Glucose is an important preferential substrate for energy metabolism during acute coronary syndrome (ACS) attack, although insulin resistance (IR) increases during ACS. Increasing evidence indicates that natriuretic peptides (NP) regulate glucose homeostasis. We investigated possible compensatory actions of NP in collaboration with other neurohumoral factors that facilitate glucose utilization during ACS. The study population consisted of 1072 consecutive cases with ischemic heart disease who underwent cardiac catheterization . Among ACS subjects, biochemical data after acute-phase treatment were available in 91 cases, defined as ACS-remission phase (ACS-rem). Path models based on covariance structure analyses were proposed to clarify the direct contribution of B-type NP (BNP) and noradrenaline to glucose and HOMA-IR levels while eliminating confounding biases. In non-ACS and ACS-rem subjects, although noradrenaline slightly increased glucose and/or HOMA-IR levels (P < 0.03), BNP did not significantly affect them. In contrast, in ACS subjects, high noradrenaline was a significant cause of increases in glucose and HOMA-IR levels (P < 0.001), whereas high BNP was a significant cause of decreases in both parameters (P < 0.005). These findings indicate that BNP and noradrenaline coordinately activate glucose metabolism during ACS, with noradrenaline increasing glucose levels, as an energy substrate, while BNP improves IR and promotes glucose utilization. Insulin plays a pivotal role in accelerating the uptake and utilization of glucose in the skeletal muscle as well as in the myocardium. However, we and others have recently reported that insulin resistance (IR) increases in the acute phase of ACS attack6. The activation of various counter-regulatory neurohumoral factors, such as catecholamines, cortisol and cytokines, during ACS attack antagonizes the action of insulin, which leads to high hepatic glucose output and IR and eventually causes the development of stress-induced hyperglycemia7.Although fatty acid oxidation is the predominant metabolic pathway in the normal adult heart, glycolysis and subsequent glucose metabolism become critical for ATP generation and cardiomyocyte survival during ischemic attack of acute coronary syndrome (ACS)8. NPs regulate blood pressure and fluid homeostasis through vasodilatory and diuretic actions and improve cardiac remodeling, mainly through the inhibition of renin-angiotensin-aldosterone and the sympathetic nervous systems9. In addition to its classical action of hemodynamic regulation, there has been increasing evidence to indicate that NPs also regulate energy balance and glucose homeostasis14 as well as thermogenesis in adipose tissues, as recently reported by us and others16. Furthermore, one study investigating the direct effects of NPs on systemic glucose regulations in humans showed that B-type NP (BNP) administration reduces the plasma glucose concentration17. In addition, another in vitro study showed that NP increases the glucose uptake during hypoxia in cardiomyocytes18. Furthermore, Coué et al. very recently reported that NPs promote the uptake of glucose and enhance glucose metabolism in human adipocytes19.Natriuretic peptides (NPs) are produced in the heart and classically act on the renal and cardiovascular systems20. A covariance structure analysis is therefore useful for eliminating confounding biases and clarifying possible cause-and-effect relationships25.Given these previous findings, we hypothesized that the compensatory activations of neurohumoral mechanisms, such as NPs and noradrenaline, facilitate glucose utilization, which overwhelms the IR condition during ACS. One of the difficulties faced in researching the correlations between NPs or noradrenaline and glucose is the intractableness of the statistical approach, as these factors are associated with each other  as well as numerous other factors , namely confounding variables. Although there are several ways of eliminating these confounding biases, it is still difficult to control for them if multiple potential confounders are present or if the study population is of insufficient sizeUsing a covariance structure analysis together with conventional single and multiple regression analyses, this study was designed to evaluate the possible regulation of plasma glucose levels as well as the degree of IR by noradrenaline and BNP in patients with ACS compared to those with non-ACS.26. The plasma BNP was only slightly higher, while the plasma noradrenaline was markedly higher in the ACS subjects than in the non-ACS subjects. The LVEF was lower, while the blood pressure was higher in the ACS subjects than in the non-ACS subjects. Among ACS subjects, biochemical data after acute-phase treatment were available in 91 cases, defined as the ACS-remission phase (ACS-rem) (see Methods section). The levels of glucose, insulin, HOMA-IR, BNP and noradrenaline were all decreased during the remission phase of ACS compared to during ischemic attack  various other clinical parameters were also included as potential factors affecting plasma glucose levels and HOMA-IR. The paths between variables were drawn from independent variables to dependent variables (cause-and-effect relationships) with directional arrows for every regression model.The precise results of the path models are shown in Tables To visualize the relationships among pairs of estimands, bivariate marginal posterior distributions are shown Fig. . The neg19. Using covariance structure analyses, the present study showed that noradrenaline increases plasma glucose levels (possibly supplying energy substrate), while BNP decreases glucose levels (possibly promoting glucose utilization) and substantially improves IR in ACS subjects but not in non-ACS or ACS-rem subjects. These findings suggest that BNP and noradrenaline act cooperatively to facilitate glucose utilization during an ACS attack, but those collaborative actions become ambiguous during the stable ischemic phase.In addition to the classical actions of NPs on cardiovascular systems, such as hemodynamic regulation, recent studies have indicated that NPs also regulate energy balance and glucose homeostasis25. We successfully proposed a path model based on a covariance structure analysis in order to clarify the causative role of either BNP or noradrenaline in the regulation of plasma glucose and HOMA-IR levels with consideration of the inherent correlation between BNP and noradrenaline themselves. In fact, no significant correlation was observed between BNP and glucose in our simple regression analysis. However, when using a covariance structure analysis, which eliminates potential cofounding factors, the negative correlations between these two factors were clearly delineated only in the ACS subjects , with a significant positive correlation preserved between BNP and noradrenaline in both ACS and non-ACS subjects , particularly during ACS attack –cyclic GMP (cGMP)-PKG-Akt-dependent pathway and subsequently promote lipolysis through the same GC-A-cGMP-PKG pathway, as reported previously29. They also reported that the activation of the NP receptor-cGMP pathway in skeletal muscle reduces lipotoxicity and improves the sensitivity of insulin-Akt signaling in IR model mice, although these are relatively chronic effects11. The activation of Akt signaling, a major downstream target of insulin, by NPs was also previously reported by others31. Interestingly, those studies showed that NPs induce Akt signaling activation in cardiomyocytes and exert cardioprotective effects. Previous studies also suggested that NPs improve the glucose metabolism and IR via AMPK signaling pathway in failing heart tissues32. Meanwhile, we and others reported that NPs reduce oxidative stress and inflammatory activity both in vitro and in vivo, leading to an improvement of IR35. Finally, two studies investigated the direct effects of NPs on the regulation of the cardiac glucose metabolism by showing that A-type NP increases myocardial glucose uptake during hypoxia through the upregulation of glucose transporters 1 and 436. Thus, in the present study, it is possible that BNP also promoted the uptake of glucose and its metabolism in the myocardium during an ACS attack directly and/or indirectly through the decrease in IR and the improvement of insulin sensitivity. Although the underlying mechanisms by which NPs reduce the glucose level only during the acute phase of an ACS attack remain incompletely understood, the present study at least shows that BNP is able to induce temporary shifts in the whole-body response to glucose and insulin during ACS. Future research is warranted to further explore the mechanistic link between NP signaling and glucose metabolism .In the meantime, increasing attention is being paid to the possibility that NPs stimulate triglyceride lipolysis, induce adipose tissue browning and activate the thermogenic programThe sample size was somewhat small in the present study. However, a covariance structure analysis as well as a conventional multiple regression analysis consistently showed the contrasting influences of noradrenaline and BNP on glucose and HOMA-IR only in the ACS subjects, with comparable standard regression coefficients. Furthermore, only a limited number of patients actually showed significantly increased BNP levels during ACS in the present study. However, we applied a Bayesian SEM using a program embedded in the IBM SPSS AMOS software program . Although SEM applies the robust-weighted least-square approach, the Bayesian SEM implements the Gibbs sampler algorithm. Bayesianism permits uncertainty despite little information being available, and Bayesian approaches allow us to incorporate background knowledge into the analyses. We believe that additional testing with Bayesian SEM would be rationalized and helpful for reassessing our data from a completely different angle of statistics. The selected two-dimensional contour line was used in this study because it was easily visualized Figs .There is another limitation of this study. Although all patients with ACS in the present study fulfilled the diagnostic criteria, in some cases, the clinical conditions of cardiac catheterization were slightly different between acute myocardial infarction and unstable angina pectoris. Namely, short-term stabilization with medical treatment, such as the use of anti-angina agents, was preferentially performed prior to emergent catheterization in some  cases, particularly in cases of unstable angina. Thus, the clinical and pathophysiological conditions at the time of emergent catheterization in patients with unstable angina were more diverse than those in patients with acute myocardial infarction, for which coronary angiography tended to be performed in time-sensitive situations. Accordingly, covariance structure analyses using path models A or C revealed that high BNP was a significant cause of the more pronounced decreases that were observed in the glucose and HOMA-IR levels of patients with acute myocardial infarction , whereas the finding that high noradrenaline was a significant cause of the consistent increases in both parameters . These data reinforce the hypothesis that collaborative activities of BNP and noradrenaline are involved in glucose utilization, particularly during the acute phase of an ACS attack.37.The chronic persistent action of NPs has been shown to