Katzmann et al. Recent clinical trials, however, show that PCSK9 inhibitors can also effectively reduce plasma lipoprotein(a) (Lp(a))concentration, which—when increased—shows a strong association with increased risks of coronary heart disease and cardiovascular disease as demonstrated in epidemiological, genome-wide association, and Mendelian randomization studies. The mode of action on how PCSK9 inhibitors affect Lp(a) depends on co-treatments and has been reviewed by Ying et al..Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) is involved in cholesterol homeostasis. After binding to the complex low-density lipoprotein (LDL)-receptor, PCSK9 induces its intracellular degradation, thereby reducing serum LDL clearance leading to increased circulating LDL concentrations which contributes to cardiovascular disease development and progression. Therefore, circulating PCSK9 has become a novel drug target in lipid-lowering therapy as reviewed by Kucsera et al.), and comorbidities  and circulating PCSK9 levels are positively correlated to these confounders in patients.PCSK9 is almost ubiquitously expressed in different cell types throughout the body and cellular expression levels are affected by age, sex .Although PCSK9 is now been recognized as a ubiquitously expressed modifier of cellular function and signaling molecules, its physiological role in different organs such as the brain, skeletal muscle, kidney, and the intestine is not well-understood and our current knowledge of its function in these organs is reviewed in detail by Andreadou et al.; Schlüter et al.). Clinical data support the notion that inhibition of extracellular PCSK9 is associated with a reduction in the incidence of myocardial infarction and coronary revascularization in subjects with increased cardiovascular risk . The mechanisms by which extracellular PCSK9 impact on cardiomyocytes are incompletely understood but involve receptors other than the LDL-receptor which are affected by PCSK9 such as the LDL receptor-related protein 1 (LRP1). Treatment of adult rat cardiomyocytes with recombinant PCSK9 but not knockdown of endogenous PCSK9 results in a strong reduction in LRP1 protein expression and as a consequence the activation of protein kinases, the mitochondrial biogenic effect and the impact on glucose metabolism induced by adipocytokines are blunted . Thus, PCSK9 affects intercellular communication (from adipocytes toward cardiomyocytes) but, importantly, under stress conditions, signals from organs such as the kidney and the heart affect hepatic PCSK9 expression and thereby the plasma concentration of PCSK9 . Similarly, disease states such as non-alcoholic steatohepatitis impair local PCSK9 expression and the potential of the liver to take up cholesterol a process showing sex differences . Previous studies in PCSK9 knock-out mice on a high-fat diet demonstrated more excessive hepatocyte lipid droplet formation, augmented inflammatory gene expression, and more pronounced liver fibrosis, suggesting that PCSK9 deficiency could promote hepatic steatosis at least in preclinical models. Whether the pharmacological inhibition of PCSK9 either by monoclonal antibodies or silencing RNAs has similar effects in morbidly obese patients with non-alcoholic fatty liver disease or non-alcoholic steatohepatitis has not been studied yet.In the heart and especially in cardiomyocytes, PCSK9 is up-regulated and released during ischemia and decreasing extracellular PCSK9 expression impacts on infarct size and cardiac dysfunction  and systemic (endocrine) effects of PCSK9 exist .All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.PF is the founder and CEO of Pharmahungary, a group of R&D companies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Axon regeneration is orchestrated by many genes that are differentially expressed in response to injury. Through a comparative analysis of gene expression profiling, injury-responsive genes that are potential targets for understanding the mechanisms underlying regeneration have been revealed. As the efficiency of axon regeneration in both the peripheral and central nervous systems can be manipulated, we suggest that identifying regeneration-associated genes is a promising approach for developing therapeutic applications in vivo. Here, we review the possible roles of stem cell marker- or stemness-related genes in axon regeneration to gain a better understanding of the regeneration mechanism and to identify targets that can enhance regenerative capacity. Understanding which genes are switched on/off in nerve cells in response to injury may help develop new treatments to repair nerves. Nerve cells can be cued to regenerate in both the central  and peripheral nervous systems. Studying patterns of post-injury gene activation may allow therapeutic boosting of injury-responsive genes to cue nerve regrowth. Jinyoung Lee and Yongcheol Cho at Korea University, Seoul, South Korea have reviewed those genes identified as markers of stem cells (cells with high regenerative capacity) that are differentially regulated in nerve cells. They present several promising target genes for further study, noting that little is known of the mechanisms underlying regeneration or the interacting genetic pathways. These results point the way to identifying genetic targets to improve treatment for nerve damage. As the regenerative potential of injured neurons is distinctively regulated by both intrinsic and extrinsic factors7, differentially expressed genes under regenerative or nonregenerative conditions are considered potential targets for understanding the mechanism of axon regeneration that is orchestrated by injury-responsive genes10. Therefore, manipulating regeneration-associated genes (RAGs) is a potential method for developing approaches that promote regeneration. Diverse experimental designs have been utilized for comparative investigations of differential gene expression, revealing core genes that regulate regenerative capacity.Neuronal competence of axonal growth is regulated by transcriptional changes in response to injury11. They made a comprehensive list of genes that are differentially regulated based on the environmental conditions and are potential targets for manipulation that promotes axon regeneration. They identified Cacna2d2 as a negative regulator of regeneration and presented a new method for promoting axon regeneration in vivo, as the pharmacological inhibition of Cacna2d2 resulted in significantly improved regeneration in vivo; this work suggests that manipulating the expression of identified genes is a suitable strategy for promoting axon regeneration.Tedeschi et al. reported three paradigms that produce the conditions for differential axonal outgrowth at different embryonic development stages, different regenerative stages of adult DRG neurons, and preconditioned adult DRG neurons12. Stemness is a manipulatable physiological characteristic, and the use of engineered pluripotent stem cells has ushered in a new era of regenerative medicine14. In addition, direct reprogramming technologies are emerging as novel approaches for treating neurological disorders and neural injury17. The elucidation of cellular stemness has resulted in the identification of genes that are specifically expressed in diverse types of stem cells. However, understanding how these genes regulate cellular stemness remains unclear. In addition, it remains unknown whether manipulating stem cell marker genes in nonstem cells might potentiate particular biological functions, such as regeneration and how they might be used to understand the mechanism by which stem cell marker genes mediate their effects.Stemness refers to the ability of a cell to perpetuate its lineage, produce differentiated daughter cells, and self-regulate its proliferation or regenerationProm1, a marker of stem cells, is expressed by dorsal root ganglion (DRG) neurons and is downregulated when neurons mature in adult mice18. Prom1 overexpression in the DRG of adult mice in vivo resulted in the promotion of axon regeneration after sciatic nerve injury, implying that stem cell marker genes are potential candidates for developing new methods that enhance neuronal regenerative capacity. Here, we review neuronally expressed stem cell marker genes and their roles in axon regeneration and propose a new idea for establishing neuroregenerative applications related to cellular stemness . Tamoxifen-inducible Sox9 deletion in adult mice has been reported to result in reduced CSPG production and small scar sizes in the spinal cord after injury, subsequently resulting in improved motor function recovery23  that regulates the translation of target mRNA and is known to be enriched in the CNS28. Musashi1 is required for the proliferation of neural progenitor cells, including CNS stem cells29; it binds to Robo3 to regulate posttranscription, a phenomenon that is essential for the midline crossing of pre-cerebellar neurons30  levels upregulation after injury. However, overexpressing NICD inhibits regeneration via transcriptional functions. These results indicate that the target genes regulated by NICD, as well as the molecular mechanism activating Notch in response to injury, must be identified. Moreover, because the Notch pathway is regulated by multiple ligands77, injury-dependent Notch ligands must also be identified. Importantly, Notch1 mRNA and protein were upregulated in an injury-responsive manner exclusively within neurons in a mouse spinal cord injury model80, suggesting that the role of injury-responsive Notch1 upregulation needs to be investigated  analog and baclofen (a GABA receptor agonist) treatments promoted axon regeneration after spinal cord injury (SCI)82. Differential gene expression analysis was utilized to understand the molecular mechanism, and the results showed that HESB expression in the brainstem was significantly reduced by GABA or baclofen treatment 29 days after SCI82. HESB is one of the primary target genes of the Notch signaling pathway and functions as an effector of NICD83, implying that NICD negatively regulates axon regeneration after spinal cord injury via its downstream targets, including HES genes. This research group also showed that gamma-secretase inhibitor PF-3804014 treatment reduced the expression of HESB in the brainstem and enhanced axon regeneration83. This recent result shows that HESB is a potential downstream effector of Notch pathway-mediated axon regeneration. In addition, the target genes regulated by HES in response to injury need to be identified to understand the molecular mechanisms of NICD-regulated axon regeneration. In addition, the proteins interacting with NICD need to be investigated in axon injury models to identify their unidentified roles in regulating axon regeneration.Direct evidence of NICD-mediated regulation of axon regeneration was reported by a study using a spinal cord injury model of sea lampreyCD44 is a transmembrane glycoprotein regulating cell-cell interactions. CD44 is a stem cell marker of many types of cancer, such as bladder, breast, colorectal, gastric, head and neck, liver, lung, ovarian, pancreatic, and prostate cancers84. CD44 participates in diverse cellular processes, such as cell adhesion and cell migration, by interacting with the extracellular matrix. CD44 mRNA and protein were detected in the adult rat brain, and they colocalize with neuronal markers and astrocytes and regulate the structures and functions of dendritic spines85. CD44 is involved in the regulation of cytoskeletal reorganization in the nervous system via small GTPases86. CD44 inhibits retinal axon growth in vitro in an embryonic retinal explant model87. The role of CD44 in axonal regeneration is not yet understood, although CD44 expression is upregulated by axonal injury in a c-jun-dependent manner88. Recent transcriptome data indicate that CD44 is a DLK-dependent injury-responsive gene, as indicated by the genetic deletion of DLK in sensory neurons in mouse DRG impairing the upregulation of CD44 mRNA24. Notably, CD44 is highly upregulated in the sciatic nerve quickly, within 2 h of injury, and its levels are sustained for as many as 72 h25. Furthermore, identical marked injury-responsive upregulation of CD44 in both the proximal and distal parts of severed mouse sciatic nerves has been observed, suggesting that the role of CD44 in regenerating and degenerating nerves must be considered25 92. Moreover, the Msi-1 protein was present in an oligomeric state in AD brains, forming aggregates93. In addition, Notch1 expression was significantly higher in the hippocampus of patients with amyotrophic lateral sclerosis (ALS), although the immunoreactivity of NICD was lower, suggesting that the Notch pathway is inactivated94. However, the Notch pathway was abnormally overactivated in the spinal cords of sporadic ALS and ALS model mice95, suggesting that aberrant Notch pathway activation contributes to ALS pathogenesis. These reports clearly show that the Notch pathway and its related effectors are potential targets for understanding axon regeneration and degeneration96. CD44 expression in astrocytes and microglia is also known to be associated with ALS progression in a mouse model97, suggesting that the molecular interplay between stem cell marker genes from the nervous system needs to be investigated.Here, we review a group of genes involved in cellular stemness as target genes for studying axon regeneration. A set of genes that are significantly upregulated in stem cells have been identified and categorized as stem cell markers. However, the molecular functions of the individual genes in this set are still under investigation to understand the roles of stemness regulation. By recognizing that a group of stem cell marker genes shows developmentally decreased levels of expression81. Although more research needs to be performed to gain a full understanding of the molecular connections between cellular stemness, the expression of stem cell marker genes, and neuronal regenerative potential, stemness and its related genes are potential targets to study molecular mechanisms regulating axon regeneration. Manipulating the expression levels of stem cell-associated genes that are selected from comparative transcriptomic data is one way to initiate this research. As a number of stem cell marker genes are transmembrane proteins, including Prom1, CD44, and Notch1, understanding the molecular mechanisms of membrane-associated stem cell markers that may lead to therapeutic applications may be realized by identifying ligand-like molecules regulating axon regeneration via membrane proteins.As indicated above, stem cell-associated genes are known to be differentially expressed in injured neurons or to display functions that regulate axon regeneration. Therefore, stem cell marker genes are potential candidates to manipulate for determining axon regenerative capacity in vitro and in vivo and to find downstream effector molecules that regulate regeneration, similar to the study on the Notch pathway in axon regeneration


Blunt chest trauma related acute thoracic aortic injury (TAI) is a life-threatening condition that requires prompt diagnosis and appropriate management because of high mortality. Computed tomography angiography (CTA) is the imaging of choice for evaluation of patients with major chest trauma findings suspicious of TAI on chest radiography. This case series describes the CTA findings in four high-velocity incident survivors with associated TAIs, discusses the injury type and treatment, and reviews the literature. The descending thoracic aorta is only involved in 1% – 12% of cases.2The site of attachment of the thoracic aorta determines the severity of rupture, with the isthmus being involved in 90% of cases because of its immobile position, fixed by the ligamentum arteriosum, a congenital remnant of the ductus arteriosum.As mentioned earlier, most vascular injuries sustained to the thoracic aorta are fatal. This case series reveals the findings in four survivors of high-velocity incidents with associated TAIs, including a review of the type of injuries, computed tomography angiography (CTA) findings and a review of the literature.Four male patients, aged between 22 years and 45 years, presented to the emergency unit of our hospital, and were suspected of having acute aortic injury on the basis of a widened mediastinum on chest radiography. There was a history of two of them being involved in a motor vehicle accident (MVA), one in a pedestrian vehicle accident (PVA), whilst the fourth patient fell from the fourth floor.Signs of mediastinal haemorrhage on chest radiography were observed in the first patient . ComputeCase 2 relates to a patient involved in a PVA, with multiple other injuries, including intra-cranial haemorrhage and lower limb fractures. The initial chest radiograph revealed signs of aortic injury , which wThe third patient presented with a type 2 aortic injury , developThe fourth patient fell from the fourth floor and sustained multiple additional injuries. He was stented for a type 2 aortic injury , which wThis case series describes three different types of blunt chest trauma  complicated by type 2 traumatic aortic injury, an area amenable to surgical or endovascular treatment. Because of the unstable condition of the patients and the complexities of additional injuries, EVAR was considered as a therapeutic option. Although two of the cases in this case series developed endoleak complications and one further reported complications of local and distal thrombosis, all four patients recovered completely.1Thoracic aortic injury secondary to blunt chest trauma is a life-threatening emergency with a very high morbidity and mortality rate, which requires urgent medical attention and management. In missed or untreated cases, the mortality has been estimated to be 80% – 90%.2 The first two portions are intra-pericardial; hence, their involvement results in haemopericardium.2The thoracic aorta is anatomically divided into four segments .2 The fi4There are several mechanisms that act either in isolation or in combination in TAI, one of the main mechanisms being deceleration with displacement of the heart and displacement, torsion and shearing forces acting on the immobile aorta. Other proposed methods include associated osseous pinch and hydrostatic forces, also known as the water-hammer phenomenon.3 Ninety per cent of the aortic injuries have shown the involvement of the isthmus.2Rapid deceleration including the associated shearing forces exerted, particularly within the frontal and lateral direction, affects front seat passengers in high-velocity MVAs, resulting in injury to the relatively immobile aortic isthmus (ligamentum arteriosum), aortic root and aorta at the region of the diaphragm, contributing to the majority of cases reported.4 proposed the theory regarding the ‘osseous pinch’ based on the compression of the aorta between the spine and manubrium, first rib, and/or the medial aspects of the clavicles. The water-hammer effect is secondary to a sudden increase in intra-thoracic pressure that results in a sudden increase in intra-aortic pressure with subsequent rupture of the pericardium and cardiac