data and ethnographic studies , 75, 102Finally, we provided additional insights on how the colonial era impacted the genetic diversity of southern Africa, in particular among the groups that trace part of their ancestries to the Hessequa people of the southern Cape coastal region.The Hessequa descendant samples were collected in Western Cape, South Africa, after in-depth field work by an anthropologist (MDJ) on the ethnographic background of the communities in the region. Written informed consent was obtained from all 162 participants included in the study before saliva samples were collected. Sample collection of Coloured, Khoe-San and Khoe-San descendent groups were approved by the University of the Witwatersrand Human Research Ethics board, clearance numbers M980553, with renewals M050902, M090576, M1604104. This specific project was approved by University of the Witwatersrand Human Research Ethics board, clearance number M180655 and the National Ethics review board of Sweden, clearance number Dnr 2021–01448. The biological material was collected in 2 mL Oragene saliva kits (DNA Genotek) and DNA was extracted using the prepIT L2P extraction protocol. The 162 samples were genotyped on H3Africa Consortium SNP panel implemented in Illumina Infinium assay (H3Africa_2017_20021485_A2 BeadChip). The data were generated by the SNP&SEQ Technology Platform in Uppsala, Sweden. The data were analyzed using GenomeStudio v.2011.1 and aligned to the Human Genome built version 37. A total of 2,267,346 genomic markers were obtained.Data management and quality filtering were carried out using the PLINK v.1.90 software . Of the The assessment of the allele frequencies for the lactase persistence mutations was performed exclusively among Hessequa descendants using PLINK on the raw data in order to control for possible errors due to flipping between forward and reverse strands while merging with other datasets.K, was set from 2 to 10, replicated 50 times with random seeds. The cluster-inference and visual inspection was made with Pong v.1.4.5 [We ran initial population structure analyses and estimated admixture fractions with ADMIXTURE . The dat v.1.4.5 .K = 5, were kept. The filtering strategy is meant to minimize the impact of admixture in recent generations (last 2-to-3 generations), resulting, for example, from increased mobility and the imposed social restructuring during Apartheid. However, the same analysis was performed on each separate Hessequa descendant group for support information . To minTo test whether the arrival of the East African component in southern Africa was sex-biased, we estimated the X chromosome/Autosomal ratio for admixture proportions. The tested populations were merged with a comparative dataset of 20 Yoruba (YRI), 20 Amhara, 20 Central Europeans (CEU), 20 Sri Lankan Tamil (STU) and 17 Ju|’hoan, which were used in the supervised admixture to have known ancestries. X chromosomes from both males and females were included and the data was filtered using the same criteria settings as the autosomes described above, excluding HWE filtering.To avoid discrepancies in resolution power between the X chromosome and autosome sizes, we selected chromosomes 7, 10 and 12 since they have approximately the same genetic length as the X chromosome (180 cM). Furthermore, we cut the first 180 cM of chromosomes 1 to 6 as well. For each of the selected autosomes, we randomly down sampled SNPs to the same number present in X chromosome. The “shuf” command in bash was applied for random down-sampling. We ran supervised admixture for chromosomes 1 to 7, 10, 12 and X separately, replicated 20 times each. The cluster-inference and visual inspection was made with Pong v.1.4.5 , and aveN = 10), Melkhoutfontein (N = 10), Railton (N = 8), Riversdale (N = 12), Rotterdam Farm (N = 7), Slangrivier (N = 8), Stormsvlei (N = 10), Suurbraak (N = 11) and Swellendam (N = 11). We amplified two fragments of 7.7 kb and 9.2 kb , following the protocol proposed by [We selected 7–12 Hessequa descendants randomly from the nine sampling sites (in accordance with sample availability), resulting in 87 individuals to sequence the complete mtDNA: Heidelberg , Y chromosome-specific markers were extracted, and 2538 SNPs were retained. The number of males (N = 58) were not the same individuals that were selected after recent admixture removal  nor were they the same individuals carrying the L0d mtDNA haplogroup —the re-occurrence of the number 58 for these individual groupings was a chance event. Y chromosomal markers were used to assign Y haplogroups using SNAPPY [For each self-identified male in the Hessequa descendant groups . Table S3. Allele frequencies of LP associated variants present in the H3Africa SNP array. Table S4. Relevant admixture time estimates based on pairwise coancestry curves. Table S5. X-chromosome to autosomal ratio based on the average ancestry proportion. Table S6. Summary results for the mitochondrial haplogroup assignment for the 87 sub-sampled Hessequa-descendants. Table S7. Summary results for the Ychr haplogroup assignment for the 58 male Hessequa-descendants. Table S8. List of populations included in the dataset.


