Atopic dermatitis (AD) is a common skin disease affecting 20% of the population beginning usually before one year of age. It is associated with the emergence of allergen-specific IgE, but also with autoreactive IgE, whose function remain elusive. This review discusses current knowledge relevant to the mechanisms, which leads to the secretion of autoreactive IgE and to the potential function of these antibodies in AD. Multiple autoantigens have been described to elicit an IgE-dependent response in this context. This IgE autoimmunity starts in infancy and is associated with disease severity. Furthermore, the overall prevalence of autoreactive IgE to multiple auto-antigens is high in AD patients. IgE-antigen complexes can promote a facilitated antigen presentation, a skewing of the adaptive response toward type 2 immunity, and a chronic skin barrier dysfunction and inflammation in patients or AD models. In AD, skin barrier defects and the atopic immune environment facilitate allergen sensitization and the development of other IgE-mediated allergic diseases in a process called the atopic march. AD is also associated epidemiologically with several autoimmune diseases showing autoreactive IgE secretion. Thus, a potential outcome of IgE autoreactivity in AD could be the development of further autoimmune diseases. Atopic dermatitis (AD) is a chronic relapsing systemic skin disorder characterized by an intense pruritus and the development of eczematous lesions to particular sites. It usually begins before one year of age, and fades after childhood in ~80–90% of the cases. Adult-onset has been observed as well. The adult form of the disease is more severe, and associated with a high morbidity. It is the most common skin condition showing up to 20% lifetime prevalence in some countries ,2. AD haichthyosis vulgaris, which is a condition where about half of the patients develop AD [stratum corneum (the outer layer of the epidermis), and the skin barrier function. Its deficiency causes the Netherton syndrome, characterized by dry scaling skin, and atopic symptoms resembling AD [A skin barrier dysfunction can be observed on both lesional and non-lesional sites on AD patients’ skin. Around 20% of AD patients show mutations in Filaggrin, which is a critical epidermal pro-barrier gene. Its deficiency permit a systemic barrier dysfunction and velop AD ,7. Deficvelop AD . SPINK5 bling AD . GeneticSkin barrier function emerges from the controlled process of keratinocyte death by cornification. Injury, infection, and inflammation promote keratinocytes proliferation and differentiation and disrupt this tightly regulated process, which leads to skin barrier dysfunction ,11. InjuStaphylococcus aureus over other species. Other members of the Staphylococcus sp. have been shown to secrete specific AMPs to keep S. aureus in check in healthy individuals. S. aureus colonization is frequent in AD patients and is associated with the severity of the disease both in adults and children . It could be a secondary cause of AD, and of the chronification of the disease since dysbiosis and S. aureus colonization occur after the first AD symptoms in infants [S. aureus and other bacteria found on the skin of AD patients (such as Roseomonas mucosa) induce a skin barrier dysfunction and an IgE secretion after inoculation on the skin of rodent AD models, while a healthy microbiome is protective [S. aureus products alone can induce an epidermal barrier dysfunction and skin inflammation, the development of specific IgE, skin mast cell activation, and eosinophils and basophils recruitment, which supports the fact that S. aureus colonization is an integrant part of the AD pathogenesis [Epidermal inflammation and barrier dysfunction are associated with an altered lipid composition, an increased pH, and a reduced secretion of antimicrobial peptides (AMP) in the skin of AD patients. These factors are important for the homeostasis of the skin commensal microbiome. In AD, a decreased diversity of the skin microbiome has been observed at lesional sites ,2. As an infants . Indeed,otective ,24. Furtogenesis ,26.Malassezia sp. genus, which require exogenous lipids to grow. As such, they thrive more on adults at the sites of sebum secretion than on children prior to puberty. Healthy individuals show natural IgG and IgM specific of these fungi, but only AD patients show Malassezia sp. sensitization, specific IgE, and positive skin prick tests. At least 13 allergens from this yeast have been described, but several Malassezia specific IgE and T cells show a strong crossreactivity to identified autoantigens. Thus, it is not currently known if the Malassezia sensitization seen in AD arises from autoimmunity or if the sensitization to these commensals leads to the emergence of autoreactive T cells and IgE [Fungi are also an unrecognized part of the human skin commensal microbiome. They mainly comprise lipophilic yeasts of the  and IgE ,28.Both a mechanical or chemical disruption of the epidermal layer and a colonization by pathogenic microbes promote skin inflammation. Epidermal inflammation observed in AD is linked to the expression of epidermal derived alarmins or cytokines, such as TSLP , IL-18, IL-33, IL-25, IL-1, and artemin. These mediators can activate immune cells locally in the skin (Type 2 innate lymphoid cells (ILC2), dermal dendritic cells (DC), Langerhans cells, mast cells), or systemically  . In AD mbona fide Th2 cells) or a follicular (Tfh2 cells) phenotype [Professional APCs such as dermal DCs exposed to this atopic environment, and particularly to TSLP, will migrate more to skin draining lymph nodes to present antigens . The skewing of DC activation during allergic sensitization can involve various signals and very heterogeneous transcriptional responses. Different subsets of dermal DC participate to initiate specific Th2 response in several settings of skin allergic inflammation . Type 2 henotype . Tfh2 cehenotype . DOCK8 dhenotype ,21, and henotype . Thus, iMalassezia antigens. Furthermore, several Malassezia sp. epitopes show a strong sequence homology with human proteins ), and induce T cells and IgE responses that cross-react with auto-antigens [AD symptoms appear primarily in children before two year of age, and the severity of the disease is associated with high IgE titers and sensitization to allergens. However, at this age, increase in total IgE, or allergen sensitization, is observed in less than half of AD mild to moderate cases. More than 40% can outgrow their symptoms before three years old . Around antigens ,46,47. Nantigens . The preantigens . Thus, tstratum corneum antigens or FcεRIα, which is contrary to conditions such as psoriasis or chronic urticaria, respectively. IgG against CCL3 and CD28, alongside anti-phospholipid antibodies, were commonly found in AD. Anti-nuclear antibodies (ANA) were increased in eight distinct studies. Pooling data from these studies showed a significantly higher frequency of ANA positivity in AD patients  [Autoreactivity to autologous dander, sweat, or epithelial cells has been observed for a long time in AD. It is thought to be an essential part of its pathogenesis or of its chronification ,40. Two 1–3.64}) . EvidencThe observation of allergic symptoms to autologous material led to the description of AD as an “autoallergy.” This was supported by the discovery of autoreactive IgE targeting several auto-antigens in