induce adipose tissue browning, lipolysis and thermogenesis as well as muscle mitochondrial biogenesis, which leads to the reduction of IR and the improvement of insulin sensitivity. However, we showed in the present study that the acute action of BNP, in collaboration with noradrenaline, promotes glucose utilization with substantially improved IR in patients with ACS. These findings highlight a previously underappreciated role of NPs in glucose metabolism, which overwhelms the increased IR during ACS attack. The administration of agents that increase circulatory natriuretic peptides levels during the acute phase of ACS may provide therapeutic benefitsPatients with IHD who underwent cardiac catheterization in our institution for the evaluation of coronary organic lesions from May 2015 to March 2018 were included in this study.20. Organic lesions producing ≥75% luminal stenosis of the coronary arteries on coronary angiography were defined based on the modified American Heart Association coronary tree segment classification. Patients with coronary spastic angina were also included. ACS was defined as the presence of myocardial infarction (MI) or unstable angina pectoris, as described in detail previously26. All patients with ACS underwent cardiac catheterization within 24 hours of the onset. Patients were excluded if they were receiving or beginning to receive dialysis, were in cardiogenic shock (Killip grade IV) or had been treated with insulin. Based on these selection criteria, 1072 consecutive cases, including 216 cases with ACS (112 with MI), were enrolled in the present study , and we complied with the routine ethical regulations of our institution. All patients provided their written informed consent before undergoing the procedure, and all clinical investigations were conducted in accordance with the principles expressed in the Declaration of Helsinki. According to our routine ethical regulations, we also posted a notice about the study design and contact information at a public location in our institution.20. Some of the patients had comorbid cardiovascular diseases, such as valvular disease, arrhythmia, cardiomyopathy and other conditions. Hypertension, diabetes mellitus (DM) and dyslipidemia were defined as described previously20. HOMA-IR and the estimated glomerular filtration rate (eGFR) were calculated as described previously26. The left ventricular ejection fraction (LVEF) was measured at the time of left ventriculography20.The clinical characteristics were collected retrospectively from the hospital medical records. We collected blood samples and hemodynamic data during cardiac catheterization. Plasma and serum biochemical analyses, including the levels of glucose, BNP, and noradrenaline, were performed in a central laboratory of our hospital during the study periodContinuous variables are expressed as the mean ± standard deviation. To compare the clinical characteristics between the ACS and non-ACS subjects, the statistical analyses were performed using the chi-square test for categorial variables and the Mann-Whitney U test for continuous variables. The correlation between the plasma glucose level, BNP and noradrenaline in each group was investigated by a simple regression analysis and expressed as Spearman’s correlation coefficient. Plasma glucose levels were compared among four groups divided by the indicated BNP levels with the Mann-Whitney U test. A multiple regression analysis was performed to compare multiple values. The above-mentioned statistical analyses were performed using the SPSS Statistics software program . P-values of < 0.05 were considered to indicate statistical significance.25. The causality model defines some hierarchical regression models between clinical factors and plasma glucose level or HOMA-IR. For every regression, the total variance in the dependent variable is theorized to be caused by either independent variables that are included in the model or by extraneous variables (e)25. The path analysis was performed using the IBM SPSS AMOS software program . The obtained structural equation models (SEMs) were tested and confirmed at a significance level of P < 0.05.A path analysis based on a covariance structure analysis was used to elucidate the contribution of BNP and noradrenaline to the plasma glucose level and HOMA-IR, respectively, in patients with and without ACS. This analysis compares the power among multiple independent variables that confound each other, and specifically identifies probable causal effects on either the plasma glucose level or HOMA-IRIn addition, we applied a Bayesian SEM using a program embedded in the IBM SPSS AMOS software program . The frequency polygon was described with the marginal posterior distributions of the estimands. The selected two-dimensional contour line was used in this study because it was easily visualized.Supplementary Information


Tight regulation of cellular processes is key to the development of complex organisms but also vital for simpler ones. During evolution, different regulatory systems have emerged, among them RNA-based regulation that is carried out mainly by intramolecular and intermolecular RNA–RNA interactions. However, methods for the transcriptome-wide detection of these interactions were long unavailable. Recently, three publications described high-throughput methods to directly detect RNA duplexes in living cells. This promises to enable in-depth studies of RNA-based regulation and will narrow the gaps in our understanding of RNA structure and function. In this review, we highlight the benefits of these methods and their commonalities and differences and, in particular, point to methodological shortcomings that hamper their wider application. We conclude by presenting ideas for how to overcome these problems and commenting on the prospects we see in this area of research. They interact with proteins, RNA, and DNA and thereby interconnect different regulatory systems. The elucidation of the underlying networks is thus of central importance for a holistic understanding of cellular regulation. Computational prediction based on sequence alone turns out to be unsatisfactory, and, with the availability of second- and third-generation sequencing technologies, data-based inference of regulatory networks involving ncRNAs is the more promising approach. RNA–protein interactions are more or less routinely studied using crosslinking immunoprecipitation-sequencing (CLIP-seq)6, represent an important first step toward this goal. In the following, we will discuss their pros and cons and present our view on what future improvements may look like and what their impact might be.A large fraction of ncRNAs carry out their function through complementary base pairing to other RNAs, often mRNAs. This may involve proteins, as in the case of the RISC complex for microRNA (miRNA)- or small interfering RNA (siRNA)-mediated post-transcriptional gene silencing, but the ncRNA is the main factor that specifies the target RNAs via base complementarity. Thus, unbiased methods for the transcriptome-wide discovery of direct RNA–RNA interactions are needed to decipher RNA regulatory networks. Approaches developed in 2016, described as Direct Duplex Detection (DDD) methodsin vitro. Second, there is no technology available that can sequence interacting RNA strands and maintain their relationship. The first approach to tackle these problems (in a rather coarse way) was described by Ramaniet al.7. They presented RNA proximity ligation (RPL), in which interacting RNA strands are ligated to each other in a standard ligation reaction using RNase inhibitor-treated crude cell extracts followed by RNA-seq. The resulting sequencing reads contained chimeric reads but to a rather low fraction (0.28%). Nevertheless, the proximity ligation approach presents a solution to the abovementioned second problem because it allows one to trace back RNA–RNA interactions in data sets obtained by next-generation sequencing technologies.RNA–RNA interactions pose two basic problems when analyzing them. First, they are commonly disrupted during the process of RNA extraction and cannot be reconstituted to their native statein vivo crosslinking of RNAs has been known since the 1970s and successfully used to interrogate the structure of ribosomal RNAs, interactions between small nuclear RNAs, and several other RNA structures and interactions20.The remaining problem of losing RNA–RNA interactions due to the harsh RNA extraction protocols is as long-standing as its solution: direct RNA crosslinking via psoralen derivatives. Following irradiation, psoralens covalently and reversibly connect two pairing stretches of RNA. Thus, duplexes are maintained during RNA extraction, which enables their enrichment, for example, by nucleases that digest single-stranded RNAs. The psoralen-mediated21, psoralen analysis of RNA interactions and structures (PARIS)22, and sequencing of psoralen-crosslinked, ligated, and selected hybrids (SPLASH)23. Although they differ in several steps of their experimental protocols, they all share the same principal design, which isin vivo RNA crosslinking followed by RNA extraction, enrichment of crosslinked RNAs, proximity ligation, and sequencing via RNA-seq. A comparison of the individual methods is shown inThe combination of the two aforementioned strategies is the hallmark of the currently available DDD methods, namely ligation of interacting RNA followed by high-throughput sequencing (LIGR-seq)24, meaning that interactions in GC-rich regions may be under-represented. Nevertheless, the chance that uracil residues are neighboring another base pair is 25%, assuming a uniform distribution of all possible pairs of bases.A common bias that occurs when psoralen is used to crosslink double-stranded RNA (dsRNA) is its preference to intercalate into adjacent opposite pyrimidine bases, preferably uracilThe major problem of all of the abovementioned methods is that the proximity ligation step is highly inefficient and that it is not possible to perform an RNA-seq library preparation that enriches for or specifically targets the ligated RNAs. Furthermore, the proximity ligation step does not exclusively ligate interacting RNA strands but also all kinds of single-stranded RNAs in a random fashion. As a result, the yield of RNA–RNA interaction informative reads is very low, as shown in21. Data from PARIS support the results of Linet al.25, who discovered that the interactions within the long ncRNA NEAT1 have an important architectural function in paraspeckle formation and thus impact the regulation of gene expression. In addition, PARIS could detect inter-repeat duplexes in the long ncRNA XIST, which is essential for X chromosome silencing22. Furthermore, the use of SPLASH has uncovered the RNA–RNA interactome of influenza A viruses required for virus growth26; this might facilitate the prediction of the emergence of new pandemic influenza strains.Nevertheless, the presented approaches provided interesting and deep insights into the RNA–RNA interaction networks of the studied organisms and the respective growth conditions. With the help of these DDD methods, already experimentally verified RNA structures could be accurately recapitulated; in addition, novel RNA structures as well as new RNA–RNA interactions were revealed. For instance, LIGR-seq identified novel interactions between small nucleolar RNAs (snoRNAs) and