A-α5 receptor is proposed as a mechanism to attenuate GABAergic function and restore the excitatory/inhibitory balance.There are currently no pharmacological therapies to address the intellectual disability associated with Down syndrome. Excitatory/inhibitory imbalance has been hypothesized to contribute to impairments in cognitive functioning in Down syndrome. Negative modulation of the GABAA-α5 negative allosteric modulator, was evaluated at 120 mg or 240 mg BID (80 or 160 mg for 12–13 years) in a 6-month, randomized, double-blind, placebo-controlled phase II trial (Clematis) for efficacy and safety in adolescents and young adults with Down syndrome. The primary endpoint was based on a composite analysis of working memory  and independent functioning and adaptive behavior . Secondary measures included the Behavior Rating Inventory of Executive Functioning-Preschool (BRIEF-P), Clinical Evaluation of Language Fundamentals (CELF-4), and Pediatric Quality of Life Inventory (Peds-QL). EEG was conducted for safety monitoring and quantitatively analyzed in adolescents.Basmisanil, a selective GABABasmisanil was safe and well-tolerated; the frequency and nature of adverse events were similar in basmisanil and placebo arms. EEG revealed treatment-related changes in spectral power (increase in low ~ 4-Hz and decrease in high ~ 20-Hz frequencies) providing evidence of functional target engagement. All treatment arms had a similar proportion of participants showing above-threshold improvement on the primary composite endpoint, evaluating concomitant responses in cognition and independent functioning . Further analysis of the individual measures contributing to the primary endpoint revealed no difference between placebo and basmisanil-treated groups in either adolescents or adults. There were also no differences across the secondary endpoints assessing changes in executive function, language, or quality of life.Basmisanil did not meet the primary efficacy objective of concomitant improvement on cognition and adaptive functioning after 6 months of treatment, despite evidence for target engagement. This study provides key learnings for future clinical trials in Down syndrome.The study was registered on December 31, 2013, at clinicaltrials.gov as NCT02024789.The online version contains supplementary material available at 10.1186/s11689-022-09418-0. Down syndrome (DS), the triplication of whole or part of chromosome 21, is the most common identifiable cause of intellectual disability with an incidence of 1 in 650 to 1 in 1000 live births per year worldwide , 4, whicA-α5 negative allosteric modulators (NAMs)—such as α5IA [A NAMs [A-α5 receptors may represent an attractive mechanism to enhance cognition in individuals with DS.Although the etiology of the cognitive disability in people with DS remains unclear, cellular and anatomical abnormalities in the prenatal and perinatal forebrain and cerebellum suggest that early brain development is altered in individuals with DS –7. Simil as α5IA , RO49385 as α5IA , and bas as α5IA —improved [A NAMs , 18. InhA-α5 subunit-containing receptors and has been shown to improve cognition in rats and monkeys [A-α5 hippocampal receptor occupancy between 30–65% was required for efficacy in preclinical studies [Basmisanil  is a potent NAM, which combines both binding and functional selectivity at GABA monkeys . GABAA-α studies . BasmisaGiven the absence of any effective therapy for the intellectual disability associated with DS, the supportive 5-week safety and tolerability profile established in individuals aged 18–30 years with DS  with DS  were included. Minimum verbal abilities were required to participate in the study, as defined by a minimum raw score of 7 for adults, or 4 for adolescents, on the Clinical Evaluation of Language Fundamentals Preschool-2 (CELF-P) Word Classes subtests . The IQ Individuals with a diagnosis of autism spectrum disorder, major depressive disorder, a history of infantile spasms or epileptic encephalopathy, or a history of seizures within 2 years prior to the screening visit were not included in the trial. Participants consented or assented to participate, and written informed consent was obtained from their caregiver.clinicaltrials.gov as NCT02024789, approved by local ethics committees, and conducted in accordance with the principles of the “Declaration of Helsinki” and Good Clinical Practice. A Roche-independent safety committee was responsible for the monitoring of safety data on a regular basis.BP27832 (Clematis) was a randomized, double-blind, placebo-controlled, multi-country phase II study to investigate the efficacy and safety of basmisanil in adults (18–30 years) and adolescents (12–17 years) with DS  over 6 months (26 weeks). The low dose of basmisanil was 120 mg and the high dose was 240 mg, except for participants below 14 years where the low dose was age-adjusted to 80 mg and the high dose to 160 mg. Dose selection was based on an integrated evaluation of pharmacokinetics (PK), pharmacodynamics, PET  on a composite endpoint, concomitantly evaluating cognition and adaptive functioning, after 6 months of treatment. Above-threshold improvement on the composite endpoint was defined as (1) a relevant increase in raw scores from baseline in at least two out of three tasks from the Repeatable Battery for the Assessment of Neuropsychological Status ; and (2) either an increase from baseline in the Vineland Adaptive Behavior Scales-II (VABS-II) composite standard score of ≥ 7 or a Down syndrome-specific Clinical Global Impression-Improvement (DS-CGI-I) score of ≤ 3 . The DS-CGI-I evaluation was based on scoring DS-specific anchors: communication/speech, activities of daily living, social functioning, and stubbornness/non-compliance .Fifty subjects per treatment group provide a power of 80% to detect a difference between each active dose and placebo when the frequency of participants with above-threshold improvement is 30% on active dose and 5% on placebo. This calculation was based on the two-sided The proportion of participants with above-threshold improvement was analyzed by means of a logistic regression model. This included treatment and visit and treatment by visit interaction, age, sex, and IQ at baseline as covariates, participant as repeated effect. The selected covariates were defined a priori in a statistical analysis plan, as sex and age may have an impact on drug pharmacokinetic properties, and age and IQ are expected