AD, and suggested an autoimmune origin to this pathology. However, several autoreactive IgE were also found to be crossreactive with common allergens or microbes ,40,50. Summaries of the prevalence of autoreactive IgE in AD can be found in the literature ,5. Most Aspergillus fumigatus and Malassezia sp. allergens , and elicit specific IgE responses in 42% and 8% of AD patients, respectively. Human thioredoxin is highly homologous to Asp f28, Asp f29, and to Mala s13 and elicit specific IgE responses in more than 20% of AD patients [Homo sapiens allergen), which is a mitochondrial protein [C. albicans and S. cerevisiae allergens), which is highly crossreactive with the human cyclophilins, to which AD patients are commonly sensitized [Blatella germanica, or the dust mite Dermatophagoides farinae (Der f2), respectively [Autoreactive IgE found in AD patients frequently crossreact with environmental allergens. Molecular mimicry is thought to be a key mechanism for the development of autoimmune diseases, and several allergens show an important homology to human proteins. Human manganese superoxide dismutase  and ribosomal P2 protein (RPLP2) share similarities with patients ,28,48. S protein . Cyclophnsitized . Other pnsitized . Calmoduectively ), which ectively . DNA reaectively .Recently, the crossreactivity of glycans specific IgE was involved as the origin of the red meat allergy “epidemic.” Galactose-α-(1-3)-galactose is a common crossreactive carbohydrate determinant (CCD) found in mammals, but not in humans. Tick bites are thought to induce the secretion of IgE specific to this CCD, which are able to trigger fatal anaphylactic reactions to red meat and to the glycosylations of the murine chains of the hybrid monoclonal antibody Cetuximab . CCD areIgE are heavily glycosylated immunoglobulins, and their glycosylation has been associated with their biological activity and their capacity to induce degranulation . Non-speHomo sapiens allergens” Hom s1-5, and to the identification of self-antigens involving at least 102 proteins by phage display [-HBEGF  is a keratinocyte growth factor -Galectin 3 is important for atopic inflammation, binds IgE, and activates basophils ,62,63,64-Periplakin is a component of the cornified envelope of keratinocytes and of desmosomes important for the epidermal barrier -Syk is an essential intracellular mediator of IgE induced signaling, and of basophils and mast cell activation -LAMC2 is a component of laminin 5, which maintains skin integrity, (Laminin 5 α chain single nucleotides polymorphisms are associated with AD )In early studies, AD patients IgE-reactive autoantigens were detected in all subcellular fractions from epithelial cells with a predominance for nuclear and microsomal antigens. However, auto-antigens were not expressed only in organs showing atopic manifestations (such as skin lesions) but in every organ analyzed. IgE reactivity was also extended to basophils, mast cells, platelets, and T cells . Human c display ,48,59. ZThe fact that the targets of autoreactive IgE are potentially involved in AD pathogenesis reinforces the idea that autoreactive IgE levels play an important role in AD. The prevalence of the 102 targets identified by Zeller et al. remains to be investigated, but their relevance to the AD pathophysiology and to patients’ subtypes would be interesting to understand .Autoreactive IgE are also specific of other targets in AD, such as some nuclear components. Dense fine speckle 70 is a protein of 70kd whose detection by immunofluorescence show a nucleolar localization on epithelial cells during interphase, and an increased chromosomal localization during mitosis. This protein corresponds to a nuclear transcription coactivator called p75, which is the target of specific IgG and IgE of AD patients , and chrSpecific “autoallergens” were validated using various techniques, including indirect semi-quantitative measures by Western blots, immunofluorescence, and home-made ELISAs or functional assays, such as skin prick tests and basophil activation tests ,57,59,73Thus, multiple targets of autoreactive IgE have been identified in AD, including crossreactive epitopes, non-peptidic epitopes, and epitopes that do not share any homology with known allergens. A causality link between the appearance of autoreactive IgE and the pathogenesis of AD cannot be ascertained currently. However, their high prevalence, their association with AD severity, and their functional role in mediating basophil/mast cell activation and epidermal hyperplasia strongly support that they represent a unique therapeutical target for subsets of patients. Consequences of an IgE reactivity to self in AD are still unknown but can be appreciated by the knowledge of the various receptors for IgE, and their expression patterns, by the function of IgE in allergic disease, and of autoreactive IgE in autoimmune diseases.IgE binds through its Fc portion to two conventional receptors. A receptor of high affinity, FcεRI, shows a restricted pattern of expression on mast cells, basophils, and to a limited extent on plasmacytoid dendritic cells (pDCs), type 2 conventional BDCA1+ DCs, and CD1a+ epidermal Langerhans cells and specific neurons ,77. In AFcεRII (or CD23), which is the low affinity IgE receptor, is a transmembrane glycoprotein showing similarities with C-type lectins. It is expressed by lymphocytes, monocytes, granulocytes, follicular dendritic cells, intestinal epithelial cells, and in the bone marrow. It has two isoforms produced by alternative splicing in humans, CD23a and CD23b, which are associated with distinct functions. CD23 is particularly expressed by follicular B cells during their maturation in the presence of IL-4, and promote their differentiation. CD23 is frequently shed by proteolytic cleavage. Soluble CD23 (sCD23) show a range of sizes but all bind IgE. They are thought to promote or inhibit IgE synthesis, depending on their oligomerization state. They could be a biomarker of disease activity in various inflammatory settings including in auto-immune diseases . Galectin 3 is an “unconventional” low affinity IgE receptor. It is found in a monomeric or pentameric state in the cytosolic and the extracellular spaces. It can crosslink both FcεRIα and IgE (and binds differently distinct IgE glycoforms) to induce basophil or mast cell degranulation. It is known to be mainly produced by macrophages, especially in the presence of type 2 cytokines, but it can be found on most immune cells and epithelial cells ,79,81. SGalectin 9 has been shown to bind IgE glycosylations with a high specificity (binding that was dampened by lactose) through their lectin domain and represents a second IgE unconventional receptor. Galectin 9 inhibits IgE-allergen complexes formation, and basophil or mast cell degranulation ,83. HoweSeminal work on IgE showed that antigen-IgE complexes were orders of magnitude better at eliciting T cells and antibody responses in vitro and in vivo than antigens alone or antigen-IgG complexes. This was shown to be dependent on IgE-antigen complex endocytosis through CD23 expressed by B cells (and later through FcεRI expressed by DC as well), and is known as IgE-facilitated antigen presentation (FAP). FAP is thought to occur during most allergic and atopic diseases involving specific IgE secretion, and to contribute to relapses and maintenance of atopy ,87. IncrIn an “auto-allergy” context, autoreactive