mRNAs, including the snoRNA SNORD83B that controls the steady-state levels of its target mRNAsin vivo. Nevertheless, there is still room for improvement. A stricter enrichment of crosslinked RNAs by combining enzymatic digestions (LIGR-seq) with two-dimensional gel-electrophoretic separation (PARIS) or biotinylated-psoralen selection (SPLASH) or both could eliminate the undesired fortuitous ligation of single-stranded or non-crosslinked RNA duplexes.Even based on the highly inefficient proximity ligation, the currently available DDD methods have been able to capture and analyze crucial RNA–RNA interactions in a transcriptome-wide fashion andThe efficiency of the proximity ligation could be enhanced with non-complementary overhangs. These may be either left over by a limited nuclease digestion or introduced synthetically before ligation, for example, by polyA tailing or by using different terminal transferases. Such duplex overhangs are more likely to be ligated and could lead to improved yields of interaction-informative sequencing reads.27. However, to the best of our knowledge, the results have never been reproduced, nor was the method used in further studies. In addition, future discoveries might provide engineered DNA ligases with substrate promiscuity to increase the dsRNA ligation efficiency. Eventually, a direct ligation of dsRNA adapters would pave the way for novel and hopefully more-efficient DDD protocols.The most straightforward approach to turn RNA duplexes into sequencing templates would be a direct dsRNA ligation method. So far, only one report has proposed using RNA and DNA ligases for the enzymatic joining of two or more dsRNA moleculesLikewise, the bioinformatics pipelines for basic data analysis and statistical inference of interactions need to be improved. Currently, none of the existing software packages considers the actual complementarity of the putative interaction partners but relies only on statistical measures. Furthermore, RNA–RNA interactions are driven by base-pair formation, for which thermodynamic parameters and kinetic models exist. Integrating these aspects into the analyses may additionally improve the reliability and robustness of data analysis.28, schizophrenia29, and various cancer types30 such as leukemia31, breast cancer32, or lung cancer33). Thus, there is and will be a wide application range for first- and next-generation DDD methods that have the potential to provide important contributions in areas of basic and applied sciences.In conclusion, the currently available DDD methods pave the way to a deeper understanding of the so-far little-studied RNA–RNA interactome. Crucial for their future acceptance and success will be to increase the yield of informative reads, which would allow RNA regulatory networks to be deciphered in greater depth. Results from such studies could have a great impact on many research areas. For example, a lot of human diseases are associated with ncRNAs (for example, Alzheimer’s disease


Engineering cell surfaces with natural or synthetic materials is a unique and powerful strategy for biomedical applications. Cells exhibit more sophisticated migration, control, and functional capabilities compared to nanoparticles, scaffolds, viruses, and other engineered materials or agents commonly used in the biomedical field. Over the past decade, modification of cell surfaces with natural or synthetic materials has been studied to exploit this complexity for both fundamental and translational goals. In this review we present the existing biomedical technologies for engineering cell surfaces with one important class of materials, polyelectrolytes. We begin by introducing the challenges facing the cell surface engineering field. We then discuss the features of polyelectrolytes and how these properties can be harnessed to solve challenges in cell therapy, tissue engineering, cell-based drug delivery, sensing and tracking, and immune modulation. Throughout the review, we highlight opportunities to drive the field forward by bridging new knowledge of polyelectrolytes with existing translational challenges. The term “cell surface engineering” refers to the modification of cell surfaces with synthetic or natural materials to modulate cell function. The purpose of this review is to describe how polyelectrolyte materials can be used in cell surface engineering to address some of the clinical challenges in translational ideas from the benchtop to the clinic. We begin with a brief background on cell surface engineering, along with highlights of the current clinical opportunities. Next we introduce polyelectrolytes and present current and future opportunities for this class of materials to be exploited in overcoming challenges in the biomedical field by pushing the forefront of cell surface engineering.Many biological surfaces are composed of combinations of lipids, proteins, and carbohydrates that serve as the primary means by which cells transmit environmental cues for signaling. These cascades signal to and between cells to perform specific functions. The rapid development of new biological tools has helped to identify a plethora of pathways that can be harnessed to direct such processes. Cell receptors and their respective ligands, downstream signaling pathways, and external stimuli can be exploited to regulate cellular functions spanning survival, proliferation and differentiation, and metabolism. Cell surface engineering utilizes engineering, chemistry, and biology techniques to conjugate cell surface molecules with natural ligands, functional biological components, or synthetic materials.Surface-engineering is already being applied to many clinical areas using approaches that include polyelectrolytes and many other strategies. Conjugating exogenous ligands  onto cell surfaces can alter molecular pathways within cells, causing changes in functions such as cell adhesion, migration, and proliferation ,2,3. ForDespite the potential of cell surface engineering, enormous challenges remain in designing effective and clinically-translatable technologies and therapies. Surface engineering should not impact the viability of the cells, and surface grafting or conjugation procedures must be performed without hindering normal cell functions or interactions between surface proteins or ligands. Surface modifications should also minimize altering cell membrane properties, such as the viscosity and elasticity of lipid bilayers, unless this is the goal. In addition, the technologies need to avoid exposure of cells to harsh conditions such as heat, extreme pH, shear force, and organic solvents. Unfortunately, these challenges are exacerbated by the diverse conditions that cells function in, the varied membrane compositions, and the contrast between the synthesis conditions of synthetic materials and cells. Thus, one of the major challenges in cell surface engineering is modifying specific cell surface molecules without compromising the normal functions or properties of other molecules, and the cells more generally.As already alluded to, cells have a sophisticated dynamic structure, creating additional challenges for cell surface engineering . For exaGenetic engineering is broadly studied as a technology for modulating cell functions and involves introducing or knocking out specific genes or target receptors ,25. SomeMany classes of materials have been explored for engineering cell surfaces, ranging from biological molecules such as proteins, to synthetic materials including scaffolds, magnetic or drug-carrying nanoparticles, and polyvalent molecules or polymers . These mPolyelectrolytes are materials that contain a high level of ionizable groups along a polymeric backbone. These materials can be anionic, cationic or amphiphilic in charge, and include both synthetic and naturally occurring cargos. Due to their unique properties, polyelectrolyte materials have been widely used in the biomedical field, but this review is focused on their use in cell surface engineering ,30. Layel-lysine (PLL)) onto cell surfaces. By alternating exposure of cells to polyanions and polycations, cells can be deposited with a controlled number, thickness, and concentration of cargo layers. However, direct deposition of polycations onto the cell surfaces can result in cell membrane damage that hinders the translational potential . D. Dl-lysidiabetes B. To unddiabetes B. The auN-vinylpyrrolidone) (PVPON) and tannic acid (TA)  C. Interecid (TA) D, top. Icid (TA) D 44]. T. TN-vinycid (TA) D, bottomKeeping cells alive during cell surface modification is a prerequisite for most biomedical applications. The above studies demonstrated that polyelectrolyte materials provide a material library for engineering cell surfaces while maintaining viability. Additionally, recent studies reveal polyelectrolyte structure and molecular weight can influence performance for in vivo applications, especially in the immune system. For example, Andorko et al. demonstrated that poly(β-amino esters)—polycationic materials—activate dendritic cells and influence antigen presentation in a molecular weight-dependent fashion in both cells and mice . While tIncorporating functional materials into PEM shells can alter cell growth and lead to unique capabilities that cannot be achieved with unmodified cells. For example, incorporating silica nanoparticles into PEM shells slows cell growth ,50. YangCell therapy is a quickly expanding field in translational medicine ,76. ClinClinical and preclinical diabetes trials have sought to replace conventional insulin injections with islet implantation. While islet transplantation has shown promising results, a significant barrier is that the infused islets do not maintain long term function. This is due to a combination of delayed revascularization, host inflammatory response, and innate and adaptive immune responses ,83. As dEncapsulating islet cells in bulk materials can hinder transport through the portal vein of the liver—another challenge in pancreatic islet transplantation . CompareThe idea of implanting islet cells for diabetes treatments has existed for over 30 years. Most techniques have focused on developing a semipermeable passive membrane that can protect the islets from the complicated in vivo environment within the recipient . PolyeleAlong these same lines of control, the Jewell Lab has developed novel PEMs composed entirely from immune cargos, such as antigenic peptide and toll-like receptor ligands—RNA or DNA that exists in virus or bacteria but not in human—to promote or block specific immune functions ,39,76,90Beyond islet cells, surface engineering is being explored to modify stem cells for treating central nervous system disorders . In one In another recent report, Zhao and coworkers incorporated pluripotent stem cells (PSCs) into a matrix composed of alginate and chitosan for myocardial infarction  treatment. They found that the polyelectrolyte matrix reduced cell damage during injection partially due to the enhanced cell surface modulus from the polyelectrolyte matrix. In mice, cells protected with polyelectrolyte matrix maintained higher survival levels than non-protected cells (50% vs. 20%). Importantly, the polyelectrolyte-modified PSCs significantly reduced the fibrotic area that often thickens as a result of damage during heart attack C. FurtheEarly clinical studies have made significant progress in using