to influence cognition, language, and adaptive behavior in individuals with DS. For all endpoints normally distributed a mixed model analysis of variance was applied to change from baseline scores, where applicable, with baseline, age, sex, and IQ at baseline as covariates, treatment and treatment by visit interaction, with visit as repeated measurements and participant as random. Inferential findings are provided for descriptive purposes only and without any confirmatory meaning. Multiple endpoints and multiple treatment comparisons were analyzed; however, due to the exploratory nature of the study, multiplicity was not statistically adjusted for, and the risk of false positive results should be taken into consideration in the interpretation of the results.Blood samples were collected for determination of plasma concentrations of basmisanil. Concentrations were measured by a specific liquid chromatography-mass spectrometry/mass spectrometry method. The following time points were included prior to dosing to assess trough concentrations of basmisanil at weeks 2, 6, and 12.Safety surveillance of participants included adverse event (AE) reporting, physical examinations, vital signs including 12-lead ECG recordings, clinical chemistry, hematology, and urinalyses. Comorbidities were monitored, such as ADHD (Conner’s questionnaire); sleep problems (Children’s sleep habits questionnaire); anxiety and depression (ADAMS questionnaire). As per regulatory guidance, suicidality monitoring was implemented using the pediatric and adult C-SSRS version.EEG recordings were primarily included to monitor the emergence of epileptiform abnormalities in adolescents and participants with a medical history of epilepsy, to confirm the favorable safety profile of basmisanil previously established in adults with DS . A 30-min EEG recording was performed at baseline (pre-dose), week 2, and week 20. Recordings from adolescents were used for the exploratory quantitative EEG analyses reported here. The exploratory quantitative analyses of the EEG data were restricted to spectral power, which provides a macroscopic measure of synchronized neuronal activity. No assumptions were made about spectral or spatial properties of possible treatment effects. The statistical analysis accounted for multiple comparisons across frequencies and electrodes using a cluster randomization approach.n = 37, low dose: n = 14, high dose: n = 23). Full details on the EEG acquisition and analysis can be found in Additional file To test for a PK-PD relationship we performed non-parametric correlations  between individual measured trough exposure levels and theta and beta-band EEG power from the identified clusters both measured at week 2. Although the EEG was recorded 4–5 h post administration, and the PK sample before administration, the measured trough concentrations at steady state are considered as a reasonable proxy for the individual basmisanil concentration at the time of the EEG recording. For this analysis we used all dosed participants but only included participants with a PK sample and an EEG recording at week 2 , French (20%), and Spanish (13%) sites. For adolescents, the majority were recruited at Spanish (42%) and US (31%) sites.A total of 155 participants (91%) completed the study and were included in the analysis .At 3 months there was no statistically significant difference in improvement overall Fig. C, howeveAdditionally, no differences in the proportion of participants with above-threshold improvements were detected between placebo and basmisanil-treated groups on any of the individual components of the composite endpoint  and frequencies (2–32 Hz). The analysis identified two clusters, i.e., differences between the combined dose groups and placebo that extended across frequencies and electrodes. A “positive cluster” in the theta-frequency range  and a “negative cluster” in the beta-frequency range  but not for the beta band (p = 0.27).There was no difference between the low or high dose . Numerically, the correlations were in the expected direction  but lacked significance.Neither the theta-band nor the beta-band EEG pharmacodynamic effects correlated with exposure .Five serious AEs, reported in five participants, were considered not related to treatment Table , and oneVital sign monitoring did not reveal changes in heart rate and blood pressure  and could thus be expected to be efficacious. The lack of differentiation between doses limits meaningful interpretations of dose-dependent treatment effects from both safety and efficacy perspectives in the overall population. In adolescents, there was a higher proportion of participants showing improvement on the primary endpoint after 3 months of treatment . This effect was not maintained after 6 months of treatment despite stable exposures and was not reflected in any of the secondary measures. The absence of differences in exposure-response relationships between participants with and without above-threshold improvements, across ages and doses (data not shown), corroborate a true lack of effect of basmisanil.Clematis was the first phase II trial performed in the DS population with a compound specifically designed to address excessive inhibition in limbic brain areas, hypothesized to contribute to the intellectual disability associated with DS , 15. OveThe primary endpoint was designed to capture potential improvements in intellectual functioning from multiple perspectives by combining direct measures of cognition (RBANS memory tasks), clinician ratings (DS-CGI-I), and caregiver-reported measure (VABS-II). These measures were selected based on their suitability for the population, reliability, stability over time, and feasibility of implementation, as previously determined in a 6-month observational study with a comparable study