IgE-autoantigen complexes should facilitate auto-antigen presentation and the maintenance of an adaptive autoimmune response. In SLE, IgE-dsDNA complexes are endocytosed through FcεRIα faster than IgG-dsDNA complexes by pDCs. IgE complexes are also superior to IgG complexes to induce pDC activation, expression of costimulatory molecules, and antigen presentation to T cells in co-culture experiments . IgE alsMast cells can directly present antigen in the context of MHCII molecules, but are not considered to be professional antigen presenting cells (APCs), and this role was not observed during AD . HoweverBasophils have been shown to express MHCII molecules and to induce specific Th2 responses in various allergic settings due to their innate high expression of IL-4, including after incubation with IgE-allergen complexes. More specifically, basophils were found to be critical for the induction of type 2 responses upon intravenous administration of IgE-antigen complexes or basophils coated with IgE-antigen complexes ,97. BasoWhether basophils would initiate a Th2 adaptive response without DCs proved to be controversial, and allergen-dependent or model-dependent. Thus, basophils are considered atypical but not professional APCs . Otsuka The IgE high affinity receptor is dominantly expressed by mast cells and basophils. These innate cells produce high amounts of type 2 cytokines such as IL-4 and IL-13 upon crosslinking of their surface IgE by a multi-valent (auto)allergen encounter and can migrate to secondary lymphoid organs where the adaptive immune response takes place. Basophils are known to be recruited into the skin draining lymph nodes in various models of AD, where they can help DC to prime CD4+ T cells toward a Th2 phenotype ,99,101. IL-4 is critical to skew the development of an adaptive T cell response toward a Th2 response. It is also important for B cell maturation and class switching toward an IgE or IgG1 phenotype in mice and an IgE or IgG4 phenotype in humans  ,105. BasAutoreactive IgE could also induce a direct effect of basophils on B cells. Human basophils were shown to promote B cell proliferation, maturation to plasma cells, class-switching, and antibody production in vitro in an Il-4 dependent way, both in the presence and in the absence of T cells . In vivoSimilarly, IgE dependent or independent basophil activation has been shown to lead to the secretion of basophils extracellular traps, which are similar to mitochondrial-derived neutrophils extracellular traps (ETs) . BasophiIn addition to the FAP, IgE mediated activation of professional APC can participate in skewing the development of a T cell response in an AD context by directly stimulating their cytokine secretion or their expression of costimulatory molecules. The IgE crosslinking of AD patients’ monocyte-derived Langerhans cells or IDECs by specific allergens resulted in the generation of a preferentially Th2 or Th1 CD4+ T cells, respectively . This efThe affinity of the IgE for an (auto)allergen, or the nature of the (auto)allergen, could also be very important in determining the outcome of IgE binding on professional APCs. The peripheral blood mononuclear cells from patients reactive to both the grass allergen Phl p1 and the autoantigen Hom s4 show more secretion of IFNγ and IL-10 after stimulation with the auto-antigen Hom s4 than with Phl p1, whereas the secretion of the type 2 cytokine IL-5 did not show this bias. Stimulation with the autoreactive allergen also induced more IgG1 and IgG2 secretion (associated with type 1 responses in humans), while the grass allergen induced more IgG4 (associated with type 2 responses) ,105. AnoMultivalent allergens or auto-allergens can bind several IgE molecules to induce the crosslinking of FcεRI on the surface of mast cells and basophils and their degranulation. Histamine is a potent vasodilatator released during mast cell or basophil degranulation, which immediately increase local inflammation and the recruitment of pro-inflammatory cells in a skin-prick test reaction or allergen challenge. Both mast cell, basophil, and eosinophil infiltration were observed on the site of the allergen challenge. Immediate type hypersensitivity reactions to autoantigens, autologous serum, or autologous sweat were observed frequently in AD as well ,102,117.stratum corneum filaments and transmembrane proteins [Are IgE reactive auto-antigens available to activate basophils and mast cells during AD chronic skin inflammation? Autoreactive IgE were found to commonly target epithelial cell lines or primary keratinocytes in AD, particularly intracellular peptides, but also proteins . Chronicproteins . Keratinproteins . Damage proteins . This suproteins ,110). Anproteins ,56,113. Malassezia globosa antigen MGL_1304. It induces AD patient basophils’ degranulation frequently, but no auto-antigen has been identified to crossreact with MGL_1304 [Are auto-antigens able to induce a direct IgE-mediated degranulation in controlled settings? Of note, hypersensitivity reaction to autologous sweat is very common in AD  and primarily involves an IgE reactivity to the commensal fungi MGL_1304 ,122. AutMGL_1304 . HoweverMGL_1304 . AutoreaMGL_1304 . HoweverMGL_1304 . KnowledIgE-induced anaphylactic degranulation of mast cells or basophils is highly pro-inflammatory and results in the secretion of stored and newly synthetized vasoactive and pruritogenic compounds including histamine, eicosanoids, proteases, and the type 2 cytokines IL-4 and IL-13. IgE also triggers ETosis, and the release of ROS-producing enzymes associated with oxidized DNA, which are highly pro-inflammatory and can induce chronic tissue damage ,113.Histamine is the main pruritogenic molecule found during skin allergic inflammation. Antihistamines were not found to be efficient in controlling AD associated pruritus so far, which could be due to the chronic nature of the disease. However, specific histamine receptors still represent unique therapeutic targets in AD ,2. SinceThe prostaglandin D2 (PGD2) is a potent vasodilatator, mainly produced by mast cells, but also by eosinophils and basophils, and by epidermal keratinocytes in AD . PGD2 leOther eicosanoids can be released by IgE stimulated basophils or mast cells, which express a diverse array of lipoxygenases, and especially ALOX5 (5-lipoxygenase). ALOX5 allows the production of cysteinyl leukotrienes, which are one of the first known bioactive pro-inflammatory eicosanoids released upon mast cell or basophil degranulation . They arThe crosslinking of IgE induces the release of IL-4 and IL-13 by human mast cells and basophils. The type 2 cytokines IL-4 and IL-13 are secreted in the lesions of AD patients, and are known to have a negative effect on keratinocyte differentiation and epidermal barrier function . Autorea“Natural” immunoglobulins are produced by B1 cells. They are usually of low affinity, can be polyreactive to numerous auto-antigens, and have been mainly described for the IgM, IgG, and IgA isotypes . NaturalAtopic dermatitis is associated with the development of chronic rhinosinusitis, asthma, and food allergy, which is a phenomenon called the atopic or “allergic march” . The sysSince AD is a gateway for the generation of allergen-specific IgE and allergic diseases, it could also promote the development of autoreactive IgE-associated “autoallergic” diseases. In fact, recent epidemiological data show a significant co-occurrence of autoimmune diseases in patients suffering from AD. A cross-sectional study of 87 million US inpatients from 2002 to 2012 revealed that AD was associated with auto-immune disorders affecting the skin , the joints (rheumatoid arthritis and SLE), or the nervous system (multiple sclerosis) , which cMaurer et al. recently reviewed the presence of auto-reactive IgE in chronic spontaneous urticaria, rheumatoid arthritis, multiple sclerosis, and SLE, which are diseases associated with AD . Thus, aOne of the main effector cell bearing auto-reactive IgE in AD are basophils, which show important functions in both the skin and the secondary lymphoid organs. Evidence for a role of basophils in models of diseases associated with both AD and IgE autoreactivity  has alreFurthermore, since human basophils are an important innate source of IL-4 and IL-13 able to promote adaptive cellular and humoral type 2 immunity ,163, the