different types of stem cells to address a spectrum of diseases. For example, Limbal stem cells are registered in Europe for the treatment of eye burns . In anotMany tissue engineering applications involve assembling cells into solid supports or scaffolds that promote adhesion, proliferation, and in some cases, polarization toward desired functions. Hydrogels are one of the most commonly used supports, but surface modification is often necessary for cell adhesion. Despite their wide-spread use, a potential issue with hydrogel-based tissue engineering is the failure to promote the effective diffusion of the necessary chemical and biological cues into the core part of the gel . The inhl-lactic acid) (PLLA) were surface modified to promote cell attachment  and biocompatible polyelectrolytes B 57]. U. U57]. UCell surface engineering with polymers also presents a powerful approach to promote or control cell aggregates and adhesion for tissue engineering. Pasparakis et al. used a thermo-responsive polyelectrolyte to assemble cells into cellular aggregates/spheroids in a kinetically-controlled manner A 61,62],6261,62]Cell surface engineering can be used to label cells for the purpose of tracking and sensing both in vitro and in vivo. Zhang et al. functionalized cell surfaces with a disk-shaped PEMs patch composed of polyelectrolyte and gold nanoparticles for surface-enhanced Raman scattering (SERS)-based cell tracking . Gold naIn clinical trials, several types of cells are being investigated as drug delivery vehicles, such as erythrocytes , for example ,97,98. CSome of the same PEM cell modification technologies being explored for cell adhesion see , are alsZhang et al. developed thermoplastic-PEM hybrid particles that were attached onto cell surfaces for unidirectional drug delivery to cells . In thisWhile the sections above used a PEM patch on a portion of a cell, fully encapsulating cells with PEMs shells loaded with drugs are also being explored for delivery. Lin et al. employed PEM shells to encapsulate dermal papilla cells—highly specialized mesenchymal cells located in hair follicles—for hair regrowth . The PEMIn the area of cancer, cell-laden microcapsules have also been explored as therapeutics. Sakai et al. for example, engineered cells to express an enzyme that activates a cancer therapeutic . These cThe above studies illustrate the use of polyelectrolyte materials to engineer cell surfaces for drug delivery purposes. PEM patches or shells can also be used to control drug diffusion from cells compared to cells being used as carriers after loading with a drug. Importantly, entrapping the therapeutic cargos in the PEM patches or shells can help minimize the alteration of cell functions that occurs when drug is loaded directly in cells. Further, PEM structures can incorporate multiple therapeutic cargos and potentially generate synergistic therapeutic effects, with improved targeting through cell homing.The immune system can be activated with stimulatory cues  to generate efficient and specific responses against infectious diseases and cancer. In recent years the use of biomaterials for immunotherapies and vaccines has dramatically increased. Many studies demonstrate polymeric materials can activate pro-inflammatory pathways  as a result of their intrinsic physicochemical properties ,103,104.In addition to emerging technologies such as iPEMs, recent studies have also demonstrated that polyelectrolytes and other materials can assist in avoiding host immune rejection. Both Wilson et al. and Kozlovskaya et al. used PEM shells to protect islet cells from immune rejection during diabetes treatment . In diabOne of the most exciting ideas for cancer therapies is a vaccine like-approach in which a patients’ immune system is exposed to fragments of tumor cells to generate T cells that can destroy tumors. For an effective targeting of tumor tissues, tumor associated antigens need to be processed by the immune cells to activate this anti-cancer immunity. However, many cancer antigens are still unidentified and will mutate continuously, making this idea difficult to implement thus far. Toward addressing this hurdle, the De Geest group used PEM-coated melanoma cells (B16-F10) for modulating immunogenicity against cancers. Prior to cellular encapsulation, the cells were exposed to heat to enhance the expression of heat shock proteins—signals that then increased the immunogenicity of the tumor cells, and upon injection, might lead to enhanced anti-tumor immunity . Thus, tWays to manipulate less specific mechanisms of the immune system are also being developed, for example, the complement system—a series of pathways that help remove bacteria, toxins, and other harmful agents. Leung et al. demonstrated that engineering red blood cells with different concentrations of hyper-branched polyglycerol (HPG) can be used to control which parts of the complement system are activated . In one Together, these studies highlight some of the opportunities to use polyelectrolytes to modulate immune function. This concept can be expanded to broader applications as well. For example, intra- lymph node injections of myelin self-antigen and a regulatory signal (rapamycin) was recently shown to permanently reverse paralysis associated with Multiple Sclerosis (MS) after just one treatment at the peak of disease . Polyelen-hydroxysuccinimide (NHS) ester. This strategy has been used to couple polyethylene glycol (PEG) onto islet surfaces to prevent the cells from the host immune reactions [x, a selectin glycoprotein ligand, is reacted with stem cell surface proteins that control cell homing through a covalent streptavidin-biotin bridge to improve in vivo targeting of stem cells [In addition to polyelectrolyte-based cell surface engineering, other strategies including chemical modification and genetic engineering are also being used to engineer cell surfaces for translational applications ,108. Celeactions . Similarem cells . Using aem cells . While tGenetic engineering is also used to modulate cell surfaces by introducing or knocking out specific genes or cell receptors ,111; thiThe examples above demonstrate the potential polyelectrolyte-based cell surface engineering has for many translational areas. Compared to genetic-based cell surface engineering, polyelectrolyte strategies avoid tedious protocols; instead relying primarily on facile chemical and engineering techniques. Additionally, most polyelectrolyte materials can be used in aqueous solution, thus avoiding the exposure of cells to harsh organic solvents. Polyelectrolytes also offer easy-to-tailor molecular structures, for many material options and flexibility for specific applications. However, realizing these opportunities will require greater emphasis on developing and testing polyelectrolyte cell modification in clinically-meaningful models, and ultimately in human settings. In this article we have highlighted areas where these investments might lead to the biggest gains.


Streptococcus (GBS) vaccine for maternal immunization constitutes a global public health priority, to prevent GBS-associated early life invasive disease, stillbirth, premature birth, maternal sepsis, adverse neurodevelopmental consequences, and to reduce perinatal antibiotic use. Sample size requirements for the conduct of a randomized placebo-controlled trial to assess vaccine efficacy against the most relevant clinical endpoints, under conditions of appropriate ethical standards of care, constitute a significant obstacle on the pathway to vaccine availability. Alternatively, indirect evidence of protection based on immunologic data from vaccine and sero-epidemiological studies, complemented by data from opsonophagocytic in vitro assays and animal models, could be considered as pivotal data for licensure, with subsequent confirmation of effectiveness against disease outcomes in post-licensure evaluations. Based on discussions initiated by the World Health Organization we present key considerations about the potential role of correlates of protection towards an accelerated pathway for GBS vaccine licensure and wide scale use. Priority activities to support progress to regulatory and policy decision are outlined.The development of a group B  According to recent global disease burden estimates, GBS causes 319,000 cases of early life invasive bacterial disease and 90,000 infant deaths annually. While data gaps hinder precise estimates for other disease syndromes, a conservative assessment suggests that yearly, 57,000 stillbirths are caused by GBS and 33,000 pregnant or puerperal women have GBS sepsis. There is also a significant, unquantified burden of GBS-related prematurity, neurodevelopmental impairment and maternal deaths. The African continent accounts for an estimated 54% of cases of GBS invasive disease and 65% of all foetal/infant GBS deaths The reservoir for GBS in humans is the gastrointestinal tract, and maternal recto-vaginal colonization can lead to ascending foetal infection, stillbirth and early onset neonatal sepsis. Maternal colonization in pregnancy (ranging from about 10% to 40%) has been found in women in all geographical settings evaluated 1.2Panel 1Two large pharmaceutical companies, GlaxoSmithKline (GSK) and Pfizer, are building on their experience in polysaccharide-protein conjugation technology to develop GBS vaccines. Ten GBS capsular envelope polysaccharide (CPS)-based serotypes have been described, however, the majority of invasive disease in young infants is caused by a few of these serotypes  A BMGF-funded partnership has also been announced between PATH and The Biovac Institute, a South-African government-private partnership, aimed at developing in-country manufacturing capacity. This vaccine development programme will aim for the development of a low-cost conjugate GBS vaccine against serotypes causing >90% of invasive GBS disease in Africa.Minervax, in contrast, is developing a protein-based GBS vaccine, targeting the N-terminal domains of the Alp-protein family. The induction of functional activity and protection from protein-based GBS vaccines have been investigated in preclinical infection models, and further sero-epidemiological studies are required to confirm whether immunity targeting these proteins derived from natural exposure is associated with protection against early life invasive disease A summary of the GBS vaccine development landscape is presented in 1.3The conduct of double-blind placebo-controlled randomized trials to assess vaccine efficacy against clinically relevant, pre-defined, endpoints constitutes the gold-standard approach to generate evidence for vaccine licensure and policy decisions. However, a pivotal GBS vaccine efficacy trial would require a very large sample size, as shown in 1.4If a pre-licensure pivotal randomized controlled clinical efficacy trial is not possible, on grounds of cost or feasibility, alternative strategies to support licensure need to be considered. The aim of this report is to highlight the potential role that immune correlates of protection may have for an accelerated GBS vaccine development pathway to licensure and policy decision. Related, priority activities to are outlined in General WHO considerations on methods leading to definitions of correlates of vaccine-induced protection have been presented elsewhere 1.5in vitro bacterial killing assays; and ultimately human