tamponade.3Crass et al.5The survival of these patients mainly depends on the site of rupture; the extent of the rupture did not have any bearing on survival. A periaortic haematoma contained by other mediastinal structures and development of a false aneurysm may prolong survival from months to years. Only 2% of the patients live to develop a chronic false aneurysm.6Initial imaging in these patients at our institution is a trauma series of radiographs, which includes a chest, abdominal and pelvic radiograph. Chest radiographic signs of TAI have low specificity and include the following: mediastinal widening > 8 cm, abnormal aortic contour, shift of the endotracheal tube and/or trachea to the right, depression of the left or right main stem bronchus, deviation of the nasogastric tube to the right, presence of an apical pleural cap, first rib fracture, acute left-sided haemothorax, retro-cardiac density, loss of the aorto-pulmonary window, widening of the right paraspinal interface, right paratracheal stripe (> 4 mm) and a pneumothorax.Furthermore, if clinically indicated in a polytrauma patient, the emergency physician usually requests a non-enhanced cerebral and cervical computed tomography (CT) scan together with a thoraco-abdominal and pelvic CT scan in the arterial phase followed by an abdominal-pelvic CT scan in the portal phase after injection of 100 (mL) of iodinated contrast agent (Omnipaque 300) at a rate of 4 mL/s – 5 mL/s. Currently, scans at our institute are acquired with a 128-slice Phillips Ingenuity CT scanner and multiplanar reconstruction (MPR), maximum intensity projection (MIP), curved MPR and volume rendering (VR) 3D reconstruction are also used to analyse the available imaging.8 In the case of equivocal findings, it becomes a diagnostic challenge for the radiologist if the direct and indirect signs of TAI are absent; in such cases, direct catheter angiography is warranted. Periaortic haematoma in the absence of associated vascular injury may represent injury to the aortic adventitia.7The CTA findings of acute TAI have been categorised into direct and indirect or associated findings. Direct findings include intramural haematoma formation, an intimal flap as well as pseudoaneurysmal formation. Indirect or associated findings include periaortic haematoma formation, change in the normal aortic calibre and normal aortic contour.1 pathologically classified traumatic aortic injuries into six groups: (1) intimal haemorrhage, (2) intimal haemorrhage with laceration, (3) medial laceration, (4) complete transection, (5) pseudoaneurysmal formation and (6) periaortic haemorrhage. Heiberg et al.9 were the first to describe the appearance of traumatic aortic injury on CT in 1983. Goarin et al.10 later classified traumatic aortic lesions into three groups (In 1958, Parmley et al.e groups  based on10 All of the patients in this case series sustained Grade 2 injuries and survived.Grade 2 lesions, in which both the intima and media are involved, define injuries that include pseudoaneurysm formation and haemomediastinum. Grade 3 lesions are fatal involving all three layers of the tunicae with rapid extravasation of blood.11Thoracic aortic injury is uncommon in the paediatric population and accounts for only 2% of paediatric deaths from trauma. This is because of the increased compliance of the chest wall in children, elasticity of the tissues, decreased body mass and lack of atherosclerotic disease.12 Aortic pulsations may simulate a dissection, which can be avoided by performing a cardiac-gated study.3 The current problem with routine cardiac gating in cases of chest trauma is the presence of ‘band artefacts’ in patients with a heart rate of over 80 beats per minute, which distorts the findings on MPR and VR images. In the case of ascending aortic pulsatile artefact, a second gated scan after a routine trauma CT chest with 60 mL of intravenous contrast can be performed.The pitfalls of CTA in trauma patients include artefacts related to patient movement and breathing, cardiac motion and aortic pulsation, partial volume averaging and support devices. These may cause difficulty in the interpretation of findings in some cases.13 Anatomical variants that a reporting radiologist should be aware of include a ‘ductal remnant’ located at the inferior surface of aorta near the isthmus, as well as the ‘infundibulum’, which is a cone-shaped lesion found at the origin of bronchial or intercostal arteries.3Axial scans are best to exclude ascending aortic injury.6 Advantages of EVAR include a lower mortality rate, a lower risk of paraplegia and no need for invasive open thoracic surgery. However, disadvantages include regular imaging follow-up to asses for graft complications, which include graft migration, graft fracture, endoleak, graft infection and complications related to vascular access.14 The patients described in this case series underwent successful endovascular repair with an uneventful follow-up.Treatment options include three categories, namely, open surgical repair, endovascular repair and medical management. Although open repair of injuries to the aortic root, ascending aorta and aortic arch remains the procedure of choice in recent times, injuries to the aortic isthmus, descending and abdominal aorta have shown successful outcomes with EVAR.15 Chronic untreated false aneurysms eventually undergo extensive dense calcification. Densely calcified, pain-free aneurysms detected years after the initial trauma have a stable course and may not require surgical intervention.16Medical management with frequent imaging follow-up is regarded as safe in cases with minimal aortic injuries, defined as intimal flaps measuring < 1 cm with no or minimal periaortic haematoma formation.Thoracic aortic injury is a serious complication with devastating consequences in untreated patients. A high index of suspicion and careful evaluation are needed for accurate diagnosis of TAI. Computed tomography angiography is the gold standard for diagnosis and -post-treatment monitoring. CT with its high accuracy, negative predictive value, and multiplanar and volumetric abilities can clearly display the aortic and associated injuries and any anatomical variations that may simulate pathology.


Dosing regimens guided by therapeutic drug monitoring (TDM) may be able to improve penicillin exposure in patients. Improved penicillin exposure could result in improved patient health outcomes. This systematic review aims to describe the impact TDM in penicillin treatment has on health outcomes, including the emergence of antimicrobial resistance (AMR).A search of four databases was conducted and supplemented with a hand search of the literature. Studies that measured concentrations of penicillins in patients, adjusted dosing regimens according to the result and reported health outcomes were selected. The risk of bias was assessed according to study type: randomized controlled trials (RCTs) were assessed with the revised Cochrane risk-of-bias tool for randomized trials (RoB2) assessment tool, the risk of bias in non-randomized studies (ROBINS-1) assessment tool was used to assess observational studies, and the Office of Health Assessment and Translation (OHAT) tool was used to assess case studies. The study characteristics were tabulated and described using a narrative synthesis.1–5 bacterial persistence,1,4,5 ICU length of stay,4 treatment efficacy,1,4,6 and suppression of AMR.4 However there was a high risk of bias in all of these studies for health outcomes. Most had pharmacological primary outcomes and were underpowered to detect health outcomes. All three RCTs and four (25%) observational studies found that the use of penicillin TDM was associated with improved pharmacological target attainment. Only one study was found that assessed the impact of β-lactams TDM on AMR and found that target attainment of 100% ƒT>4×MIC was significantly associated with suppression of resistance. No studies found a detrimental effect of penicillin TDM on health outcomes. A meta-analysis was not performed due to the heterogeneity of the included studies.Three RCTs, 16 observational cohort studies and 9 case studies were included. None of the studies showed statistically significant improvements in health outcomes, when comparing groups receiving TDM and standard care. Five observational studies showed improvement in at least one health outcome statistically significantly associated with target attainment: mortality,There is little evidence to suggest that TDM improves health outcomes, however neither health outcomes nor reductions in emergence of AMR were adequately addressed by currently published studies. The evaluation of health outcomes was not the primary aim of the majority of the included studies, consequently those that did include these outcomes were underpowered to detect a difference. Observational studies often did not take into account co-interventions, or confounding factors, resulting in a high risk of bias. Large variations in how TDM of penicillins was implemented and the different populations used meant that a quantitative synthesis of results was not suitable. Recommendations to standardize penicillin TDM are a challenge currently, as there is no clear evidence of optimal conditions. Suitably powered studies designed to assess clinical outcomes are required to resolve the ambiguity surrounding the impacts of TDM on clinical outcomes, and to address the gap of the impacts on AMR. Further, appropriate standardized protocols and concentration targets for penicillin TDM in humans need to be identified for it to be implemented successfully.