design and population , 23. In These changes were more pronounced in the adolescent population and are in line with published placebo response rates of 10–30% described in DS  and otheThe threshold for improvement on the primary composite endpoint combined improvements on RBANS memory tasks and global functioning on either the VABS-II or the DS-CGI-I. Because the DS-CGI-I anchors were mainly derived from the VABS-II domains, DS-CGI-I scores may not be independent of the caregiver perception captured by the VABS-II. The increases over time in VABS-II scores observed across groups may reflect treatment expectancy effects and directly (or indirectly via the DS-CGI-I) drive improvements on the primary endpoint. The composite endpoint is a multidimensional measure which increases the complexity of the analysis and interpretation and requires consistent effects to reach statistical significance. The choice of a composite endpoint, although a high bar objective, is unlikely to have masked effects as no beneficial treatment effects were detected on any of the individual components of the primary endpoint. Consistent with these findings, the analysis of secondary outcome measures did not show any beneficial effects of basmisanil over placebo after 6 months of treatment. Importantly, scores from the direct performance-based evaluations of cognition assessing memory (RBANS) and language (CELF), thought to be less sensitive to treatment expectancy bias, remained generally stable across age and treatment groups over the 6-month study duration, with the exception of the RBANS learning task. The small improvements observed in RBANS learning are in line with previous data from our observational study  and possA function through pharmacology [A receptor genes [Exploratory quantitative analysis of EEGs recorded in adolescents was performed to test for effects on brain function. The absence of an alpha peak in the baseline EEG power spectrum is in line with previous findings in adults with DS reporting a shift to lower frequencies –31. The macology , 33, in macology , 35 and or genes , 37, andor genes , 39. CorA receptor positive allosteric modulators after long-term use [A function did not significantly decline over time and no withdrawal effects were observed when the administration of basmisanil was stopped. Finally, there is no preclinical evidence suggesting that α5 subtype-selective compounds, such as basmisanil, lead to tolerance [While basmisanil exposure remained stable, the EEG effect in lower frequencies was weaker at week 20 compared to week 2, while remaining significantly higher than at baseline. The decrease in EEG power at lower frequencies may indicate compensatory or adaptive neuronal mechanisms that could result in tolerance. Tolerance is a well-described phenomenon for non-selective GABAterm use . Howeverolerance . ToleranSome study limitations should be noted. The detection of significant treatment effects of basmisanil may have been limited by the small sample size. Indeed a potential selection bias cannot be controlled for, albeit random treatment group assignment. Cognitive and behavioral measurements were not assessed during the first month of treatment; we are therefore unable to interpret potential improvements in relation to the early pharmacodynamic EEG changes observed. This would have also been helpful to interpret the trend observed after 3 months in adolescents, as well as the trends observed after 5 weeks of treatment on the RBANS tasks in a small exploratory phase IB trial in young adults with DS  [It is also conceivable that selective modulation of the GABA (~ 40%)  may not  (~ 40%) . Alterna (~ 40%) , and the (~ 40%) . Future Here we have described some of the challenges, and potential strategies to address them, from the perspective of investigators experienced with research in this population . The lowAdditional file 1. Previous clinical information. Summary of PET and MAD data from Study BP25611 and Study BP25543.Additional file 2. Study design.Additional file 3. Primary and secondary assessment scales. Provides more detailed information on the scales, including the DS-CGI-I.Additional file 4. EEG supplementary methods. Provides detailed methodology.Additional file 5. Percent of Participants with Relevant Improvements for each Assessment of the Composite Endpoint by Age Group at 3 and 6 months. A table showing percent of participants with above-threshold improvements for each assessment by age group and time point.Additional file 6. VABS-II: Change from baseline at 6 months. Figure showing VABS-II data: composite and individual scores for socialization, communication, and daily living skills.Additional file 7. Quantitative EEG. Figures showing further analysis of EEG to support the EEG data in the main manuscript.Additional file 8 Estimated Receptor Occupancy and Pharmacokinetics: Table 1: Estimated Receptor Occupancy from Geomean Trough Basmisanil Plasma Concentrations (ng/mL) by Age Group and Dose. Table showing trough concentrations and estimated receptor occupancy by age group and dose. Table 2: Geomean Trough Basmisanil Plasma Concentration (ng/mL) and Receptor Occupancy by dose. Table showing trough concentrations and estimated receptor occupancy by dose. Table 3: Geomean Trough Basmisanil Plasma Concentrations (ng/mL) in Adolescents and Adults by visit and dose. Table showing trough concentrations by age group, dose, and timepoint.Additional file 9. Diastolic and systolic blood pressure. Table summarizing the change from baseline data at 2 weeks, 3 months and 6 months.Additional file 10. ECG QTcF changes from baseline. Table summarizing the change from baseline at 2 weeks, 3 months and 6 months.Additional file 11. Co-morbid Symptoms: Change from Baseline at 6months. Table summarizing Conner’s, ADAMS and CSHQ data.Additional file 12. Change from baseline score for each assessment by age group and timepoint. Table summarizing change from baseline score by age group and timepoint.