efficacy, effectiveness studies.Correlates of protection have played an important role in supporting the licensure of several conjugated vaccines targeting encapsulated bacteria Panel 2Haemophilus influenzae type b (Hib) infections showed that serum bactericidal activity (SBA) increased with age in vitro, and a lower antibody threshold of protection than for the polysaccharide vaccine. Post-immunization with a protein conjugate Hib CPS vaccine, a peak antibody concentration of 1 μg/mL is considered as a marker of long-term protection. Prevalent concentrations above 0.15 μg/mL are considered as protective against Hib bacteraemia and are accepted as such by regulators for licensure of new vaccines or for bridging studies As early as 1933, epidemiological studies of Epidemiological studies in the 1960′s similarly showed an association between age and anti-capsular groups A, B and C meningococcus serum complement-dependent bactericidal assay (SBA), and between the latter and the risk of meningitis More recently, a non-capsular, outer membrane protein (OMP)-based meningococcal group B vaccine was licensed, based on indirect evidence of efficacy. Vaccine efficacy had been demonstrated in the 1990s for two OMP-based vaccines derived from outer membrane vesicles (OMV), and the association with hSBA responses (h indicative of human complement use) had been shown Randomized controlled trials initially established the protective efficacy of the 7-valent pneumococcal conjugate vaccine (PCV), which allowed the derivation of serotype-specific serum antibody protective thresholds for some serotypes, but not for the less common ones. Although there was no evidence demonstrating that protective thresholds were the same for different serotypes, it was considered acceptable to estimate an aggregate threshold through data pooling, serving as target for all serotypes Correlates of protection were initially established in efficacy studies of PCV against all invasive pneumococcal disease (including bacteraemia and meningitis), but true threshold might differ according to disease entities. Protective levels associated with protection against acute otitis media may be higher When putting into perspective past experience with other vaccines and considerations on substitute immunological endpoints for GBS, important similarities and differences can be highlighted. Often, the mechanism of immunity derived from natural exposure and vaccine-induced immunity is similar, and based on functional antibodies, but protective thresholds can differ according to antibody qualitative characteristics. Thresholds may also vary by pathogen strain/serotype and clinical syndrome. In all the non-GBS examples discussed in 22.1in vitro killing assays were used. While antibody-mediated risk reduction estimates have been reported from different studies for the most frequent serotypes , rigorous estimates of protective thresholds have not been established. There were important differences in the design of the various studies, which were conducted across diverse populations and epidemiological settings, with different standards of care, studying different target antigens, using different reference sera and other control reagents, assay methodologies, analytical methods, and length of follow-up. An inverse association between levels of antibodies and neonatal risk of invasive disease has also been shown for some protein candidate antigens, but not others The first suggestion that it may be possible to establish a serological correlate of protection for GBS was presented four decades ago 2.2In South Africa, a prospective, observational cohort of 35,000 mother-newborn pairs was enrolled between 2015 and 2017. Enrolment was re-initiated in 2019, to include a further 15,000 pairs. Infants were followed-up by hospital-based surveillance for invasive GBS disease. In parallel, infants with invasive disease that were not enrolled into the birth cohort were, and will further be, identified across multiple hospitals. The study aims to be powered for a case-control evaluation of sero-correlates of protection for serotype Ia and III, the most common serotypes causing invasive disease in South Africa. Cases will be matched to controls for maternal colonization status with a homotypic serotype during labour, maternal age, birth weight, newborn gender and maternal HIV status .A case-control study with similar objectives is also planned in the United States by the Centers for Disease Control and Prevention (CDC). The association between GBS serotype-specific capsular IgG antibody concentrations at birth and odds of invasive GBS disease will be estimated, for serotypes Ia and III separately. Cases will be identified in 2019–2022 from CDC’s Active Bacterial Core surveillance program. Controls will be babies of GBS-colonized mothers identified at participating clinical centres performing routine antenatal screening for GBS colonization. Remainders of the newborn screening dried blood spot (DBS) samples will be used for antibody measurements. The study aims to enrol approximately 500 cases and 3000 controls .Pilot studies are underway both in the UK and Uganda to assess the feasibility of prospectively enrolling a high number of mother-infant pairs and collect maternal and cord sera , using similar methodologies. All pairs are planned to be followed up to 90 days after birth. In case of disease, another maternal and infant sample will be taken and antibody titres compared to those at birth. Controls will be selected from both GBS-colonized and GBS non-colonized women matched on gestation and gender, aiming for at least 3 controls for every case. A further retrospective study from 2014, based on stored DBS samples, is also underway, using serum samples from 150 cases and three times as many controls .2.3In addition to sero-epidemiological studies, studies in relevant experimental animal models can contribute to establishing evidence of a protective role of antibodies induced by vaccination. Such models have provided useful mechanistic insights into the pathogenesis and the impact of antibody-mediated responses on GBS disease. Passive transfer of antibodies targeting the polysaccharide envelope has provided serotype-specific protection from experimental GBS challenge 3Panel 3The most important determining factor for the accelerated approval of a GBS vaccine based on a non-clinical endpoint will be how strongly the available package of evidence is graded as reasonably likely to predict clinical benefit.The Prentice criteria have been proposed to assess the validity of proposed substitute endpoints The Bradford-Hill criteria have been proposed to support causality assessments: 1. Temporal sequence of association ; 2. Strength of association ; 3. Consistency of association (shown through repeat studies in different settings); 4. Biological gradient ; 5. Specificity ; 6. Plausibility ; 7. Coherence ; 8. Experimental reversibility (upon waning of immunity acquired post exposure); 9. Analogy .Based on this analysis framework, a functional antibody assay would seem preferable as a scientifically justifiable substitute endpoint to just a quantitative, non-functional antigen-binding assay. Alternatively, strong evidence of the correspondence between functional and non-functional quantitative assays should be demonstrated. Animal models of vaccine-induced protection would also add to the evidence that the measured antibodies are clinically relevant.Upon demonstration that a traditional clinical development pathway is unfeasible at a reasonable cost, alternative approaches should be considered. Whatever the approach, strong evidence establishing the validity of substitute endpoint(s) as reliable predictors of clinical benefit will be required to support regulatory and policy decisions. Important consensus recommendations about the use of an immunological substitute endpoint are highlighted here, and an analysis framework for critical appraisal of evidence is presented in 3.1The safety evaluation of a candidate vaccine needs to be at least as rigorous as that required for vaccines licensed by traditional clinical development pathways. When considering risk-benefit, the uncertainty about the benefit derived using a substitute endpoint for licensure might justify setting a higher safety profile expectation. Considerations about pregnancy immunization safety characterization are presented elsewhere 3.2Standard case definitions and ascertainment methodologies are needed to produce clinically relevant estimates of effect for both the evaluation of vaccine efficacy against a clinical endpoint, and for the evaluation of association between antibody levels and clinical outcomes in a sero-epidemiologic study. WHO preferred case definitions and ascertainment methodologies are described elsewhere The role of GBS maternal or neonatal colonization as a potential substitute endpoint has been discussed elsewhere and is outside the scope of this report 3.3High quality standards in assay quantification of antibody level and function should apply. Characterization of the performance of the assay need to be adapted to the vaccine development status, noting that an immunological substitute endpoint should ultimately be based on fully analytically validated assays. International agreement on standards and methodologies will provide the necessary support for comparison and bridging studies. A collaborative initiative towards the development of validated standardized immunoassays is ongoing 3.4The choice of sampling timepoints in both mothers and offspring will be a key determinant of the evaluation of the association between antibody levels and protection. Samples from both the mother and the offspring should be analysed. Several options could be considered to define the primary time-point used to assess protection thresholds. The analysis of a maternal post-immunization serum sample may constitute a pragmatic approach to capture an overall target level that would be associated with a reduction of the overall risk of adverse outcomes, including stillbirth, maternal and infant outcomes. However, sources of heterogeneity need to be considered, as the gestational age at vaccination, prematurity and co-morbidities affect placental transfer of antibodies. Thresholds may also vary according to clinical outcomes considered. Antibody levels in cord blood provide an opportunity to define, possibly with more specificity and precision, a functional protective antibody threshold – the level of antibodies that is required to prevent invasive disease in the offspring, upon initial bacterial penetration into the blood stream. Further research is necessary to understand whether neonatal antibody levels measured may be lowered, in the context of invasive infection, by bacterial binding of antibody, and implications for data interpretation. The characterization of antibody persistence over time up to the end of the at-risk period may also contribute to assess the relationship between antibody levels and disease risk, especially for late-onset disease.In vaccine studies, antibody levels at birth should be evaluated as a function of timing of maternal immunization during pregnancy, for vaccination program planning.3.5Important statistical considerations driving the prospective development of analysis plans need to be defined. WHO has expressed a preference for an 80% level of protection