Proper pedicle screw placement is an integral part of spine fusion requiring expertly trained spine surgeons. Advances in medical imaging guidance have improved accuracy. There is high interest in the emerging field of robot-assisted spine surgery; however, safety and accuracy studies are needed. This study describes the pedicle screw placement of the first 100 cases in which navigated robotic assistance was used in a private practice clinical setting. A single-surgeon, single-site retrospective Institutional Review Board-exempt review of the first 100 navigated robot-assisted spine surgery cases was performed. An orthopaedic surgeon evaluated screw placement using plain film radiographs. In addition, pedicle screw malposition, reposition, and return to operating room (OR) rates were collected. Results demonstrated a high level (99%) of successful surgeon assessed pedicle screw placement in minimally invasive navigated robot-assisted spine surgery, with no malpositions requiring return to the OR. Pedicle screw constructs are widely used for posterior fixation in spinal surgery because of their biomechanical superiority and significant correction. However, safe pedicle screw placement is paramount to achieving successful spine surgery . SpecialVarious techniques have been used to guide and confirm pedicle screw placement . The useAn Institutional Review Board-exempt retrospective chart review was conducted from October 2017 to September 2018 on the first 100 navigated robotic-assisted spine surgeries. The demographic, intraoperative, and perioperative data of 100 patients who underwent lumbosacral pedicle screw placement with minimally invasive navigated robotic guidance using preoperative or intraoperative CT were analyzed. Pedicle screw malposition and reposition rates based on the surgeon’s intraoperative radiographic observations were collected. Secondary outcome measures included patient demographics, robot workflow, length of surgery, length of hospital stay, and intraoperative blood loss.®; Globus Medical, Inc. Audubon, PA, USA)  Fig.  uses eitA computed tomography (CT) scan of the spinal levels in the operative field was taken prior to the patient entering the operating room (OR) and screw placement planning was completed. The CT data set was transferred into the robotic positioning system, and then, registration was completed for vertebral levels.In intraoperative CT mode, the image coordinate system was obtained from a portable intraoperative CT  or standard CT scan was taken at the time of surgery, with the patient already in position on the OR table. Spinal levels were identified and a CT scan was taken. Pedicle screw trajectories were planned and saved.A surgeon-controlled foot pedal activated and positioned the robot arm to the planned pedicle trajectory. Stab incisions were made on the skin using a scalpel. Pedicle screws were inserted under neuromonitoring using navigated instruments guided by the robotic arm. This sequence was repeated until all pedicle screws had been placed. Rods were then placed in a standard fashion. Locking caps were set once the rods were in their proper position. Intraoperative fluoroscopy images were taken to verify the screw and rod position. Pedicle screw placement was qualitatively assessed using postoperative X-rays. Following screw and rod placement, lumbar interbody fusion was performed using 1 of 3 approaches: lateral, anterior, and posterior. The endplates were prepared and the interbody spacer was manually inserted. In the lateral approach, pedicle screws were placed, while patient remained in the lateral decubitus position. In ALIF, patient was repositioned from supine to prone position after interbody placement from the anterior approach. Screws and rods were then placed for posterior supplemental fixation.p < 0.05 for all statistical analysis.Statistical analysis was performed using SPSS Statistics Version 25 software . Data were presented as mean ± standard deviation. The level of statistical significance was set to 2 (range 17–44 kg/m2). Twenty-five percent of patients were either current or former smokers. Forty-two percent of patients were retired  were female. The average body mass index was 30 kg/mNavigated robotic-assisted spine procedures are in the early development . In thisTechnological advances including navigation have improved the safety and accuracy of pedicle screw fixation. In a meta-analysis by Mason et al. , data weThis initial study of the first 100 cases at a single institution in the clinical use of navigated, robot-assisted spine surgery demonstrated a high pedicle screw placement success rate. There were no postoperative screw malpositions requiring a return to the OR. While this is a single-surgeon, single-site retrospective study, the pedicle screw placement success rate is better than the rates reported in the literature using robot-assisted techniques.Navigated robotic guidance provides successful pedicle screw placement at a rate of 99% at this single institution, with a 0% return to OR rate.


Although the relationship between allergic sensitization and increased respiratory symptoms of chronic obstructive pulmonary disease (COPD) has been suggested, which allergen has a significant effect on COPD pathology is unclear. This study aimed to identify the specific IgE related to clinical features of COPD and the diagnosis of asthma-COPD overlap (ACO).