In flowering plants, precise timing of the floral transition is crucial to maximize chances of reproductive success, and as such, this process has been intensively studied. FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) have been identified as closely related eukaryotic phosphatidylethanolamine-binding proteins (‘EuPEBPs’) that integrate multiple environmental stimuli, and act antagonistically to determine the optimal timing of the floral transition. Extensive research has demonstrated that FT acts similar to hormonal signals, being transported in the phloem from its primary site of expression in leaves to its primary site of action in the shoot meristem; TFL1 also appears to act as a mobile signal. Recent work implicates FT, TFL1, and the other members of the EuPEBP family, in the control of other important processes, suggesting that the EuPEBP family may be key general regulators of developmental transitions in flowering plants. In eudicots, there are a small number of EuPEBP proteins, but in monocots, and particularly grasses, there has been a large, but uncharacterized expansion of EuPEBP copy number, with unknown consequences for the EuPEBP function.To systematically characterize the evolution of EuPEBP proteins in flowering plants, and in land plants more generally, we performed a high-resolution phylogenetic analysis of 701 PEBP sequences from 208 species. We refine previous models of EuPEBP evolution in early land plants, demonstrating the algal origin of the family, and pin-pointing the origin of the FT/TFL1 clade at the base of monilophytes. We demonstrate how a core set of genes  at the base of flowering plants has undergone differential evolution in the major angiosperm lineages. This includes the radical expansion of the FT family in monocots into 5 core lineages, further re-duplicated in the grass family to 12 conserved clades.We show that many grass FT proteins are strongly divergent from other FTs and are likely neo-functional regulators of development. Our analysis shows that monocots and eudicots have strongly divergent patterns of EuPEBP evolution.The online version contains supplementary material available at 10.1186/s12915-021-01128-8. Arabidopsis thaliana (‘Arabidopsis’), these signaling pathways regulate the expression of a small number of ‘floral integrators’ including SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT). FT belongs to a family of highly conserved eukaryotic phosphatidylethanolamine-binding proteins (‘EuPEBPs’) in plants (Interpro classification IPR035810). These are quite distinct from ‘bacterial’ PEBPs of the YbhB/YbcL class (Interpro classification IPR005247), which are also found in plants, and which will not be discussed further here. FT and its orthologues have been identified as key regulators of flowering time in many angiosperm species . Ho. Ho39]. Consistent with the analysis of , our datOur most remarkable finding is the dramatic expansion of the FT lineage across the monocot group. At the base of monocots, an initial duplication led to two FT sub-clades (FTU and FTV), before an expansion to five clades in most monocot orders. Due to the still-fragmentary genomic/transcriptomic resources available in basal and medial monocot lineages, we have been unable to precisely pinpoint the timing of the original duplication in the FT lineage, nor the points at which the lineage expanded to five members. Nevertheless, the gradual increase in the size of the lineage can be clearly inferred. The lineage then further expanded to seven clearly defined FT clades within the Poales , and then to twelve conserved clades within the Poaceae. Interestingly, FT1, FT2 and FT10 remained unduplicated during the evolution of the Poales and Poaceae, suggesting strong purifying selection to maintain them as single copy genes, while the FTX and FTY lineages acted as a ‘sandpit’ for sub- or neo-functionalization in grass genomes. The expansion to five FT lineages in most monocots implies a radical change in the way that FT proteins were used to regulate development, as does the subsequent increase in the Poales/Poaceae. An interesting question is whether this represented a partitioning of the prototypical FT function between multiple proteins  or whether FT proteins acquired new functions .One way in which function and neo-function can be inferred is through analysis of protein motifs and domains. Our analysis identified key conserved residues that define each the MFT, TCB and FT proteins  at the base of flowering plants has undergone differential evolution in the major angiosperm lineages. At its most dramatic, this differential evolution includes the radical expansion of the FT family in monocots into 5 core lineages, further re-duplicated in the grass family to 12 conserved clades. Our analysis shows that many grass FT proteins are strongly divergent from other FTs and are likely neo-functional regulators of development, and provides a platform for the functional characterization of this fascinating family of regulatory proteins in the key crop species wheat, rice and maize.https://phytozome.jgi.doe.gov). BLAST searches were performed using the FT, TCB and MFT coding sequences from Arabidopsis thaliana. In the majority of cases, searching with any of these sequences returned the same top hits in each species, including all bona fide FT, TCB and MFT, with very low E values (1 × 10− 50 or below); some transcriptomes however returned no hits. Reciprocal BLASTs were performed with recovered sequences to confirm that only true homologues of the target gene had been identified.EuPEBP family members were identified by BLAST searches against complete genomes from Phytozome (https://db.cngb.org/onekp). For transcriptome datasets, we BLASTed each major taxonomic group separately. Generally, only closely related sequences were identified through this approach, and no cut-off was used. For non-annotated sequences from transcriptome datasets, we searched translations across all 6 reading frames to identify ORFs, and the longest ORFs were extracted for alignment. All sequences are listed in Supplementary File 1.Preliminary trees were assembled and used to guide the iterative interrogation of other genome and transcriptome databases, particularly those generated by the 1KP project (Alignments were initially performed in BioEdit  using ClFor each alignment, we trimmed the alignments to remove poorly conserved regions and then performed preliminary phylogenetic analyses to explore the topology of the tree and the effect of inclusion or exclusion of various groups of sequences. We found that using amino acid alignments generated extensive polytomies, so we restricted ourselves to nucleotide-level analyses. Once we were satisfied with the initial alignment, we used jModelTest 2.1  to identAdditional file 1:.Additional file 2:.Additional file 3:.Additional file 4:.Additional file 5:.Additional file 6:.Additional file 7:.Additional file 8:.


FLT3 mutations are the most frequently identified genetic alterations in acute myeloid leukemia (AML) and are associated with poor clinical outcome, relapse and chemotherapeutic resistance. Elucidating the molecular mechanisms underlying FLT3-dependent pathogenesis and drug resistance is a crucial goal of biomedical research. Given the complexity and intricacy of protein signaling networks, deciphering the molecular basis of FLT3-driven drug resistance requires a systems approach. Here we discuss how the recent advances in mass spectrometry (MS)-based (phospho) proteomics and multiparametric analysis accompanied by emerging computational approaches offer a platform to obtain and systematically analyze cell-specific signaling networks and to identify new potential therapeutic targets.  Over the last decades, molecular biology has witnessed tremendous technological and scientific breakthroughs. This progress has advanced our understanding of cell molecular functioning. We are now aware that signals sensed by a cell do not necessarily propagate in linearly. Signaling networks are rather assemblies of intricate highly connected modules controlling key biological processes in a context-dependent manner. It has also become clear that diseases are often due to perturbation of molecular networks, rather than consequences of single or few gene dysfunctions. In this context, the analysis of networks rewiring in healthy and diseased conditions is critical to understand the properties of the system and to identify new therapeutic strategies. However, because of their complexity and size, the dynamic characterization of signaling networks in health and during disease onset and progression often turns out to be a daunting task ,2,3.Different computational strategies have been developed to model signaling networks and predict their behavior upon environmental or genetic external or internal  perturbations. Most of these approaches take advantage of high throughput proteomic or transcriptomic data. Although both data-types are necessary to build models faithfully mimicking a system behavior, mass spectrometry (MS)-based proteomics is unique for this scope as it enables to measure both the concentrations and the activation levels of thousands of proteins in different conditions . Over reAcute myeloid leukemia (AML) is characterized by a dysfunction of the hematopoietic process resulting in the perturbation of the balance between stem cell proliferation and differentiation. In the last decade, genomic studies identified many different genetic alterations in AML patients, revealing the complexity and heterogeneity of this cancer type ,9. ThankFLT3 belongs to the class III receptor tyrosine kinase (RTKs) group of receptors, characterized by an N-terminal extracellular region consisting of five immunoglobulin-like domains, a juxtamembrane domain (JMD) followed by two tyrosine kinase domains (TKDs). In physiological conditions, FLT3 is mainly expressed in hematopoietic stem cells (HSC) and B-cell progenitors. Its expression decreases during hematopoietic differentiation, with the exception of a subpopulation of monocytes that maintain high levels of FLT3 expression after differentiation . In the Over the past years, several systems of classification of AML patients have been proposed. The European Leukemia Net (ELN) risk stratification guidelines recommended to stratify patients into three groups with different inferred clinical outcome: favorable, intermediate and adverse. This classification system is based on cytogenetic and molecular criteria ,16,17. AAmong the 1540 AML patients, about 500 carry FLT3 mutations. Indeed, it has been estimated that about 30% of AML patients have their FLT3 gene mutated at diagnosis . FLT3 muthe TKD1 beta-sheet1 (amino acids 610 to 615) in 24.6% of FLT3-ITD positive AML patientsin the nucleotide binding loop (NBL) (amino acids 616 to 623) in 2% of FLT3-ITD positive AML patientsin the TKD2 beta-sheet2 (amino acids 624 to 630) in 1.3 % of FLT3-ITD positive AML patients.ITDs are characterized by in-frame duplications 3 to 1236 bp in length, mostly in exons 14 and 15, which correspond to the juxtamembrane domain (JMD). The crystal structure of autoinhibited FLT3 leads to direct evidence of how the JMD region may exert its autoinhibitory effect on the catalytic activity of FLT3. More specifically, the insertion of ITD sequences disrupts the autoinhibitory interaction between the juxtamembrane domain and the kinase domain, allowing FLT3 to switch from the inactive to the catalytically active conformation in absence of the FLT3 ligand . Such coInterestingly, these observations are clinically relevant as the ITDs in the kinase domain have been associated to unfavorable patient prognosis in terms of complete remission, relapse-free survival and overall survival. It has been observed that the ITD size is also extremely variable from patient to patient and that the ITDs in the JMD and TKD regions are highly variables in length ranging from a few to hundreds of nucleotides ,24,25,26Given the importance of FLT3 mutations in AML onset and development, over the past decade, therapeutic protocols based on FLT3 inhibitors have been tested in clinical trials more or less specific kinase inhibitors have been tested either in monotherapy or in combination with chemotherapy. Protocols based on first-generation inhibitors, in monotherapy, are often clinically inefficient possibly because of low potency and selectivity. Thus, these inhibitors are commonly used in combination with standard chemotherapy  ,28. In rOther than the efforts to develop targeted therapies to block or attenuate the activity of the FLT3 receptor, increasing evidence have shown that cancer cells treated with tyrosine kinase inhibitors (TKIs) tend to acquire additional genetic alterations to escape inhibition. Although some patients obtain great benefit from the addition of FLT3 inhibitors  to standard chemotherapy , a signiMutations in the FLT3 receptor have been associated with the development of innate or acquired resistance. For example, Linardopoulos’s team generated resistant cells by long-term exposure of FLT3-mutant human cell lines with selective inhibitors . The resulting resistant cells harbored an additional mutation (D835Y) in the tyrosine kinase domain on the gene with the FLT3-ITD (+) allele. Consistently, the acquired FLT3 D835Y mutation has been recently identified in relapsed FLT3-ITD positive AML patients . These oWorth to mention, as in other types of leukemia, AML cells harboring pre-existing resistance-mediating mutations can be selected by chemotherapeutic or TKIs treatment. Interestingly, FLT3-ITD mutation has been recognized as a relapse-related genetic marker . Here weThe reduced sensitivity of TKD-ITD cells is still poorly understood. It is clear, however that the molecular mechanisms underlying the different sensitivity cannot be simply explained by the hyperactivation of the usual culprits, such as AKT, STAT5 and ERK1/2 b. The TK2 beads affinity purification . The resulting high content phosphoproteomic datasets allow to obtain a comprehensive overview of a cell phosphoprotein profile. Additionally, the described workflow enables to simultaneously process multiple samples, allowing to characterize the cell phosphoproteome in a time and space-resolved manner.In principle, MS-based phosphoproteomics is the best possible technology to elucidate how signaling pathways are rewired in AML cells harboring the different ITD mutations ,43,44. HDifferent network-based approaches have been developed to obtain from phosphoproteomic measurements information about signaling pathway modulation in disease. It has emerged over the years that diseases are often caused by genetic alterations that trigger a significant rewiring of the protein interaction network rather than by the simple alteration of the activity of a single protein. Thus, the characterization of a disease state, diagnosis and prognosis requires a proteome wide characterization of protein activities. As phosphorylation is the main post translational mechanism modulating protein activity, MS based phosphoproteomics plays a prominent role in the characterization of the modules of the signaling network that are altered in a disease condition. Phosphoproteomics-based network medicine applies high-throughput phosphorylation profiling in the classification of cancers, therapy planning and prediction of drug response . In a reHere we review this approach and discuss its application to elucidate signaling networks rewiring in drug resistant and sensitive FLT3-positive AML cells. The strategy takes advantage of SIGNOR, a manually curated database capturing more than 26,000 causal interactions. Every entry is SIGNOR is a binary, signed and directional interaction between proteins or other biological entities , supported by literature evidence. In addition, entries are linked to additional metadata such the molecular mechanism involved in the regulation  and, when available, the amino acid position of post-translational modifications (PTM). At the data of writing SIGNOR stores approximately 9800 regulatory phosphorylation reactions and information for 8800 phosphorylated sites ,59. The (a)Network template: the file should contain a table with at least five columns listing the source nodes, the target nodes, the causal effects (up- or downregulation) and the information about the amino acid position of the phosphorylated site as well as the amino acid sequence context of the phosphosite (sequence window). In our case, this file is the complete list of causal interactions available from the SIGNOR database.(b)Node experimental attributes: a table containing the protein expression levels in specific experimental conditions, as revealed by MS-based proteomic experiments.(c)Edge attributes: a table listing the phosphorylation level of the regulatory phosphopeptides involved in each activation/inactivation reaction, as revealed by the MS-based phosphoproteomic experiment.(d)Cytoscape software installed. For the scope of this example, we used default options. Alternatively, a plethora of adds on applications developed to visualize and analyze networks and omics data are made available at the Cytoscape App store .To reproduce the workflow, the following material is required:These are the steps required to obtain a FLT3 specific-causal network.https://signor.uniroma2.it/downloads.php accessed on 24 April 2021).Download the complete list of interactions from the Download all data section of the SIGNOR database  ≥ 0 to remove unconnected nodes.Use the “style” tab to modify the layout of the network, e.g., the size of the nodes to reflect protein expression level c.Import phosphoproteomic data as attribute of the edges, using the 15mer sequence in SIGNOR as key (see field SEQUENCE).Use the “style” tab to modify the visual properties of the edges. Use arrow style to show effect and directionality and modify color according to phosphoproteomic data d.The result of this pipeline is a signed directed graph that includes only proteins (nodes) expressed in a specific context, as per proteomic data, and where edges represent phosphorylation events at specific sites detected by phosphoproteomics.The resulting network still lacks a crucial piece of information as the activation level of each node is not annotated. Converting phosphorylation data into qualitative information on protein activity is fundamental to interpret signaling network rewiring. Phosphorylation of one or more residues in the activation loop is the most common mechanism to regulate kinase activity. Thus, for many signaling proteins, it is possible to estimate activity from the phosphorylation levels of its regulatory sites. However, there are also pitfalls to consider. Although annotation of the relation between site phosphorylation and kinase activity is annotated in databases such as SIGNOR and PhosphoSitePlus ,59,60, iThe resulting network offers a static snapshot of how the FLT3-ITD mutations impact signaling network and determine a different sensitivity to chemotherapeutic treatments. The goal is that of obtaining a predictive network which can be used as a framework to infer potential therapeutic targets to revert drug resistance. Boolean approaches have been widely applied to obtain context-specific predictive signaling network models . HistoriThe CNO software takes as input a prior knowledge network (PKN) and a training data set. The PKN could be either downloaded from the many pathway or model databases or curated from causal databases as we have described for the FLT3-ITD specific signaling networks e. The trTo obtain the training dataset, it is necessary to select a panel of proteins, often dubbed “sentinel proteins”, whose activity can be monitored under different perturbation conditions. System perturbations can be obtained by treating cells with small-molecule inhibitors and/or cytokines, whereas the activity of the sentinel proteins is analyzed by experimental approaches such as the Luminex xMAP sandwich assay, which permits to measure multiple protein concentrations or modifications in up to 96 samples in a single run.In conclusion, this approach enables to obtain dynamic networks describing the different response to perturbations in sensitive and resistant cells.Only few studies addressed the impact of FLT3-ITD TKD mutations on patients’ survival and therapy resistance. These studies have contributed to raise new fundamental and applied questions: (i) How do the TKD-ITD mutations alter the structure of the FLT3 receptor? (ii) Can the size and the position of ITD trigger the activation of different signalling pathways? (iii) Can the different ITD localization be considered as a patients’ stratification feature? (iv) Which is the role of co-mutations of AML patients in the TKD-ITD resistance? We have described here a general strategy to address such questions. Understanding the molecular changes occurring in a perturbed system provides the means to build predictive models and to set a rational basis to formulate new hypothesis. This approach can also have a translational impact as the optimized model may be used to test new strategies to revert the drug resistance phenotype in AML ITD patients. AML patients carry a complex mutational landscape which can be explored by taking advantage of the recent technological advances in single-cell techniques. Over the last years, mass cytometry has played a crucial role in the single cell characterization of AML patient-specific signalling ,74,75,76


Oxalate is an ionic form of a potentially toxic oxalic acid that is formed in the human body from a combination of food sources and their absorption in the gastrointestinal tract (approximately 30%), as well as the endogenous synthesis of glyoxylate (40%) and ascorbic acid (30%) . The balGiven that oxalate is mainly excreted by the kidneys, dialysis seems to be the main approach for oxalate removal in patients with ESRD . NeverthSlc26a6, is expressed at high levels in the intestine and proximal renal tubules and plays a major role in controlling systemic oxalate metabolism  dietary restriction; 2) high uremic toxin concentration; 3) malabsorption; 4) low blood concentrations of calcium, magnesium, and fiber that may affect the oxalate absorption; 5) use of antibiotics, phosphate binders, or other medications that can influence the quantitative and qualitative composition of gut microbiota; and 6) oxalate-degrading activity (ODA) of gut microbiota .O. formigenes degrade oxalate in the intestine and stimulate its endogenous secretion  were identified . Both uremia- and oxalate-induced molecular mechanisms initiated in the gut can be simultaneously involved in the activation of oxidative stress, chronic inflammation, and atherogenesis. However, the role of gut microbiota in oxalate homeostasis and CVD is still far from being fully understood. Thus, we believe that our hypothesis would be reflected in further experimental and large-scale research to clarify the interaction mechanisms and the role of impaired oxalate homeostasis in CVD risk in patients with ESRD.