provided by GBS vaccination Geographical heterogeneities should be considered, as the protective threshold may differ according to various host and pathogen factors. Statistical evaluations should also be undertaken in consideration of other variables that may impact the validity of the assumptions. The number, intensity and duration of prior GBS infections, comorbidities, the microbiome, host genetics, or pathogen characteristics may impact the type of immune response generated after natural infection as compared to a response to vaccination, with potential consequences on antibody transfer. Statistical models supporting the assessment of the role of multiple variables may contribute to a better characterization of the strength of the assumptions. Exploratory evaluations can contribute to characterize the role of phenotypic and genotypic differences in the pathogen with protection, thereby producing valuable knowledge to inform vaccine development pathways and decision-making.If the vaccine-induced antibody levels are far above the antibody protective threshold defined by studies in conditions of natural exposure, the likelihood that the vaccine is protective will be higher. A risk management plan should be considered if a significant proportion of vaccinated individuals are categorized as low responders, which may justify investigating a two-dose vaccine regimen, possibly including a pre-pregnancy priming dose.in-vitro demonstration that the measured antibody is functional, through measurement of bacterial killing assays, and by animal models of protection.A concern, regarding the validity of extrapolating a threshold indicative of antibody-mediated immunity resulting from natural exposure to post vaccination levels, relates to the possibility that other protective immune mediators, activated by natural exposure (ie antibodies targeting other antigens), are transferred to the foetus, but may not be induced by vaccination. In theory, this effect could be complete, or only partial, if the measured antibody is just one mediator of protection amongst others. Such a situation may lead to an overestimation of the likely vaccine efficacy based on the substitute immunological endpoint. A way to mitigate this concern would be to build a strong data package indicative of the direct protective role of the substitute immune endpoint effector , or of the close association between the measured endpoint and the causal mediator of protection. For instance, confidence in the extrapolation of immunity from natural exposure to immunity resulting from vaccination can be increased by 3.6Even if it were possible to conduct a large randomized controlled trial testing the efficacy of a multivalent polysaccharide-protein conjugate candidate vaccine, such a trial may generate an overall vaccine efficacy estimate, and possibly serotype-specific efficacy estimates for the most frequent serotypes. It is unlikely that reliable vaccine efficacy estimates would be produced for less common serotypes. Similarly, epidemiological studies in conditions of natural exposure may generate estimates of protective serological thresholds against the most frequent serotypes, but not the less common ones. As has been done for other multivalent vaccines, an aggregate threshold may need to be derived, based on a weighted average, observations about threshold differences across common serotypes and some plausible assumptions. Later, subsequent post-licensure investigations may allow more detailed characterization.3.7An inherent disadvantage of an approach based on a substitute endpoint relates to the imprecise characterization of the individual and public health impact of the vaccine, better estimated through direct evidence generated in a large double blind randomized controlled trial. In the absence of such evidence pre-licensure, impact estimates must rely on mathematical models prone to imperfect assumptions.In such a context, regulators are likely to consider sero-correlate-based indirect evidence of protection only in the context of agreed post-approval studies for further evaluation of effectiveness and impact. Licensure may be conditional, subject to review after the conduct of such studies. Policy makers would also likely require studies to assess effectiveness and impact before advising in favour of prioritizing resources for large-scale implementation. Post-licensure studies offer the possibility to further assess the public health impact of the vaccine, including its preventive role against less common endpoints, such as mortality or multi-factorial syndromes like prematurity. Such studies may include cluster-randomised trials, stepped-wedge designs for vaccine introduction, case-control studies or ecological studies monitoring changes in population disease burden. They may be linked to evaluations of vaccine programme implementation feasibility, as these are increasingly seen as necessary on the pathway to vaccine availability, especially in resource-limiting settings with frail health systems, where deployment of new interventions is most challenging, and where competition for scarce resources is most prevalent 4There is an urgent need for new tools to prevent the morbidity and mortality associated with GBS disease, and GBS vaccine development is a leading priority for protection of early life. While randomized controlled clinical efficacy trials constitute the gold standard for evidence generation, there are significant obstacles to conducting such trials to assess efficacy against GBS invasive disease.Alternative strategies to accelerate licensure of candidate vaccines must be explored, including efforts to define serological correlates of protection as acceptable substitute endpoints, derived from studies of immunity resulting from natural exposure. Existing evidence suggest this may be a viable approach. Vaccine development sponsors, advised by regulators and policy makers, will need to decide on their preferred vaccine development pathway for licensure and policy decisions, through a careful analysis of risks and opportunities. Multiple stakeholders should be engaged early to promote strategic alignment and agree prospectively on methodological preferences, data package requirements and success criteria, tackling priority activities outlined in Any evaluative approach is likely to include important assumptions and gaps. For instance, it will be hard to generate evidence about less common serotypes. Sources of geographical heterogeneities should be considered. A global strategy should be developed, and similar studies should be undertaken in high-, low- and middle-income country settings. Protective thresholds may differ according to local epidemiology and host and pathogen factors.From an early stage in vaccine development, regulators and policy makers should work with sponsors on possible vaccine development pathways. A pathway involving regulatory decision-making based on indirect evidence of clinical impact will require post-licensure confirmation of public health benefits associated with vaccine use in large populations, possibly during a pilot implementation stage. Early planning of confirmatory evaluation according to agreed methodologies, with identified financing, will be key to reduce delays to wide-scale use, including in low resource settings.


I could have selected my favourite flowering image from thousands of astonishing images that have captured the geometric regularity of head-like inflorescences in Asteraceae. These unique inflorescences pack hundreds of flowers into precise spirals whose numbers follow a famous mathematical rule. Meanwhile, the whole structure may mislead a layman (or a pollinator) by mimicking a single solitary flower although it consists of morphologically and functionally distinct types of flowers.per se but affects crop yield, fitness, and adaptation of plants. The flowering image I selected represents a classical example of spiral phyllotaxis found in composite inflorescences such as sunflower (Helianthus sp.). This image . Intriguingly, the number of these spirals follow two consecutive numbers of Fibonacci series . In the particular cultivar in my favourite image, there are 34 clockwise and 21 anti-clockwise spirals (you may check this!) while our standard model cultivar, Terra Regina, typically shows 55 and 34 spirals, respectively. Interestingly, although the Fibonacci numbers clearly dominate, non-Fibonacci structures such as double Fibonacci numbers , Fibonacci ±1 or Lucas numbers  do appear, as recently demonstrated in a large citizen science experiment engaging the public to grow and count sunflower spirals  has showy, large and bilaterally symmetrical marginal ray flowers, and smaller, more radially symmetrical central disc flowers. The image I selected is taken from a cultivar that only develops male sterile ray-like flowers  and UNUSUAL FLORAL ORGANS (UFO), we recently discovered that the capitulum resembles a solitary flower also at the molecular level  that mimic solitary flowers. As visible from the image, the capitulum is surrounded by involucral bracts (the bright green leaf-like organs) that perform a sepal-like, protective function. The showy ray flowers can be seen to be analogous to attractive petals in solitary flowers, and the hermaphrodite disc flowers to reproductive organs (carpels/stamens). By conducting functional analyses for the gerbera orthologues of flower meristem identity genes Although some details on the gene functions and molecular networks controlling capitulum architecture and flower type differentiation have been revealed, the future challenge is to understand the dynamics of early patterning of the inflorescence meristem and establishment of the spiral phyllotaxis. Classical experiments in sunflower already showed that patterning can be disrupted by wounding , 1990. I