®). ACO was diagnosed according to the Japanese Respiratory Society Guidelines.We recruited 76 patients with COPD and analyzed 39 IgE using panel IgE test (View Allergy 39Candida (23.7%), Dermatophagoides pteronyssinus (22.4%) and house dust (22.4%), and concerning pollen, Japanese cedar (35.5%) and Japanese cypress (22.2%) exceeded 20%. Only the positivity of IgE for Dermatophagoides pteronyssinus and house dust was significantly higher in ACO compared with that in non-ACO COPD. Moreover, it contributed to the diagnosis of ACO in an IgE class-dependent manner. Patients with cockroach IgE exhibited higher residual volume, whereas those with Japanese cedar IgE exhibited better diffusion capacity than negative patients. The contribution for ACO diagnosis by the receiver operating characteristic curve analysis was comparable among total IgE , blood eosinophil count (234/μL), and fraction of exhaled nitric oxide (31.0 ppb).As for perennial aeroallergens, the positivity for moth (31.5%), The prominent role of mite-specific IgE in the diagnosis and pathology of ACO and the potentially detrimental effect of cockroach sensitization on air trapping in COPD were suggested. The finding highlights the future development of a treatment targeting IgE as a treatable trait in COPD. Allergic sensitization is an established feature of asthma, and assessment of sensitization status is recommended for patients with asthma . In contDermatophagoides-specific IgE /forced vital capacity [FVC] < 70%) with 1 or more of the COPD features plus 2 or more of the asthma features  or Prism® 8  was used for statistical analyses.Bartlett’s test was employed to check the variances across samples. Differences between the two groups were analyzed using Student’s t-test or Mann–Whitney U-test when the variances were equal or different, respectively. The relationship between biomarkers was analyzed by Spearman’s correlation coefficient. The receiver operating characteristic (ROC) analysis was employed to find a cutoff value for biomarkers to detect ACO diagnosis. Multivariate logistic regression analysis was employed to estimate odds ratios (ORs) with 95% confidence intervals (CIs) for ACO diagnosis. Statistical significance was defined as Candida (23.7%), Dermatophagoides pteronyssinus , and house dust (22.4%) was > 20% , .5%, Cand1 tended to be lower (69.5% vs 78.7%) compared with patients with negative IgE  was significantly higher than in patients with negative IgE (92.1% vs 73.2%) . A significantly greater proportion of patients with ACO exhibited all the features of asthma, except for airway reversibility . Concerning food allergens, the overall positivity for beef-specific IgE was low (8.5%); however, the positivity was significantly lower in ACO. We also analyzed the relationship between the value of IgE class and the proportion of patients diagnosed with ACO  for blood eosinophil count, 0.665  for total IgE, and 0.703  for FeNO . The best cutoff value for diagnosis was 234 count/μL for blood eosinophils, 158 IU/mL for serum total IgE, and 31.0 ppb for FeNO.As the diagnostic criteria for ACO included FeNO and blood eosinophil count, in addition to IgE levels, we compared the utility of those biomarkers in ACO diagnosis by the ROC curve analysis. Although there were weak or no significant relationships between those biomarkers . In addition, although specific IgE for house dust (P = 0.0146) and D. pteronyssinus (P = 0.0146) significantly contributed to the diagnosis of ACO, positivity for moth (P = 0.2564), cockroach (P = 0.5653), Japanese cedar pollen (P = 0.1593), and Candida (P = 0.2917) did not exhibit significant contributions. We also analyzed the contribution of the number of sensitized allergens, and the number did not contribute to the diagnosis of ACO (P = 0.98).The contribution of blood eosinophils, FeNO, and IgE was analyzed to further determine the relative importance of the different biomarkers in ACO diagnosis. In univariate analysis, the criteria, except for blood eosinophils, significantly contributed to ACO diagnosis  and moth (31.5%) in this study was comparable to the reported positivity in asthma [A previous report using skin prick test or ImmunoCAP, wherein results were highly correlated with the current panel IgE test , revealeith COPD , 18. Conn asthma  and was [Candida 3.7% and 1 tended to be lower in cockroach IgE positive COPD . Although the use of ICS may affect the diagnosis of ACO, only three patients were treated by ICS in non-ACO COPD; therefore, the effect on the overall results was limited. Due to the nature of multiple testing, analyzing more patients is desirable for future analysis especially concerning the role of specific IgE for pollens and cockroach. In addition to our current findings, the general importance of mite-specific IgE in the diagnosis or pathology of ACO was suggested. Although geographical variation in the positivity of specific IgE may exist, sensitization to D. pteronyssinus was most prevalent among various developed countries [This study has some limitations. The importance of IgE for ACO diagnosis may vary depending on the diagnostic criteria. There are various diagnostic criteria for ACO including , 11 or nountries  and diffountries . It remaIn conclusion, by analyzing a panel of specific IgE in COPD, we revealed the specifically important role of mite-specific IgE in the diagnosis or pathology of ACO and the potentially detrimental effect of cockroach sensitization to air trapping in COPD. The finding highlights future study for developing a novel treatment strategy targeting IgE as a treatable trait in patients with ACO.Additional file 1 Features of COPD or asthma described by the Japanese Respiratory Society GuidelineAdditional file 2 The number of patients analyzed in the studyAdditional file 3 Comparison of patient characteristics based on positivity for allergen specific IgEAdditional file 4 Proportion of patients fulfilling the criteria for features of asthmaAdditional file 5 Relationships between blood eosinophil count, total IgE level, and FeNO


Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3′ and 5′ ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between Kyrpides-Ouzounis-Woese (KOW) motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively. Cdk9 (P-TEFb) and its substrate Spt5 influence events throughout the transcription cycle. Here, the authors define two switches with the potential to regulate promoter-proximal pause release and termination, respectively containing phosphatases PP4 and PP1, which are both inhibited by Cdk9, but have different specificities for sites on Spt5 and occupy opposite ends of genes. Differential phosphorylation of the CTD, which consists of heptad repeats of consensus sequence Y1S2P3T4S5P6S7, inscribes a CTD code5 that is read by factors and enzymes that preferentially bind the modified CTD, in part to coordinate RNA-processing and chromatin modification with transcription6. Concurrently, CDKs phosphorylate many other targets to control progression through the transcription cycle9.The transcription cycle of RNA polymerase II (Pol II) is divided into discrete phases of initiation, elongation, and termination. This process is regulated by cyclin-dependent kinases (CDKs) that generate stage-specific patterns of phosphorylation on the carboxy-terminal domain (CTD) of the Pol II large subunit Rpb111. This pause is established within the first ~100 nucleotides (nt) downstream of the transcription start site (TSS) by recruitment of the DRB-sensitivity inducing factor (DSIF)—a heterodimer of Spt4 and Spt5 subunits conserved in all eukaryotes—and a metazoan-specific negative elongation factor (NELF)12. In human cells, DSIF and NELF recruitment  depends on activity of Cdk7, a component of transcription initiation factor TFIIH16, whereas pause release depends on the Cdk9/cyclin T1 complex, also known as positive transcription elongation factor b (P-TEFb)17. In vitro, P-TEFb phosphorylated Pol II, Spt5, NELF, and other components of a reconstituted, paused complex, to convert it into an active elongation complex from which NELF was displaced19. P-TEFb and its orthologs in yeast are the major CDKs active during the elongation phase of Pol II transcription, phosphorylating Spt5 to enable its function as a processivity factor20, and stimulating elongation rate by three to fourfold22.A promoter–proximal pause soon after the transition from initiation to elongation is a rate-limiting step in transcription