Emerging evidence suggests that type 2 diabetes (T2D) may impair the ability to properly adjust the circulation during exercise with augmented blood pressure (BP) and an attenuated contracting skeletal muscle blood flow (BF) response being reported. This review provides a brief overview of the current understanding of these altered exercise responses in T2D and the potential underlying mechanisms, with an emphasis on the sympathetic nervous system and its regulation during exercise. The research presented support augmented sympathetic activation, heightened BP, reduced skeletal muscle BF, and impairment in the ability to attenuate sympathetically mediated vasoconstriction  as potential drivers of neurovascular dysregulation during exercise in T2D. Furthermore, emerging evidence supporting a contribution of the exercise pressor reflex and central command is discussed along with proposed future directions for studies in this important area of research. Type 2 diabetes (T2D) negatively impacts cardiovascular health, contributing to a high risk of premature mortality  to the heart facilitate increases in cardiac output and sympathetic outflow to periphery and viscera elicits vasoconstriction of inactive skeletal muscle and tissue beds, respectively. These actions contribute to elevations in BP while facilitating increases in BF to contracting muscles   in normotensive T2D patients. Consequently, leg vascular resistance was substantially greater in the T2D patients. Similarly, a more recent study  and metabolic  stimuli produced by the contracting skeletal muscle and reflexively increases MSNA, BP, HR, and respiration . This likely contributes to variations in diabetic phenotypes  attenuates increases in exercising muscle BF and augments the BP response to exercise. Nonetheless, there is a clear need for future studies to investigate the impact of T2D on neural and vascular control mechanisms and their interaction during exercise.T2D patients are at a twofold higher risk of premature mortality, contributing to a significantly shorter life expectancy (Raghavan et al., A-KG drafted the manuscript, which was critically evaluated by JK, AS, and PF. All authors approved the submitted version. All authors contributed to the article and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bordetella pertussis. All ages are susceptible. In the prevaccine era, almost all children became infected. Pertussis is particularly dangerous in young infants, who account for practically all hospitalizations and deaths, but clinical disease is burdensome at any age. Widespread use of pertussis vaccines dramatically reduced cases, but concern over adverse reactions led to the replacement of standard whole-cell by acellular pertussis vaccines that contain only a few selected pertussis antigens and are far less reactogenic. Routine administration of acellular pertussis vaccines combined with diphtheria and tetanus toxoids is recommended in infancy with toddler and preschool boosters, at age 11, and during pregnancy. Boosting in the second half of every pregancy is critical to protection of the newborn. Waning of vaccine immunity over time has become an increasing concern, and several new pertussis vaccines are being evaluated to address this problem.Pertussis (whooping cough) is a respiratory infection caused by  Bordetella pertussis bacteria. Classic pertussis is a cough illness that may last for many weeks and is marked by paroxysms of repeated coughs that end with a gasping “whoop.” Historical records describing a pertussis-like disease go back approximately 1000 years [1], and genetic analyses suggest that the bacterium has been associated with humans for millions of years [2]. Outbreaks of pertussis were recorded in Persia in the 15th century [3], and an outbreak that resulted in many deaths among infants and young children was documented in Paris in the summer of 1578 [4]. British records from the early 16th century and the 1701 London Bills of Mortality reported whooping cough [5]. , problems sleeping (77% and 84%), vomiting (56% and 54%), urinary incontinence (3% and 28%), weight loss (33% and 33%), rib fracture (1% and 4%), and loss of consciousness (1% and 6%) [27]. Other known complications of pertussis in adults include triggering of migraines [28], carotid artery dissection [29], fainting after cough [30], and loss of memory . Death is rare in adolescents and adults, but it does occur [33–35].Before widespread pertussis vaccination, the United States annually recorded as many as 270 000 pertussis cases with up to 10 000 deaths [36]. However, these only represented the cases that were reported; it is believed that almost every child contracted pertussis. The introduction of pertussis vaccine led to a dramatic decline in reported pertussis.Although it was once believed that pertussis disease produced lifelong protection, immunity after infection wanes and repeated episodes of illness can occur. Before widespread vaccination, pertussis was so common among children that adolescents and adults repeatedly had their immunity reinforced by a mild or inapparent illness. The curtailment of pediatric pertussis by vaccination meant that immunity among older individuals was no longer being boosted by community exposures and case counts began to increase. During the 1990s, numerous studies in North America, Europe, and Australia demonstrated that pertussis occurred commonly in adults . During the late 1990s and early 2000s, pertussis rates in adolescents and adults rose sharply. In the United States, rates in 2000–2003 were 5.5-fold higher for adolescents and 4.9-fold higher for adults than in 1990–1993 46,,48. PertB pertussis circulation, with 1 in 20 subjects having serological evidence of recent infection [52]. A review of pertussis in the 7 countries represented in the Association of Southeast Asian Nations found substantial variation in immunization schedules and pertussis rates, with the true disease burden remaining unclear due to lack of reliable data [53]. Another factor causing considerable variability in the pertussis epidemiology in LMICs is the fact that vaccines incorporating acellular pertussis (aP) components are available in the private market in most countries that use whole-cell pertussis (wP) vaccines in their public vaccination programs, with the relative population penetrations varying substantially from country to country [54].Since 1980, the global pertussis case count has fallen more than 90% as immunization coverage has increased [49]. However, assessment is complicated by the fact that pertussis surveillance data tend to be limited or lacking in low- and middle-income countries (LMICs) [50]. Moreover, especially in Africa, marked variations in pertussis vaccine coverage between and within nations still persist, with large areas of coverage below 50% [51]. A serosurvey conducted from 2013 to 2016 in 7 Asian countries found evidence of substantial Bordetella pertussis is a small, pleomorphic, Gram-negative bacillus. The organism contains numerous antigens including pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), several serotypes of fimbrial agglutinogens, adenylate cyclase toxin, tracheal cytotoxin, heat-labile toxin, Bordetella resistance to killing protein (BrkA), and endotoxin  had been proven effective in clinical trials and were available in combination with diphtheria and tetanus toxoids (DTP). These DTP vaccines were routinely used in the United States and many other countries, leading to dramatic declines in pertussis morbidity and mortality. These vaccines currently remain in use in most LMICs.The effectiveness of wP vaccines has been demonstrated repeatedly, beginning with studies in the Faroe Islands in 1923 and 1929 [124] and in Michigan in the early 1930s [125]. During the studies of diphtheria, tetanus, and aP (DTaP) vaccines conducted in the 1980s in Germany , England [128], Sweden [129], Italy [130], and Senegal [131], 5 DTP comparator vaccines were evaluated, and their efficacies against protocol-defined symptomatic illness ranged from 83% to 98% for 4 of the vaccines. One US vaccine was substantially less efficacious (36% to 48%) .Additional proof of the effectiveness of pertussis vaccines was provided by 3 countries that initially adopted, but then reduced or eliminated, pertussis vaccination. Japan introduced pertussis vaccine in 1949, and reported pertussis cases and deaths declined markedly . However, in 1975, widely publicized concerns about adverse events alleged to be due to DTP vaccine led to the cessation of infant-toddler pertussis vaccination [134]. Epidemic pertussis recurred; cases rose from 206 in 1971 to over 13 000 in 1979, with hundreds of childhood deaths . A similar pattern was seen in England and Wales, where pertussis vaccine acceptance rates fell to 25% during the mid 1970s. Major epidemics of pertussis returned, with numerous deaths . In 1979, Sweden suspended the use of a DTP vaccine that was considered poorly effective; the incidence of whooping cough more than quadrupled thereafter, with several major outbreaks . In each country, after eventual resumption of pertussis vaccination , pertussis rates again declined .The benefit of pertussis vaccination is not measured solely by disease avoided but also by disease ameliorated. Particularly during outbreaks, the pressure of infection is sufficiently high that properly vaccinated persons may nonetheless contract pertussis disease. Multiple studies have demonstrated that such breakthrough cases are substantially milder than cases in unvaccinated or undervaccinated persons .Whole-cell pertussis vaccines are among the most reactogenic of the routinely administered vaccines. Minor local  and systemic  reactions occur in approximately half of DTP recipients—5 times the rate seen with diphtheria and tetanus (DT) vaccine [146–149]. Unusual high-pitched crying or persistent inconsolable crying beginning 2–8 hours after vaccination and lasting 1 hour or more is reported to occur in 3% to 4% of children 149, . DiphtheThe most serious reaction that was attributed to DTP was acute encephalopathy. The identification of Dravet Syndrome, an epileptic encephalopathy due to a sodium-channel mutation [152], has broadened the understanding of encephalopathy after vaccination [153]. It is believed that the fever associated with DTP likely unmasked the syndrome and consequent seizures in such children, rather than the vaccine itself causing encephalopathy.Concerns regarding DTP grew in the early 1970s, particularly in countries where widespread vaccination had eliminated most disease and young parents and physicians had never witnessed the morbidity and mortality of whooping cough. Although school-entry immunization laws enabled US vaccination rates to be maintained, litigation alleging vaccine injury contributed to the discontinuation of pertussis vaccine production by most US manufacturers. This prompted the US Congress to pass the National Childhood Vaccine Injury Act, which limits civil suits while providing rapid and certain compensation for adverse events recognized to be vaccine-related.During the early 1990s, the Institute of Medicine (IOM) carefully reviewed the evidence for a causal relation between vaccines and various adverse events , 155–157Efforts to develop less reactogenic pertussis vaccines began in Japan after their cessation of DTP vaccination in the mid-1970s. The initial aP vaccines were composed predominantly of FHA along with smaller amounts of inactivated PT and, often, traces of fimbrial proteins and PRN [134]. Other aP vaccines containing equal quantities of PT and FHA soon followed. Acellular pertussis vaccines have been used exclusively in Japan since 1981; household-contact studies and pertussis surveillance after implementation of the acellular vaccines demonstrated their effectiveness .During the 1980s, some 2 dozen acellular vaccines were developed in various countries and demonstrated much lower rates and severities of adverse reactions. Diphtheria, tetanus, and aP vaccines have entirely replaced DTP in the United States, Canada, Australia, most of Europe, and in some Asian and Latin American countries and are available in private markets in almost every country.B pertussis proteins, the amount of each included protein, the methods of manufacture and detoxification of the proteins, and the choice of adjuvants and excipients . The DTaP vaccines differed with respect to the number of included cipients . Antibodcipients .Between 1985 and 1993, 9 large studies of aP vaccines were conducted in Europe or Africa in areas not routinely vaccinating against pertussis. The trial protocols and included vaccines differed substantially . NonetheA study conducted in 1986 in Stockholm  compared 2 Japanese vaccines, one containing both PT and FHA and the other only PT, but in larger quantity. Initial efficacy results were disappointing at 69% and 54%, respectively, but later analyses using a case definition of culture-confirmed disease with at least 21 days of spasmodic cough  found efficacies of 81% and 75%, respectively [171]. The vaccine containing more PT was more effective in preventing severe manifestations of pertussis, whereas the vaccine containing both PT and the attachment protein FHA was more effective in preventing mild or moderate disease .In the early 1990s, various candidate DTaP vaccines were evaluated in 8 efficacy trials, of which 4 were sponsored by the NIH and 4 by the manufacturers of the DTaP evaluated. All of the NIH-sponsored studies were prospective, double-blinded, randomized, and contained a whole-cell control arm, a placebo group, or both. A study based in Göteborg, Sweden (1991–1994) vaccinated children at 3, 5, and 12 months (the standard Swedish schedule) with Certiva, a DTaP vaccine containing 40 mcg of peroxide-detoxified PT; no comparison vaccine was included [174].  Based on the WHO case definition, the efficacy of Certiva was 71%. A study conducted in Italy (1992–1993) compared a Connaught US DTP against Infanrix and Acelluvax, each of which contained PT, FHA, and PRN see  against Of the 4 manufacturer-sponsored efficacy trials, only 1 evaluated a vaccine that remains globally available. A study in Senegal (1990–1994) compared Triavax with that company’s French-manufactured DTP vaccine [131]. Estimates of absolute efficacy in this randomized, double-blind study were derived from a nested case-contact study that compared rates of pertussis after exposure to an index case among study subjects vaccinated at 2, 4, and 6 months of age and nonstudy children (who received either DT or no vaccine) living in the same housing compounds. The absolute efficacy estimates were 74%  for Triavax and 92%  for DTwP; the confidence limits are wide because the total number of cases was small, particularly in the group that had not received pertussis vaccine. Additional manufacturer-sponsored efficacy trials were conducted in (1) Munich, 1993–1995 , (2) Erlangen, Germany, 1991–1994 [177], and (3) Mainz, Germany, from 1992–1994 [126]. Interested readers are referred to their respective reports for details.The studied aP vaccines were associated with markedly lower rates of adverse events compared with whole-cell vaccine, while providing effectiveness that approached or exceeded that of whole-cell vaccine. Although the vaccines differed in efficacy, national surveillance data have shown that each acellular vaccine adopted for routine use has provided excellent control of pertussis during the ages covered by the childhood vaccination series . The first dose of aP vaccine provides modest but important protection [184]; the second dose, provides substantially more protection . The best protection is achieved with 3 primary doses  with a booster in the second year of life . The Scandinavian schedule  gives a little less protection between the second and third doses but better protection thereafter, compared with the other 3-dose schedules.Many studies comparing acellular to whole-cell vaccines have been conducted in infants and children [186–193] and have invariably found the acellular vaccines to be associated with lower rates of adverse reactions than whole-cell vaccine. P = .13). Swelling began on day 1–2 after vaccination and generally resolved by day 4, with no sequelae [196]. The occurrence of whole-limb swelling after the fourth or fifth consecutive dose of aP vaccine has been noted in many other studies [197–204]. In a study of 20 children who experienced large limb swelling after a fourth dose of acellular vaccine, only 4 had swelling of the entire upper arm after a fifth dose [204]. Most commonly, whole-limb swelling is not accompanied by pain, redness, or distress and resolves without sequelae in a few days; it is not known to be related to limb(s) used for prior vaccinations. Rather, there is evidence suggesting that the likelihood of this reaction is related to prevaccination antibody levels in the child and the quantity of antigen in the vaccine . Although whole-limb swelling was considered novel when first described after acellular vaccines, an analysis of VAERS (Vaccine Adverse Event Reporting System) reports published in 2003 found that large local limb swelling was reported equally often after acellular or whole-cell vaccine [205].Follow-up studies to the MAPT evaluated fourth and fifth doses of acellular vaccine given at ages 15–20 months and 4–6 years, respectively . Injection site redness, swelling, pain, and fever were seen more frequently with the fourth consecutive dose of acellular vaccine than with the primary series, but still at rates lower than seen in children given 4 consecutive doses of whole-cell vaccine. Several cases of swelling of the entire limb were observed after administration of the fourth dose of acellular vaccine. Of 1015 children given 4 successive doses of aP vaccine, 20 (2%) reported swelling of the entire thigh after the fourth dose versus none of 246 children primed with whole-cell and boosted with acellular vaccine [196]. Among children given 5 successive doses of acellular vaccine, 4 (1.5%) of 267 experienced swelling of the whole upper arm after the fifth dose  vaccines. The Tdap vaccines developed by GlaxoSmithKline and Sanofi Pasteur are available worldwide, and additional Tdap vaccines have recently been licensed in Asia (Bionet-Asia) or are under development (Serum Institute of India).An NIH-sponsored study found the the pertussis components of 1 Tdap vaccine to be 92%  efficacious in preventing pertussis, which meets the primary case definition [212]. The aP vaccine was well tolerated; fewer than 5% of subjects reported local or systemic adverse reactions. The study and control groups did not differ in the rate of severe adverse events. Likewise, a study during a pertussis outbreak among Australian high-school students found an effectiveness of 85%  for laboratory-confirmed cases [213].In 2005, shortly after US licensure of Tdap vaccines, the US Advisory Committee on Immunization Practices (ACIP) recommended a dose of Tdap in lieu of tetanus and diphtheria (Td) vaccine for all adolescents  and adults. Subsequent studies demonstrated the beneficial impact of this Tdap vaccine on the risk or rates of pertussis among adolescents [214–218]. Current recommendations are discussed below.Despite the success of the childhood pertussis vaccination program and the introduction of adolescent Tdap vaccination, pertussis deaths among infants 0–3 months of age continued to rise, from a total of 49 deaths during the years 1980–1989 to 84 during 1990–1999 to 175 during 2000–2009 [219]. The use of Tdap to create a “cocoon of protection” around newborns by vaccinating expectant or