Cladosporium species, C. sphaerospermum and C halotolerans. This study demonstrates that high-throughput sequencing of fungi indoors can be a useful strategy for distinguishing distinct microbial exposures in water-damaged homes with visible and nonvisible mold growth, and may provide a microbial means for identifying water damaged housing.Water-damaged housing has been associated with a number of negative health outcomes, principally respiratory disease and asthma. Much of what we know about fungi associated with water-damaged buildings has come from culture-based and immunochemical methods. Few studies have used high-throughput sequencing technologies to assess the impact of water-damage on microbial communities in residential buildings. In this study we used amplicon sequencing and quantitative-PCR to evaluate fungal communities on surfaces and in airborne dust in multiple units of a condemned public housing project located in the San Francisco Bay Area. We recruited 21 households to participate in this study and characterized their apartments as either a unit with visible mold or no visible mold. We sampled airborne fungi from dust settled over a month-long time period from the outdoors, in units with no visible mold, and units with visible mold. In units with visible mold we additionally sampled the visible fungal colonies from bathrooms, kitchens, bedrooms, and living rooms. We found that fungal biomass in settled dust was greater outdoors compared to indoors, but there was no significant difference of fungal biomass in units with visible mold and no visible mold. Interestingly, we found that fungal diversity was reduced in units with visible mold compared to units with no visible mold and the outdoors. Units with visible mold harbored fungal communities distinct from units with no visible mold and the outdoors. Units with visible mold had a greater abundance of taxa within the classes Eurotiomycetes, Saccharomycetes, and Wallemiomycetes. Colonies of fungi collected from units with visible mold were dominated by two  It is well established that humans spend most of their time in their homes; the estimate is 70% for residents of the United States . It is eThe health of homes can be compromised by water intrusion through leaks , floods, or condensation, because increased moisture promotes the proliferation of microbes on indoor surfaces . There hReliance on cultivation as part of the process to identify microbes is now known to be inadequate following the discovery that the vast majority of microorganisms are unculturable under laboratory conditions . InsteadAdvances in sampling indoor air have also facilitated our greater understanding of the airborne mycobiome. Indoor samples typically fall into one of two types, either surface sampling using swabs, wipes, or tape, to recover microbes from suspected colonies, or vacuum sampling of either air or dust settled on floors and shelving . Swab anPenicillium taxa.Researchers are just beginning to apply high-throughput sequencing techniques to characterize fungal communities in water-damaged housing. Recently Jayaprakash et al.  applied We have heeded the call to further study the relationship between the water-damaged built environment, microbial communities, and human occupants . We haveFungi in air and on surfaces were sampled at a 6-story, 150-unit, concrete public housing project in the San Francisco Bay Area that was built in 1966. The building remained occupied with residents despite having been declared uninhabitable in 2014. Federal reports had documented the roof leaks that over two decades of disrepair led to the formation of stalactites, as well as pest infestations, sewage problems, asbestos, and exposed electrical wire.At a Residents Council meeting, 21 households spanning the 6 stories in the building and inhabiting units with varied layouts volunteered to participate in this study. At the initial sampling visit, experienced mycologists surveyed the apartment and categorized the units  as having either ‘visible mold’ growth or ‘no visible mold’. Visible mold was present in kitchens, bathrooms, livings, and bedrooms, and appeared as green, black, or pinkish-orange growths and discoloration on walls; the extent of the visible mold was not recorded. We categorized 11 units as having ‘visible mold’ and 10 units as having ‘no visible mold’. We noted the floor level of the unit and room where samples were collected for each sample.Airborne house dust was collected from each unit using settled dust collectors  , that weFungal genomic DNA (gDNA) was extracted from settled dust samples using a phenol:choloform-isoamyl alcohol extraction protocol, followed by MoBio PowerSoil Kit, as previously described . SurfaceFungal community composition was determined by constructing amplicon libraries from gDNA isolated from settled dust samples and swabbed colonies. Briefly, PCR primers for the ribosomal DNA internal transcribed spacer (ITS) regions, ITS1 and ITS2  were adaAspergillus fumigatus. Fungal biomass for airborne fungi was estimated by dividing the qPCR fungal gene copy number by the petri dish surface area (56.5cm2 for a 10cm plate).Quantities of airborne fungi in settled dust samplers could be compared because the same time and area of collections were used for all samples. The relative amount of fungal DNA in each settled dust sample was determined using quantitative-PCR (qPCR) with the Bio-Rad CFX96 Touch Real-Time PCR Detection System. qPCR employed ITS primers  and SYBRhttps://github.com/linsalrob/EdwardsLab/) and paired using Pear ) sAspergillus, Eurotium, Chaetomium, Paecilomyces, Penicillium, Scopulariopsis, Stachybotrys, Trichoderma, and Wallemia) could be indicator species used to detect water damage in homes , ). ). CladosCladosporium spp., while settled dust samples recovered more phylogenetic diversity. Settled dust collectors were able to recover significantly greater abundance of a number of fungal classes: Agaricomycetes, Arthoniomycetes, Lecanoromycetes, Leotiomcyetes, Microbtroymycetes, Pezizomycetes, Saccharomceyes, Tremellomycetes, and Wallemiomycetes, to name a few. These taxa appear to become airborne, and over the course of one month are able to deposit in settled dust collectors. Interestingly, Basidiomycete yeasts in the Cystobasidiomycetes were recovered in greater abundance from surface samples compared to settled dust; possibly suggesting they are less likely to produce airborne spores or be inhaled by residents.In units with visible mold we collected swabs from actively growing colonies on surfaces, as well as airborne settled dust over the course of one month. The two collection methods provided different profiles of the mycobiota within homes. The dominant taxon recovered within each unit varied between collection methods. Only in two out of eleven units were the same dominant taxon identified using both collection methods. Surface samples were largely dominated by The utilization of varied collection methods provided for a composite view of fungal communities indoors. Collection by swabbing surfaces selects for live fungi that may be actively sporulating in homes at the moment of sampling. These colonies may also be very large in size and have pigmented spores, making them visually detectable. In contrast, settled dust samples are a collection of both live and dead airborne fungi that accumulate in the home over time. Settled dust samples also collect a greater abundance of fungi because they are set out for weeks at a time and can acquire fungal material from undetected colonies as well. The settled dust collection captures longer-term dynamics in the fungal community composition, while the surface sample is a snapshot of predominant taxa in the unit at a particular moment in time. Temporal, ecological, and presumable physiological differences are detected by different sampling methods, and both proved valuable to characterize fungi associated with water-damaged buildings.We collected 68 settled dust samples from outdoor air, units with no visible mold, and units with visible mold, within a chronically water-damaged 150-unit building in the San Francisco Bay Area. Although some units had visibly greater water-damage than others (demonstrated by actively growing mold on surfaces), it is possible that all units in the building experienced some level of water damage due to long-term structural issues, and thus we may actually have compared mildly and severely water-damaged units. A more comprehensive assessment of building water intrusion, including the measurement of relative humidity, temperature, and the surface area of mold, might have facilitated a more precise categorization of the level of water damage in each unit. It would have been ideal to have also surveyed a building of similar design and age with no prior history of water-damage in close proximity to our building. This survey would allow for the comparison of microbial communities within a water-damaged building and a healthy building within the same timescale, season, and weather regime. Additionally, surface samples were only collected from units with visible mold where we swabbed actively growing colonies. Though we presumed surface samples would be dominated by one taxon, sequence data recovered additional ASVs in surface samples at lower abundance. These additional taxa are constituents of the surface community that were not necessarily visible. It would be good to standardize surface collections in each unit and have background knowledge of which ASVs are found on surfaces in each unit regardless of the presence of visible mold.This study is the first to analyze the microbial inhabitants of a condemned building using high-throughput sequencing methods. This is also one of only a handful of studies to use culture-independent techniques to explore the impact of water-damage on microbial communities in buildings. The distinction between outdoor microbial communities, units with no visible mold, and units with visible mold, shows that insufficient building maintenance can drastically shift the assemblage of fungi indoors.In this study we showed that sampling replicated units in one poorly maintained structure can reveal differences among the airborne mycobiome seen outdoors and indoors, as well as in units with and without visible fungal colonies. Furthermore, sampling fungal spores, yeast cells, and hyphae, by gravity settling over a time period long enough to account for daily variation in airborne fungi and weekly variation in occupant behavior characterized airborne fungal communities that correlated with the presence of visible fungal colonies. Biomass of the settled fungi, however, did not correlate with the presence or absence of visible fungi in units. In units with visible mold, the airborne fungal communities were less diverse and dominated by a few major taxa.With the onset of high-throughput sequencing, it is no longer “impractical to measure all the molds in a home” collected in dust, as was suggested by Vesper et al. . We lookS1 FigComparison of homogeneity of variance of communities in settled dust sampled from outdoor air (red), indoor air of units with no visible mold (green), and indoor air of units with visible mold (blue). Significantly less dissimilarity (p = 0.02) is seen among communities sampled from units with visible mold compared to those sampled in units without visible mold or the outdoors (Tukey’s HSD test).(PDF)Click here for additional data file.S2 FigSequence abundance of fungal classes found in settled dust from outdoor air (top panel), units with no visible mold (middle), and units with visible mold (bottom). These twelve classes have significantly different abundance across the environments, as determined by Kruskal-Wallis test (p<0.05). Eight classes are more abundant outdoors, but Agaricomycetes, Eurotiomycetes, Saccharomycetes, and Wallemiomycetes are more abundant indoors.(PDF)Click here for additional data file.S3 FigAlpha- and beta-diversity of fungal communities in samples when analyzed by traditional count-based methods. (A) Alpha-diversity showing significant differences in OTU richness among all sample types, outdoor air (red), indoor air of units with no visible mold (green), and indoor air of units with visible mold (blue). (B) Beta-diversity showing significant differences in community composition among environments. Comparison of these results with those from analyses that treat sequence data as compositional Figs  and 4 sh(PDF)Click here for additional data file.S1 TableTable showing which ASV was identified as the most abundant taxon in each unit with visible mold, either by surface samples or settled dust collectors. There is discordance in what taxon predominates each unit depending on sampling method used to survey the community. Stars denote taxa that have not previously been reported from the indoor environment or water damaged buildings.(PDF)Click here for additional data file.