Administration of glucocorticoids might reduce mortality in patients with severe COVID-19 but have adverse cardiometabolic effects.to investigate the effect of systemic administration of glucocorticoids on cardiovascular complications and all-cause mortality in patients hospitalised with respiratory viral infections, including COVID-19, SARS, MERS and influenza.We identified randomised trials published prior to July 28th, 2021. The Mantel-Haenszel random effects method and the Hartung and Knapp adjustment were used to obtain pooled estimates of treatment effect with 95% confidence intervals.No randomised trials of glucocorticoids for SARS, MERS or influenza reported relevant outcomes. We included eleven COVID-19 randomised trials (8109 patients). Overall, compared to placebo or standard care, glucocorticoids were not associated with a reduction of in-hospital mortality (p = 0.09). In a pre-specified sub-analysis, in-hospital mortality was reduced by 19% when follow-up was restricted to 14 days from randomisation . With longer follow-up , administration of glucocorticoids was associated with a trend to benefit for those requiring mechanical ventilation  but possible harm for those not receiving oxygen at randomisation , an effect that was significantly different amongst subgroups (p = 0.0359). Glucocorticoids reduced the risk of worsening renal function by 37% ; reported rate of other cardiovascular complications was low.Administration of systemic glucocorticoids to patients hospitalised with COVID-19 does not lower mortality overall but may reduce it in those requiring respiratory support and increase it in those who do not.  Subsets of patients who might not benefit or be harmed may also exist It is also unclear if the beneficial effects of glucocorticoids on mortality are specific for COVID-19 or apply to other viral respiratory infections. Therefore, we conducted a systematic review and meta-analysis to assess the association between the use of glucocorticoids with cardiovascular complications and mortality in randomised trials that enrolled adults hospitalised with a suspected or confirmed infection caused by SARS-CoV-2, other highly pathogenic and deadly human coronaviruses  or influenza, to elucidate further their safety and effectiveness in these populations.2The protocol of this systematic review and meta-analysis is registered on PROSPERO (CRD42021270992). This study followed the Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA) reporting guideline.2.1We systematically searched for pre-prints and published peer-reviewed studies in Medline (PubMed), Embase (Ovid), the Cochrane Central Register of Controlled Trials (CENTRAL), and the Cochrane COVID-19 register to identify randomised trials that evaluated the use of glucocorticoids in patients with COVID-19, MERS, SARS and influenza. The search strategy consisted of the definition of condition, the intervention applied and the study design including Medical Subject Headings (MeSH) terms and using keyword search limited to the title and abstract. The detailed search strategy is shown in the 2.2We only included peer-reviewed or pre-print randomised trials that enrolled adults (age ≥ 18 years) hospitalised with suspected or confirmed respiratory viral infections including SARS-CoV-2, SARS, MERS, and influenza, which compared any dose or type of glucocorticoids given orally or intravenously, regardless of the duration of treatment, with a placebo or standard care group. Trials comparing only different types or doses of glucocorticoids were excluded, as were observational studies, editorials, letters, reviews, guidelines, case studies, case reports, case series, or cross-sectional studies, abstracts and conference proceedings, systematic reviews and meta-analyses, ongoing studies and articles that were not available in English.2.3The primary outcome was all-cause mortality at the longest follow-up duration. Follow-up between baseline and 14-days and between baseline and 21–28 days was also considered. The secondary outcomes were the incidence of cardiovascular and renal events, including arterial events , venous events (deep vein thrombosis and pulmonary embolism), arrhythmias (supraventricular and ventricular arrhythmias), circulatory failure , cardiometabolic events (hyperglycaemia or need for insulin therapy), and worsening renal function.2.4Included studies were selected in two stages. Initially, two review authors  independently screened all studies by title and abstract, then by full texts. Studies were only included if there was agreement between the two authors.2.5•Study design, size and countries where the randomised trial was conducted.•Baseline disease characteristics, settings and severity , and days from symptom onset to randomisation or hospitalization).•Participants’ baseline demographics .