of many Pol II-dependent genes in metazoans23. Pol II paused downstream of the CPS becomes heavily phosphorylated on Ser2 of the CTD, which appears to be a consequence—rather than a cause—of its decreased elongation rate25. Pausing and Ser2 phosphorylation (pSer2) in turn promote recruitment of factors needed for mRNA 3′-end maturation and termination26. We recently uncovered a regulatory circuit in fission yeast comprising Cdk9, the protein phosphatase 1 (PP1) isoform Dis2, and their common enzymatic target, Spt5, which switches Pol II from rapid elongation to a paused state permissive for termination27. During processive elongation, Cdk9 phosphorylates the Spt5 CTD and keeps Dis2 inactive by inhibitory phosphorylation. At the CPS, the Spt5 CTD is dephosphorylated dependent on Dis2, which is a subunit of the cleavage and polyadenylation factor (CPF)28; the drop in phospho-Spt5 coincides with an increase in pSer2 over the 3′ pause site. Inactivation of Cdk9 or Dis2 leads to opposite effects—more rapid termination, or more extensive read-through transcription indicating a termination defect, respectively27. The termination-promoting effect of Cdk9 inhibition was recapitulated by an spt5 mutation that prevented Spt5-CTD phosphorylation29. Recently, human PP1 and its regulatory subunit PNUTS were implicated in Spt5-CTR dephosphorylation and Pol II deceleration downstream of the CPS31, suggesting conservation of this mechanism.Pol II undergoes a second, 3′ pause downstream of the cleavage and polyadenylation signal (CPS)9. Cdk9 inhibition diminishes phosphorylation of PP1γ on a known inhibitory site, and of Spt5 on carboxy-terminal repeat region 1 (CTR1), whereas depletion of PP1 increases steady-state levels of CTR1 phosphorylation (pCTR1). In unperturbed cells, pCTR1 drops, Pol II accumulates, and pSer2 increases downstream of the CPS—the same relationships seen in fission yeast27. The Cdk9 substrate screen also identified Spt5-Ser666, a site outside the CTRs between Kyrpides–Ouzounis–Woese (KOW) motifs 4 and 59—a region of Spt5 required for pausing, which contacts nascent RNA in the ternary complex32. Although Ser666 phosphorylation (pSer666) depends on Cdk9, it is resistant to dephosphorylation by PP1, and pSer666 and pCTR1 are distributed differently on chromatin: pSer666 increases beyond the promoter–proximal pause and is retained downstream of the CPS. We identify a second site of Cdk9-mediated inhibitory phosphorylation in PP4R2, a regulatory subunit of the protein phosphatase 4 (PP4) complex. In contrast to PP1, PP4 can dephosphorylate pSer666 in vitro, but is excluded from chromatin near the 3′ ends of genes where PP1γ occupancy is maximal, potentially explaining why pSer666 is not removed downstream of the CPS. PP4 depletion increases pSer666 and pCTR1 levels and attenuates promoter–proximal pausing in vivo. Therefore, Cdk9 phosphorylates multiple sites on Spt5 while restraining activity of two phosphatases with different site specificities and chromatin distributions, to generate diverse spatial patterns of Spt5 phosphorylation and possibly to support discrete functions at different steps of the transcription cycle.Here, we show first that the entire Cdk9-PP1-Spt5 switch is conserved in human cells. Two PP1 catalytic-subunit isoforms and two residues of Spt5 were among targets of human P-TEFb we identified in a chemical-genetic screen33 and the inhibitory Thr316 residue of PP1 isoform Dis227. As Pol II traverses the CPS, Spt5-CTD phosphorylation decreases dependent on Dis2 activity, and pSer2-containing Pol II accumulates with Spt5 in a 3′-paused complex poised for termination29. We asked if this switch is conserved in human cells, where two PP1 catalytic-subunit isoforms were identified in a chemical-genetic screen for direct Cdk9 substrates9. We validated PP1γ-Thr311 as a Cdk9-dependent phosphorylation site by two approaches. First, we treated green fluorescent protein (GFP)-tagged PP1γ, expressed in HCT116 cells and immobilized with anti-GFP antibodies, with purified Cdk9/cyclin T1, followed by immunoblotting with an antibody specific for PP1 isoforms phosphorylated on their carboxy-terminal inhibitory sites. Increased signal after Cdk9 treatment of wild-type PP1γ but not PP1γT311A suggests that P-TEFb can indeed phosphorylate this residue in vitro , whereas combined treatment with NVP-2 and RO-3306 nearly abolished the signal  in HCT116 cells9. In fission yeast, chemical-genetic inhibition of Cdk9 led to rapid, nearly complete dephosphorylation of the Spt5 CTD (T1/2 ~20 s); the rate of decay decreased ~4-fold in dis2 mutant strains, suggesting that the fast kinetics in dis2+ cells were partly due to the concomitant activation of Dis2 (PP1) when Cdk9 is inactivated27. In HCT116 cells, both pThr806 and a phosphorylation outside the CTRs, pSer666, were lost rapidly upon treatment with 250 nM NVP-2 (T1/2 ~10 min), consistent with a similar, reinforcing effect of kinase inhibition and phosphatase activation  and pSer2—which have both been interpreted as markers of elongating Pol II—had different distributions. This was most evident downstream of the CPS, where total Spt5 accumulated together with Pol II; pSer2 peaked in this region, whereas pThr806 signals were diminished—a divergence evident both in metagene plots  and MDM2 increase in time-dependent fashion ; this treatment had minimal effects on chromatin association of total PP4C , Spt5, Spt6, and multiple subunits of the PAF complex. There were 14 phosphorylations on Spt5 alone, including (1) pThr806 and several other CDK-consensus (Thr-Pro) motifs in CTR1, and (2) pSer666 and two other Ser residues in the KOW4–KOW5 linker. These phosphorylations are likely to be reinforcing, such that modification of any individual site—or even whole domains—may not be necessary to promote release from the promoter–proximal pause. To circumvent potential redundancy of modifications within Spt5 and other components of the elongation complex, we focused on defining roles for the relevant modifying enzymes. Our results indicate that two classes of Spt5 marks placed by Cdk9 near the beginning of the transcription cycle are removed at different times, by different phosphatases. This raises the possibility that they perform different roles in regulating either intrinsic properties of the elongation complex  or its interactions with other factors—evidence of an Spt5-phosphorylation code. Although both pSer666 and pThr806 are detectable over much of the gene body, pThr806 is enriched at the promoter–proximal pause, whereas the pSer666:Spt5 ratio increases further downstream, suggesting that KOW4–KOW5 linker phosphorylation is more likely to occur at or after pause release. Perhaps consistent with a causal relationship between phosphorylation and release, this region of Spt5 is essential for pausing in Drosophila nuclear extracts, and makes direct contacts with the nascent transcript32. Downstream of the CPS, where elongation is slowed, pCTR1 drops—similar to the pattern observed in fission yeast27—whereas pSer666 is retained. This difference can be explained, without invoking additional kinases, by the inability of CPF-associated PP1 to dephosphorylate pSer666.A paused complex reconstituted in vitro with purified Pol II, DSIF, and NELF was converted to an activated elongation complex by addition of elongation factors PAF and Spt6 and phosphorylation by P-TEFbspt5 that