The human papillomavirus (HPV) vaccine is an underutilized cancer control practice in the United States. Although individual contextual factors are known to impact HPV vaccine coverage rates, the impact of macro-level elements are still unclear. The aim of this analysis was to use HPV vaccination rates to explore the underuse of an evidence-based cancer control intervention and explore broader-level correlates influencing completion rates.A comprehensive database was developed using individual-level date from the National Immunization Survey (NIS)-Teen (2016) and state-level data collected from publically available sources to analyze HPV vaccine completion. Multi-level logistic models were fit to identify significant correlates. Level-1  and level-2 (state) correlates were fitted to a random intercept model. Deviance and AIC assessed model fit and sampling weights were applied.The analysis included 20,495 adolescents from 50 U.S. states and the District of Columbia. Teen age, gender, race/ethnicity, and maternal education were significant individual predictors of HPV completion rates. Significant state-level predictors included sex education policy, religiosity, and HPV vaccine mandate. States with the lowest HPV coverage rates were found to be conservative and highly religious. Little variation in vaccine exemptions and enacted sex and abstinence education polices were observed between states with high and low HPV vaccine coverage suggesting various contextual and situational factors impact HPV vaccine completion rates.Given that gender, religiosity, political ideology, and education policies are predictors of HPV vaccine completion, the interaction and underlying mechanism of these factors can be used to address the underutilization of the HPV vaccine. In the United States, human papillomavirus (HPV) is the most common sexually transmitted infection causing genital warts and HPV-related cancers . HPV traEfficacy studies have indicated HPV vaccines are a highly effective prevention tool prior to HPV exposure , 6. As aAttitudes and beliefs that HPV is a low health priority and vaccination is an elective health measure have resulted in low adherence to HPV vaccine guidelines . ConsideIn order to explore opportunities to enhance HPV vaccine uptake, the aim of this paper is to study the combination of individual  and macro-level factors  potential influences on HPV vaccine completion rates as limited evidence is available. Further understanding these relationships, will not only broaden the evidence in the literature to consider influences beyond the individual, but provide guidance and support to leaderships and facilitators whom have decision-making power in HPV vaccine-related activities. These findings can potentially translate into successful implementation of HPV vaccine interventions and identify potential influences hindering their utilization. We hypothesize that broader-level factors, in addition to individual-level influences, highlights potential modifiable and non-modifiable HPV vaccine barriers influencing suboptimal HPV vaccine adherence nationally. More specifically, we seek to (1) explore possible factors of high and low HPV vaccine coverage rates, and (2) illustrate the current HPV vaccine coverage rates across the United States.For our analyses, a comprehensive database was created using individual and state-level variables from publicly available sources.The National Immunization Survey (NIS)-Teen (2016) is a cross-sectional survey which estimates vaccine coverage rates among 13–17 year old teens in the United States using random digit dialing conducted by the National Center for Immunization and Respiratory Diseases and the National Center for Health Statistics within the Centers for Disease Control and Prevention (CDC) , 22. TelFunding appropriated for HPV vaccination promotion efforts in 2017 and state and local public health governance (2015) was taken from the Centers for Disease Control and Prevention (CDC) websites , 24. TheVaccine coverage, or completion data, was based on medical records from providers and vaccines administered after the NIS-Teen interview were excluded . HPV vacCovariates at the teen and family-level were chosen from the NIS-Teen dataset based on previous literature and included teen age, gender, race/ethnicity; maternal education; family poverty status; and whether a provider had recommended an HPV vaccine series for the child. Indicator variables were used to examine race and ethnicity of the teen as Non-Hispanic White, Non-Hispanic Black, Hispanic, and Non-Hispanic other or mixed race. Poverty status of the family was categorized as above or below the federal poverty line, and maternal education was dichotomized as up to a college graduate and college graduate or higher., central, mixed, or shared [State public health departments were categorized according to their jurisdiction type as decentralr shared . FundingThe presence of an HPV vaccine and sex education mandate in state legislation, along with the type of abstinence-only policy  were also examined as potential state correlates. States that have “stressed abstinence” policies strongly emphasizes abstinence as a standard teaching . AlternaMulti-level models were fit to identify important correlates of HPV vaccine completion and quantify relationships with odds ratios and 95% confidence intervals (CI). First an unconditional logistic model was fit without any independent variables to calculate the intra-class correlation for states. Random intercept models were then fit with individual (level-1) and subsequently state (level-2) correlates. Model fit was assessed using deviance and Akaike information criterion (AIC). Sampling weights calculated for the provider-confirmed data were taken from the NIS-Teen data, which account for selection and non-response probability and were rescaled to account for the multi-level nature of the data according to best practice recommendations . Models Additionally, an analysis of education policy and broader-level factors on HPV vaccine completion rates was done. Sex and abstinence education policies were coded into four groups based on comprehensiveness of policy: (1) having both a sex and abstinence education policy (SA-YY), (2) having a sex education only policy and not an abstinence education policy (SA-YN), 3) not having a sex education policy and having an abstinence only education (SA-NY), and (4) not having either a sex and abstinence education policy (SS-NN)  and the District of Columbia (62.0%) had the highest HPV vaccine coverage rates in the United States. Those with the lowest HPV vaccine coverage rates in 2016 were Wyoming, Mississippi, South Carolina, Utah, and Texas. These states were found to be more conservative and highly religious than states with the highest coverage rates. Little variation among these states regarding education polices and vaccine exemptions was observed. . Gender, political ideology, religiosity, and sex education policy were not found to have similar impacts on Tdap and MMR vaccination rates as the HPV vaccine. See Table This multi-level analysis using state and national data found the significant predictors of HPV vaccine completion were the presence of an HPV vaccine mandate, religiosity, being female, and Hispanic or Black race/ethnicity. Although we expect race, gender, living below the poverty line, and maternal college education to be indicative of higher HPV vaccine completion rates, the effects of additional macro-level factors are important to consider in improving suboptimal HPV vaccine adherence. This suggests that a state’s sex and abstinence education policy, religiosity, and political ideology may be important correlates of HPV vaccine completion.Our analysis suggests an 11-fold decrease in HPV vaccine completion rates among states with a higher percentage of religious adults. Religion has been found to influence immunization decisions as several states have religious exemptions for school mandated vaccines. If parents are religiously opposed to immunizing their children against infectious disease such as tetanus or measles, the likelihood of vaccinating these teens for a sexually transmitted disease like HPV poses a challenge to vaccine completion rates. Although there is limited evidence, the role of religion has been found to guide decisions around certain health behaviors therefore influencing HPV vaccine acceptance and uptake , 34. As A study in Utah found that non-religious females were more likely to complete the HPV vaccine series (received 3 doses) than religious females . A similLittle is known about how strongly and consistently state-level polices impact HPV health outcomes, but these results indicate certain policies may impact HPV vaccine coverage rates. There is significant political opposition and criticism to enact legislation mandating HPV vaccination for adolescents , 37. As Despite strong support for abstinence-only education policies among some stakeholders,  it has bFurthermore, as shown in other studies, –45 our rThere are many factors that affect HPV vaccine completion rates as is not reliant on a single policy or individual-level characteristics. Gender, religiosity, political ideology, and education polices are independent predictors of HPV vaccine completion rates as they were not found to be present in Tdap and MMR vaccine completion models. Thus, further research should consider the complexity of the political and religious environment as well as other state-level policies must be considered when trying to the address the underutilization of this cancer prevention vaccine. Roberts et al. identified a combination of state-level policies associated with higher vaccine uptake, including Medicaid expansion, HPV vaccine school mandates, HPV vaccination through pharmacies, and sex education policies . This suThis analysis can also serve as an example showing how mis-implementation of an evidence-based cancer intervention is occurring. According to Padek et al., mis-implementation is the continuation of ineffective programs and policies or the discontinuation of effective ones . The impNext steps should explore current HPV vaccine-related policies and initiatives in states not having neither education policy to gain insight into other activities contributing to HPV vaccine completion rates. As states that enacted sex education policies were found to have the highest coverage rates among any policy combination, more study of the effects of comprehensive sex education to increase HPV vaccine coverage rates is supported.Although the governance of state health departments were not significant predictors of HPV vaccine completion rates, research to understand the role of state health departments on current efforts, barriers, and allocation of resources regarding cancer control programs is essential to identify the mis-implementation at a state-level. Considering and understanding the role of factors leading to mis-implementation of cancer control programs and policies regarding HPV vaccine can prevent missed opportunities to appropriately implement these efforts.Lastly, learning and further exploring elements that contribute to low and high vaccine coverage rates among states can inform the development of comprehensive HPV vaccine interventions and policies to increase the utilization of the HPV vaccine in the United States.We acknowledge that there are several limitations to this analysis. The cross-sectional nature of our study limits our ability to draw any firm conclusions on the macro-level contributing factors to low-HPV vaccine uptake. Evidence shows that provider-level recommendations has a significant influence on patient uptake of the vaccine –52.HowevDespite the study’s limitations, this analysis highlights the potential impacts of broader contextual factors effects on HPV vaccine coverage rates in the United States. found that in addition to individual-level correlates , policy, political ideology, and religiosity state-level factors are significant correlates of HPV vaccine completion rates. However, there is limited evidence on how these factors interact with each other within a state to promote HPV vaccine efforts. Further studies need to continue to identify state and local-level factors that contribute to HPV vaccine uptake and adherence as well as understand the underlying mechanisms of these factors within states of high and low HPV vaccine coverage rates. An in-depth case analysis of these states may provide insight on effective strategies, initiatives, and policies along with overcome challenges and barriers to serve as a guidance for other states and better inform HPV vaccine interventions.