•Participants’ baseline comorbidities .After selection, data were independently extracted by two review authors . Where available, for each study, we collected:Two review authors  independently assessed the quality of evidence using the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) approach. They also assessed the risk of bias on five domains  using the Cochrane Risk of Bias 2.0 (RoB2) tool. Any disagreements in study selection, data extraction, or quality of evidence assessment were escalated to a third review author (PP) who helped in reaching a consensus.2.62 was used. Due to the small number of trials included, the Hartung and Knapp (HK) adjustment was employed. Heterogeneity was firstly assessed through visual inspection of the forest plots, and then estimated using the I2 statistic: we considered an I2 of 25%, 26–50%, and > 50% as low, moderate, and high heterogeneity, respectively. Sensitivity analyses were conducted to assess for robustness of synthesised results and to assess for the impact of including trials at all risks of bias. Subgroup analyses were conducted to investigate the impact of disease severity at randomisation and treatment effect interactions. Publication bias was visually examined using funnel plot . Statistical analysis was performed using the R statistical programming environment, Version 4.02 (http://www.r-project.org). Package meta, Version 4.13 The Mantel-Haenszel random effects method was used to obtain pooled estimates of treatment effect (Risk Ratio - RR) with the associated 95% confidence interval (CI). The DerSimonian-Laird method for estimating the between-study variance t33.1After removing duplicates, we retrieved 4558 records from Embase (Ovid), Medline (PubMed), CENTRAL and the COVID-19 study registry to be screened for inclusion; of these, 321 full-text articles were examined for eligibility. We finally included 11 randomised trials in our meta-analysis (PRISMA Flow Diagram shown below); a large proportion of those excluded were observational studies, editorials, reviews or case reports. We did not identify any randomised trial assessing the use of glucocorticoids in adult patients with suspected or confirmed MERS. We identified two randomised trials assessing the use of prednisone in community-acquired pneumonia, including viral influenza PRISMA Flow Diagram for Search: The flow diagram was created in consort with PRISMA guidelines 3.2We included 11 randomised trials that enrolled 8109 patients . Eight rFive trials Five trials 3.3Overall, 89% of participants were reported to have had a positive SARS-CoV-2 PCR test . The med3.4Risk of bias was assessed for the outcomes of in-hospital mortality and worsening renal function. Five randomised trials 3.4.1Nine trials reported follow-up for at least 21 days and up to 28 days, three of which For the 14-days outcome, comparisons between patients requiring invasive mechanical ventilation (n = 440) at randomisation and those requiring oxygen support (n = 275) did not show any difference in mortality associated with glucocorticoid use (3.4.2We found a low incidence of cardiovascular events (eTable3 in the Supplement). To quantify the extent of under-reporting, we estimated the expected rate of cardiovascular events in patients with COVID-19 using a recent systematic review of prevalence studies as reference We found that, compared to other treatments or placebo, administration of glucocorticoids was associated with a 37% reduction of worsening renal function   and a hi4Glucocorticoids are often administered to patients with respiratory viral infections other than COVID-19 In this systematic review and meta-analysis, we found that administration of glucocorticoids did not significantly reduce mortality for patients hospitalised with COVID-19 overall. However, glucocorticoids may have reduced mortality for sicker patients requiring respiratory support but increased mortality amongst those who did not. This is fairly consistent with the findings of the RECOVERY trial There are many mechanisms by which glucocorticoids might have favourable effects in some patients hospitalised with COVID-19. Uncertainty exists on the extent to which organ damage reflects COVID-19 viral infection itself or the immune response to the virus Previous meta-analyses assessing the effects of systemic glucocorticoids on the mortality of COVID-19 had different designs and showed conflicting results due to substantial heterogeneity in outcomes, the inclusion of both observational studies and randomised trials, the statistical methodology and the inclusion of patients without a confirmed diagnosis of COVID-19 in some reports (eTable5 in the Supplement) We expand previous observations not only by including a greater number of randomised trials, but also reporting that glucocorticoids reduced risk of worsening renal function compared to standard care or placebo by 37%. This could be due to beneficial effects of glucocorticoids on renal endothelial inflammation. Alternatively, by increasing salt and water reabsorption, off-target mineralocorticoid effects of glucocorticoids, plasma volume and systolic blood pressure may increase, thereby maintaining glomerular perfusion and filtration rate.The rate of reported cardiovascular complications in COVID-19 trials was lower than expected based on observational studies There are several limitations to our meta-analysis. The great majority of patients included in this meta-analysis are from the RECOVERY trial. Many trials, such as CAPE COVID, CoDEX and REMAP-CAP, were stopped upon the release of the RECOVERY findings because it was no longer considered ethical to withhold steroids in sicker patients. Other trials have not been published (ie: DEXA-COVID19 (NCT04325061) 5This systematic review and meta-analysis of randomised trials of patients admitted to hospital with COVID-19 suggests that administration of systemic glucocorticoids might reduce mortality for those requiring respiratory support but increase mortality for those who do not. Premature administration of steroids may be harmful in this setting. However, further trials are required to refine the decision on whether to administer or withhold steroids for patients in whom the need for respiratory support is itself uncertain (eg: hypoxaemia that is not yet considered severe enough to require oxygen or patients who respond well to oxygen and do not require ventilator support). Uncertainty also persists about the optimal dose and length of treatment in this very heterogeneous population.EC, AOMR, PP and PM designed the study and drafted the manuscript; JGFC and TG provided supervision; EC and AOMR conducted the statistical analysis, supervised by DB. All authors critically revised and approved the final version of the manuscript.Our work was supported by the British Heart Foundation (BHF) ; the European Research Council [Project Identiﬁer: 726318]; and the Italian Ministry of University and Research (MIUR) PRIN 2017 [2017NKB2N4_003]. EC is supported by a European Society of Cardiology Research Grant.