prevent CTD repeat phosphorylation at position Thr1 (spt5-T1A) suppressed a conditional dis2-11 mutation, and mimicked termination-promoting effects of allele-specific Cdk9 inhibition29. The narrowing of the termination zone by genetic manipulations in yeast was similar to the effects of introducing an intrinsically slow Pol II mutant variant or a Cdk9 inhibitor in human cells52. Conversely, dis2 loss-of-function alleles broadened the termination zone29, as did a fast variant of human Pol II51. These results supported the idea that Spt5-CTR phosphorylation by Cdk9 is an accelerator of elongation22, whereas reversal of that phosphorylation by PP1 is a brake. Two recent reports extended this model to human cells, by showing that PP1 promotes Pol II slowing and termination through Spt5 dephosphorylation31. Another implicated PP4 in regulation of Spt5 phosphorylation and function during early stages of transcription in Caenorhabditis elegans53. The results presented here establish Cdk9 as the linchpin of this network, able to phosphorylate multiple domains of Spt5 while restraining the activity of both PP4 and PP1 through inhibitory phosphorylation.A decisive role in termination for PP1 and the Spt5 CTRs is supported by genetic interactions in fission yeast. Mutations of Based on our results we propose a phosphorylation–dephosphorylation cycle during transcription elongation, governed by Cdk9 and (at least) two opposing phosphatases, PP4 and PP1 Fig. . LocalizThe model accounts for differential distributions of pSer666, pThr806, and pSer2 on chromatin, and the biochemical relationships underpinning it have been validated by results of kinase and phosphatase assays in vitro and of enzyme inhibition or depletion in vivo. Selective inhibition of Cdk9 in human cells stimulated the phosphatase activity of PP4 complexes and diminished PP4R2-pThr173 signals on chromatin. Cdk9 inhibition also diminished phosphorylation of a known inhibitory site on PP1γ in extracts, although we were unable to detect this modification on chromatin with available phospho-specific antibodies. Finally, our studies do not rule out contributions by other kinases and phosphatases, possibly arranged in similar switch-like circuits, to the regulation of Spt5 phosphorylation or Pol II elongation.55 or PP4R245. Cdk1 inhibits PP1 during mitosis; a drop in Cdk1 activity due to cyclin B degradation at metaphase leads to PP1 activation, dephosphorylation of mitotic phosphoproteins and mitotic exit56. We proposed that an analogous mechanism controls transcription exit through PP1-dependent Spt5 dephosphorylation in fission yeast27; here we provide evidence for conservation of this mechanism. A similar interaction between PP4 and a cell-cycle CDK may regulate the nucleation of microtubules at centrosomes45; we now implicate PP4 in a Cdk9-containing circuit regulating the entry to processive elongation.We describe two regulatory circuits involving Cdk9 and distinct phosphatases subject to inhibitory phosphorylation by Cdk9. Both PP1 and PP4 are also inactivated by cell-cycle CDKs that phosphorylate either the PP1 catalytic subunit57. We propose this as a strategy to order the Pol II cycle, in which relatively few CDKs (<10) are needed to phosphorylate hundreds of substrates that act at different steps of transcription9.Analogous modules, consisting of a CDK and an opposing phosphatase that is also a CDK substrate, govern transitions in both transcription and cell-division cycles. One property conferred by this arrangement is switch like, all-or-none behavior: CDK inactivation causes rapid target dephosphorylation because it simultaneously activates the relevant phosphatase. We envision that transitions in the transcription cycle, such as changes in elongation rate dictated by template sequence elements or chromatin features, are naturally switch-like. Moreover, linking activity of a single CDK to phosphatases with different specificities can generate spatially diverse patterns, even for modifications within a single effector protein. We note that the Cdk9-phosphatase circuits described here impart positional information, differentiating a promoter-proximally paused complex (pSer666 OFF/pCTR1 OFF) from one paused at the 3′ end (pSer666 ON/pCTR1 OFF). This is a fundamental principle of cell-division control; one cyclin-CDK complex can drive the entire cell cycle, producing myriad different temporal patterns of target protein phosphorylation and function, in part through the action of multiple phosphatasesl-glutamine (Corning) supplemented with 10% Fetal Bovine Serum  and 1× Penicillin–Streptomycin (Corning). Drug treatments were performed at 50–60% confluence by substituting the growth medium with fresh medium containing DMSO, 250 nM (except where noted) NVP-2 (provided by N.S. Gray), 10 μM RO-3306 (Selleckchem), 5 μM nutlin-3 , or indicated combinations of these drugs.Colon carcinoma-derived HCT116 cells were cultured in McCoy’s 5A medium with 58, rabbit anti-PP4R2 , rabbit anti-PPP4C , sheep anti-PP4R2-pThr173 45, rabbit phospho-PP1α (Thr320) antibody , mouse anti-GFP , mouse anti-pan-PP1 , goat anti-PP1α , mouse anti-PP1β , rabbit anti-PP1γ , goat anti-PP1γ , mouse anti-tubulin , rabbit anti-GST , mouse anti-FLAG® M2 , and rabbit anti-FLAG .The antibodies used were: rabbit anti-Rpb1 , rabbit anti-Rpb1 , rabbit anti-Rpb1 CTD pSer2 , rabbit anti-Spt5 , mouse anti-Spt5 , rabbit anti-Spt5-pSer666 and -pThr806  previously described3VO4, 50 mM NaF) and 1 mM DTT. Cells were lysed in a Bioruptor (Diagenode) for 10 min with cycles of 30 s ON and 30 s OFF. The lysate was clarified by centrifugation at 4 °C at 20,000 × gav for 10 min. The chromatin fraction was prepared as described previously59.Whole-cell extracts were prepared as follows: cells were washed twice with cold phosphate-buffered saline (PBS) and collected in RIPA buffer  supplemented with protease inhibitors benzenesulfonyl fluoride hydrochloride, 1 μM Aprotinin, 1 mM phenylmethylsulfonyl fluoride), phosphatase inhibitors , PP1β (sc-36295), and PP1γ (sc-36297) were from Santa Cruz Biotechnology. For pan-PP1 depletion equimolar concentrations of each siRNA were used. HCT116 cells were transfected using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s instructions. Cells were collected 24 h post transfection for lysate preparation and immunoblotting. Human embryonic kidney (HEK293) cells cultured in Dulbecco′s Modified Eagle′s Medium, supplemented with 10% FBS and 1× Penicillin–Streptomycin, were used to generate lentivirus particles expressing PP4C-targeting shRNA obtained from Sigma: shRNA-1 (TRCN0000010737), shRNA-2 (TRCN0000272746), and shRNA-3 (TRCN0000272747). HCT116 cells were infected with pLKO.1-puro shRNA lentivirus and selected for 96 h with 2 μg/ml puromycin and protein depletion verified by immunoblot. For PP4R2 depletion, siRNA targeting PP4R2  was used. HCT116 cells were transfected using Lipofectamine RNAiMAX according to the manufacturer’s instructions. After 48 h of transfection, cells were cross-linked with 1% formaldehyde for ChIP.Phosphorylated residues of Spt5 were substituted with Ala or Asp using site-directed mutagenesis kits (Agilent Technologies), the oligonucleotides listed in Supplementary Table TM 4 Fast Flow , preblocked with 1 mg/ml bovine serum albumin  and 0.25 mg/ml salmon sperm DNA (Trevigen) was added, and the resulting suspension was incubated for 2 h at 25 °C. Beads were