new mothers and other close contacts  was shown to be effective when well implemented [220] but was very difficult to implement well [221–224]. These difficulties stimulated interest in the use of Tdap during pregnancy, but 2 concerns had to be addressed: safety (both for mother and fetus) and potential suppression of responses to infant pertussis vaccination . After initial studies assuaged both concerns , the ACIP in 2011 recommended administration of Tdap during pregnancy. Numerous subsequent studies have demonstrated the safety and effectiveness of Tdap vaccination during pregnancy 225–229, , 231–242Large clinical trials of the Tdap vaccines among adolescents and adults primed with whole-cell vaccine showed that the addition of purified aP components to Td added essentially nothing to the adverse reaction profile [247–249]. Several very large safety studies of Tdap in adolescents and adults found no evidence of important adverse events  [250–252]. Studies in adolescents and adults have not found that adverse reactions to Tdap were significantly increased by recency of prior pertussis vaccination .Since 2010, multiple large pertussis outbreaks have demonstrated increasing rates of pertussis in adolescents and older schoolchildren correlated with the transition from those whose primary vaccination was entirely with whole-cell vaccine, to those who initially received whole-cell vaccine and subsequently acellular vaccine, to those who received only acellular vaccine . It has been shown that there are persistent differences in T-cell polarization and proliferation in persons originally primed with aP, compared with wP, despite repeated aP boosters .Memory cells resident in the nasopharynx and lung seem to be important mediators of protection in the mouse, and they are stimulated much more robustly by natural infection and whole-cell vaccine than by acellular vaccine . Likewise, memory T cells resident in the nasopharynx stimulate production of secretory immunoglobulin (Ig)A that provides protection against B pertussis manifest all the findings of human pertussis. In the baboon, pertussis infection stimulates strong Th1 and Th17 responses and confers sterilizing immunity. Baboons vaccinated with wP have similar, but less robust, responses. Baboons vaccinated with aP mount strong Th2, low Th1, and no Th17 responses. When aP-vaccinated baboons are challenged with B pertussis, they have no clinical symptoms but develop persistent heavy nasopharyngeal colonization, whereas those challenged after wP vaccination are colonized less heavily and clear the organism more rapidly. Baboons vaccinated with aP who become colonized can transmit B pertussis to nearby unprotected baboons [283]. These findings in the mouse and baboon models are consistent with the observation that pertussis outbreaks are increasing in frequency and size in aP-vaccinated populations.The baboon model of pertussis has been important in validating findings in the mouse model and further advancing our understanding of pertussis infection and vaccine protection 76–78, . BaboonsA case-control study conducted during an outbreak in California in 2010 found that receipt of all 5 recommended doses of aP vaccine was 89%  effective overall; efficacy declined from 98% within 1 year to 71% 5 or more years later [284]. A similar study conducted during the 2012 Washington State outbreak found that Tdap efficacy among adolescents was 73% within 1 year of vaccination but declined to 34% 2–4 years postvaccination . LikewisMulticomponent combination vaccines are typically used for childhood vaccination programs worldwide. Public vaccination programs in Europe, North America, Australia, New Zealand, and parts of Asia and Latin America use vaccines incorporating aP components; manufacturers include GlaxoSmithKline, MCM (Merck-Connaught-Merieux) Vaccine Company, and Sanofi. Some jurisdictions allow the provider to choose from available alternative products; others specify a single product on a tender basis. The rest of the world uses multicomponent combination vaccines containing wP components, typically on a tender basis; manufacturers are almost entirely in Asia and include Biological E, LG Chemical, Panacea Biotech, PT Bio Farma, and Serum Institute of India.Pertussis vaccine is recommended worldwide for infants and young children and recommended in many countries for adolescents, adults, pregnant women, and others; details are available at WHO websites .In the United States, the ACIP recommends that every infant receive pertussis vaccine at ages 2, 4, and 6 months, 15 to 18 months, and 4–6 years . The TdaRecommendations are beneficial only if followed. Although antivaccine sentiments have circulated since the time of Jenner [290–291], the global increase in vaccine hesitancy or outright antivaccine activism in recent decades has proved challenging. Despite overwhelming scientific evidence that vaccines prevent far more mortality and morbidity than they cause, when people cannot personally see the benefits of vaccination , they may see only risks, often exaggerated or invented [291]. Numerous studies have shown that avoidance of vaccination—typically, through personal belief or unwarranted medical exemptions from school-entry vaccination requirements—results in increased risk of disease, not merely for the un(under)-vaccinated child but also for persons whom that child exposes, persons who may have medical contraindications to vaccination, immune deficits that elevate their risk despite vaccination, or simply breakthrough disease despite vaccination [292–295].The inability of current acellular vaccines to stimulate the same kinds of cellular immune responses seen with whole-cell vaccines or natural disease, and the apparent consequences of that difference with respect to durability of immunity and protection from carriage and transmission, have stimulated many efforts to develop new pertussis vaccines that preserve the safety of current acellular vaccines while improving their cellular immune response. Although some have called for a return to traditional whole-cell vaccines, that clearly would not be acceptable to those countries that previously had deep concerns about whole-cell vaccine adverse reactions.B pertussis for use in whole-cell vaccines with decreased potential for adverse reactions; enhancement of aP vaccines by inclusion of additional or different B pertussis antigens, seeking wP-like immunological responses while retaining aP-like safety; and enhancement of aP vaccines by adding an adjuvant that drives immune responses toward the pattern seen with wP vaccines. The exemplar of the first approach has been the development of the BPZE1 B pertussis variant, constructed by the genetic inactivation or removal of 3 major toxins [296–308]. BPZE1 is administered intranasally and has been shown to protect against lung and nasal B pertussis colonization both in mice and baboons. A clinical trial in human volunteers demonstrated the vaccine’s safety and ability to induce colonization in more than 80% of recipients, with seroconversion in 100% of those given the highest dose of the vaccine [309]. All vaccinees had high blood IgG and IgA titers at day 28, and specific memory B cells were detected. The antigen specificity of these antibodies, characterized by 2-dimensional immunoblotting, was broader compared with the antibody repertoire from aP-vaccinated subjects and was predominantly directed to antigens not present in the aP vaccines. Further studies with this vaccine are underway.Vaccine development efforts have mostly fallen into 3 categories: modification of B pertussis antigens , which contain multiple antigens including secreted and membrane-bound virulence factors. An OMV vaccine developed by Hozbor et al [322] induced immunity in mice that was superior to that of aP and comparable to that of wP. Also under investigation is the incorporation of new or additional antigens including biofilm-associated virulence factors , iron uptake proteins , adenylate cyclase  or other proteins [320], as well as treatment with monoclonal antibodies [326].A number of investigators have pursued the second approach of enhancing aP vaccines by adding new or different antigens , 311–322Bordetella colonization factor A [331], CpG TLR-9 agonists [328], and lipopolysaccharide [320].The third alternative being explored involves the addition of novel adjuvants to elicit a more Th1-skewed response . For example, the Mills group [330] has shown that addition of a Toll-like receptor (TLR)-7 agonist to aP vaccine enhanced Th1 and Th17 responses in mice, yielding protection after challenge comparable to that of wP vaccines. They have also developed a novel adjuvant that combines an interferon stimulator with a TLR-2 agonist and shown its ability to stimulate Th1 and Th17 responses and intranasal tissue-resident T-cells [304]. Other adjuvants under consideration include In the absence of systematic vaccination with pertussis vaccine, essentially every person will suffer whooping cough during infancy or childhood; many will suffer serious sequelae or die. No medical intervention is risk-free, but modern pertussis vaccines are both effective and safe—indeed, vastly safer than avoiding vaccination. Pertussis is an endemic and highly contagious disease; outbreaks will continue to occur. In an outbreak, vaccinated children are less likely to be infected; if infected, illness will be less severe . Unvaccinated children are at much higher risk of infection and, if infected, at much higher risk of adverse outcome. A number of approaches to new vaccines with more durable protection are under investigation.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at The complete references are available as online Supplemental Material.jiaa469_suppl_Supplementary-MaterialClick here for additional data file.