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: The aim of this study is to evaluate the association between a marker of dietary intake, the carbohydrate-to-fiber (CF) ratio, and moderate-to-severe depressive symptoms.Design: Cross-sectional study.Setting: National Health and Nutrition Examination Survey (NHANES) database from 2013-2016.Participants: Individuals 18 years and older were included. Participants with total energy intake outside of three standard deviations of the mean, pregnant or breastfeeding women, and those with missing data were excluded.Measurements: The main independent variable, CF ratio, was generated using corresponding variables in NHANES and divided into quartiles. The main outcome was depressive symptoms using the Patient Health Questionnaire-9. Unadjusted and adjusted logistic regression analyses were used to calculate odds ratios and their corresponding 95% confidence interval (CI). Results: Among all participants , 8.3% reported to have moderate-to-severe depressive symptoms (n=833). The highest proportion of depressive symptoms was reported in respondents in quartile 4 (Q4), with the highest CF ratio . After adjustment, the odds of depressive symptoms significantly increased in Q4 of the CF ratio compared with Q1 . The prevalence of depressive symptoms significantly increased in females, lower federal poverty levels, non-married individuals, smokers, and hypertension patients. Conclusion: This nationally representative sample suggests that a higher CF dietary intake ratio increases the risk of moderate-to-severe depressive symptoms. These results suggest that the CF ratio may help clinicians and patients evaluate their dietary risk for depressive symptoms. Further prospective studies are needed to validate this ratio as a dietary measurement. From 2013-2016, an estimated 8.1% of adults in the United States had Major Depressive Disorder (MDD), making it one of the most common psychiatric disorders in the United States (US) [In addition to the current standards of care, one of the most promising avenues for intervention is via the gut microbiome and gut-brain axis (GBA). Intestinal microflorae have the ability, through digestion and fermentation of the foods, to produce a vast range of neuroactive and cell signaling molecules such as short-chain fatty acids (SFCA), acetylcholine (ACh), catecholamines, gamma-aminobutyric acid (GABA), histamine, melatonin, and serotonin. The gut produces almost 90% of the body’s serotonin . MoreoveGiven this putative pathology, it is plausible that the gut-brain axis (GBA), as mediated by nutrition, may have an impact on mental health. Indeed, preliminary evidence suggests that nutrient intake correlates with mental health outcomes ,9. SpeciThe objective of this study was to investigate whether depressive symptoms may be positively associated with one’s dietary CF ratio. The hypothesis is that as the CF ratio increases, there will be an increase in the prevalence of depressive symptoms related to the pro-inflammatory effects of refined carbohydrates and the protective effects of fiber.Study design and data sourceThis is a cross-sectional study using secondary data from the National Health and Nutrition Examination Survey (NHANES) database from years 2013-2016. The NHANES database aims to determine the health and nutritional status of both children and adults in the United States. The nationally representative data is collected by conducting household visits with a team of physicians, dietary interviewers, and health technicians. NHANES includes a combination of self-reported lifestyle choices and objective medical data collected by the professionals during the visit. This well-recognized and nationally validated database is used to determine lifestyle implications and risk factors on one’s health outcomes . This suStudy populationThis subsample included adults 18 years of age and older who participated in the NHANES survey between the years 2013-2016  dietary intake was the independent variable, as measured by a validated questionnaire. The CF ratio was generated using the daily carbohydrate intake (in grams) divided by the daily dietary fiber intake (in grams). This measure was further categorized into quartiles . The first quartile was then chosen as the reference category given that the USDA recommendation for daily intake is 30g of fiber and 225-325g of carbohydrate, based on a 2000 calorie diet, which fell into the first quartile of this variable .Depressive symptomsThe dependent variable of this study was depressive symptoms and the PHQ-9 questionnaire was used to assess the severity of the depressive symptoms and the likelihood that the patients would meet the criteria for MDD . The PHQ-9 questionnaire is a validated screening tool that includes questions about different areas of mental health. The 9 items on the PHQ‐9 consist of criteria used by the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM‐V) to diagnose a major depressive disorder. Each item is ranked from 0  to 3 (“nearly every day”), with the total score ranging from 0 to 27. PHQ-9 scores greater than 5, 10, 15, and 20 represent mild, moderate, moderately severe, and severe depression, respectively [Covariates2 and categorized according to the Center for Disease Control and Prevention (CDC) criteria into normal weight (<25), overweight (25 to < 30), and obese (≥ 30.0); physical activity as measured by the hours per day of moderate and vigorous work activity and recreational activity; and total energy intake measured by kilocalories consumed [Based on previous literature, the following variables were included to control for the potential effects of confounding . Demograconsumed . Physicaconsumed . Chronicconsumed . PatientStatistical analysisX2 tests. Linear univariate regression was performed to compare the mean levels of total energy intake across quartiles and between participants with and without moderate-to-severe depressive symptoms. Unadjusted and adjusted binary logistic regression analyses were conducted to evaluate the association between CF ratios and the presence of moderate-to-severe depressive symptoms, using the first quartile of the CF ratio as the reference. The multivariable regression model was adjusted for age, gender, race, education, BMI, alcohol consumption, poverty ratio, diabetes, hypertension, marital status, work and recreational physical activity, smoking, and total calorie intake. Odds ratios (OR) and their corresponding 95% confidence intervals were presented and a two-sided p-value of less than 0.05 was considered statistically significant. Data analysis was performed using Stata 15.0 . Sampling weights were applied to generate nationally representative estimates. Baseline characteristics were presented as count and percentage (%) for categorical variables and by means and standard deviation (SD) for variables that were normally distributed; otherwise, median and interquartile range were calculated. Bivariate analysis was performed to identify the association of potential confounders with exposure and outcome variables. Percentages comparisons across categorical variables were performed using The baseline characteristics of the study participants according to the carbohydrate-to-fiber (CF) ratio (divided into quartiles) are presented in Table Unadjusted and adjusted odds ratios with 95% confidence intervals (CI) of moderate-to-severe depressive symptoms in varying CF ratio quartiles using univariate and multivariate models are shown in Table This study revealed that the carbohydrate-to-fiber dietary intake ratio was positively associated with moderate-to-severe depressive symptoms. A higher carbohydrate-to-fiber dietary intake ratio increased the odds of depressive symptoms. In addition, individuals were less likely to report depressive symptoms if they were over the age of 60 years, identified as Mexican, Non-Hispanic African American, or Non-Hispanic Asian race, or got more than four hours per day of recreational physical activity.et al. found that individuals with high dietary quality were more likely to report well-being, no associations between any dietary measures and depressive symptoms were observed [These results are generally in line with the current scientific literature showing that higher levels of nutrient intake correlate with better mental health outcomes, and that fiber is inversely associated with the prevalence of depressive symptoms ,11,16. Oobserved . Finallyobserved ,11,16, bobserved ,11,16. Tobserved .Recent evidence suggests that this relationship between carbohydrates and depression, and fiber and depression may be linked on a biochemical level via the inflammatory response. Patients with chronic inflammatory and autoimmune conditions are at a significantly higher risk of clinical depression -25. On aOne area of emerging interest is intestinal dysbiosis induced by high-glycemic/high-carbohydrate food deficit in dietary fiber . Fibers The secondary findings of this study further support these hypotheses. Notably, increased depressive symptoms were positively associated with an increased risk of developing clinical depression and mood symptoms ,42. SmokNaturally, this study has some limitations. Given the cross-sectional study design, causality cannot be determined. The PHQ-9 uses criteria from DSM-V in order to detect and determine severity of depressive symptoms, making it a widely used tool . Since tIn conclusion, given the connections between dietary fiber and carbohydrates in inducing systemic inflammation and the bi-directional link between inflammation and depressive symptoms, a metric combining these factors may be helpful in identifying a key aspect of dietary risk for depressive symptoms. The results of this study support this hypothesis, finding that participants with increasing carbohydrate-to-fiber ratios may be more likely to report moderate-to-severe depressive symptoms. Additionally, it is well established in previous literature that participants reporting modifiable factors such as sedentary lifestyles, smoking, and heavy alcohol intake are more likely to experience depressive symptoms. Reduction in these activities, in conjunction with dietary carbohydrates to fiber ratio that falls within quartiles 1 and 2, may lead to a decrease in depressive symptoms. These are promising findings as dietary intake is a readily modifiable lifestyle factor. Using a patient’s carbohydrate-to-fiber ratio may give both clinicians and patients better insight into the associations between diet and clinical depression. For clinicians offering dietary counseling, it would be beneficial to all patients, especially those with moderate depressive symptoms, treatment-resistant depression, and those opposed to medications. Future studies should further examine the links between dietary-induced inflammation and other conditions, including bipolar disorder, anxiety, and schizophrenia. Concerning clinical depression, future studies could analyze PHQ-9 scores as a categorical variable and determine the effect of increasing carbohydrate-to-fiber ratios on the severity of depressive symptoms. Since the CF ratio is a new marker, further prospective studies are needed to validate it as a dietary measurement.