washed three times with ice-cold RIPA buffer. For immunoblot analysis, proteins were separated by SDS-PAGE and transferred to AmershamTM ProtranTM 0.45 μm nitrocellulose membranes . The membranes were probed with primary antibodies at dilutions recommended by the suppliers. PP4R2 phospho-specific antibody (anti-PP4R2-pThr173) was used in the presence of a tenfold molar excess of the appropriate nonphosphorylated peptide. Immunoblots were developed with either horseradish peroxidase (HRP) conjugated donkey antirabbit , sheep antimouse , donkey antisheep , donkey antigoat , or Alexa Fluor-coupled goat antirabbit , goat antimouse , or donkey antigoat . Proteins were detected either by enhanced chemiluminescence  or with Odyssey Imaging System (LI-COR Biosciences). All primary and secondary antibodies were used for immunoblotting at 1:1000 and 1:5000 dilution, respectively. Immunoblots were quantified using Image Studio software in Odyssey CLx Imager (Version 5.2.5) and ImageJ 1.52q.To immunoprecipitate proteins, 2 mg of whole-cell protein extract was incubated with antibodies for 4 h at 4 °C in RIPA buffer. Protein G Sepharose15. Briefly, immunoprecipitated proteins were either mock treated or treated with purified Cdk9/cyclin T1 (5–10 ng) and ATP (1 mM) in kinase assay buffer  for 30 min at 25 °C. The beads were washed three times with RIPA buffer and analyzed by SDS-PAGE and immunoblotting. The PP4R2 samples (mock and Cdk9 treated) were divided into two equal parts for immunoblot analysis and phosphatase assays. To measure protein phosphatase activity, GFP-tagged PP1 isoforms , PP4C and PP4R2 immunoprecipitated from whole-cell extracts were incubated with 50 μM phosphopeptide  at 37 °C for 1 h. Colorimetric assays were performed in triplicate using BioMOL Green (Enzo Life Sciences) in 25 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM MnCl2, 1 mM DTT, in 96-well plates as described in manufacturer’s protocol. To test specificity of anti-pSer666 and anti-pThr806 antibodies, purified Cdk9/cyclin T1 was used to phosphorylate substrates purified from Escherichia coli expressing DSIF (Spt4/Spt5 heterodimer), GST-CTR1 (amino acids 720–830 of Spt5 fused to glutathione-S-transferase), and GST-CTR2 (amino acids 844–1087 of Spt5) at a kinase:substrate ratio of 1:2000 for 15 min at 25 °C in kinase assay buffer. The reactions were analyzed by SDS-PAGE and immunoblotting with anti-pSer666, anti-pThr806, anti-GST, or anti-Spt5.To detect Cdk9-dependent phosphorylations, GFP-PP1γ or PP4R2 were immunoprecipitated from whole-cell extracts. The bead-bound proteins were subjected to Cdk9 treatment as described15. In brief, HCT116 cells grown to 50–60% confluence were cross-linked with 1% formaldehyde  for 10 min at 25 °C. Cross-linking was quenched with 125 mM glycine for 5 min at 25 °C. Cells were washed twice with ice-cold PBS and collected into 1 ml of RIPA buffer supplemented with protease and phosphatase inhibitors for each 150-mm dish. Cells were lysed and chromatin sheared by sonication in a Bioruptor  at high power, for 5 × 10 min with cycles of 30 s ON and 30 s OFF. Lysates were clarified by centrifugation at 20,000 × gav for 20 min at 4 °C. Before immunoprecipitation, lysates (~5 × 106 cells per experiment) were precleared with PierceTM Protein A Agarose (Thermo Scientific) for 2 h at 4 °C. Beads were separated by centrifugation at 4000 × gav for 1 min at 4 °C. The resulting supernatant was incubated with antibodies for 4 h at 4 °C with constant nutation. The suspension was incubated at 25 °C for an additional 2 h with Protein G SepharoseTM 4 Fast Flow or DynabeadsTM Protein G (Invitrogen), preblocked with 1 mg/ml BSA and 0.25 mg/ml Salmon Sperm DNA. The beads were washed with 2 × RIPA buffer, 4 × Szak IP wash buffer  nonidet-P-40, 1% (w/v) sodium deoxycholate), 2 × RIPA buffer and 2 × TE buffer. After all wash steps, centrifugation was performed at 1700 × g for 1 min at 4 °C. Protein–nucleic acid complexes were eluted from beads with elution buffer  by incubating at 65 °C for 15 min with occasional vortexing. Reversal of cross-linking was done by incubating at 65 °C for 16 h. The un-cross-linked suspension was treated with 1 μg of RNase A at 37 °C for 30 min and with 0.8 units of Proteinase K (NEB) at 45 °C for 45 min. DNA was purified using QIAquick® PCR Purification Kit (Qiagen) according to the manufacturer’s protocol. The purified DNA was subjected to either qPCR with RadiantTM Green qPCR Master Mix (2×) (Radiant Molecular Tools) in 386-well plates, or library preparation for sequencing.ChIP-qPCR experiments were done as described previously27. In brief, the NEBNext Ultra II DNA Library Preparation kit was used to generate libraries using 5–10 ng of input or immunoprecipitated DNA and barcode adapters ). Single-end (75-nt reads) or paired-end (40-nt reads) sequencing was performed on an Illumina NextSeq 500.Preparation of multiplexed ChIP-seq libraries from purified immunoprecipitated chromatin and sequencing were performed as described60 in Galaxy . Normalization of the aligned reads was done using ‘bamCoverage’  by (1) computing and applying scaling factor obtained using aligned sequencing reads of a spike-in reference genome (for DMSO- and nutlin-3-treated samples) and (2) by computing RPKM (reads per kilobase per million) (for the untreated samples without spike-in control). Aligned sequences of each biological replicate were processed separately to identify enriched binding sites using MACS2 callpeak program61 . The resulting bedgraph files were converted to bigwig using ‘Wig/BedGraph-to-bigWig converter’ , replicates were combined using ‘Concatenate datasets’ . Matrix was computed using ‘computeMatrix’  in DeepTools62 to prepare data for plotting heat maps and/or profiles of given regions. The genome-wide distributions, heat maps and metagene plots were created using ‘plotHeatmap’  and ‘plotProfile’  tools, respectively. The phospho-over-total signal ratios (log2-ratio) were calculated using ‘bigwigCompare’ .We used ‘FastQC Read Quality Reports’  and ‘Trimmomatic Flexible Read Trimming Tool’  to check quality of the sequencing reads and for barcode trimming, respectively. Trimmed sequencing reads were aligned to the human genome  using Bowtie2P values were calculated using Student’s t test in Microsoft Excel (Version 14.7.0). The n values represent number of biological replicates, and the error bars correspond to ±standard deviation (s.d.) among biological replicates.All bar graphs were plotted using Microsoft Excel (Version 14.7.0) and Prism (Version 8.4.3). Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


This result indicated that adding more oyster mushroom restrained the expansion ratio. The extrudates had a medium density range (max of 1393.70 ± 6.30 kg/m3). By adding oyster mushroom, the extrudates attained a higher moisture content (range = 34.77% to 37.93%) compared with the extrudates containing the protein mixture only (range = 26.99% to 32.33%). The increase in screw speed and oyster mushroom significantly increased (p ≤ 0.05) the water absorption index. The increase in the texturization index was significantly influenced (p ≤ 0.05) by oyster mushroom addition rather than the screw speed. A defined fibrous structure supported the high texturization index and small shape of air cells observed in the extrudates.Single-screw extrusion of a fibrous-structured meat analog from soy proteins added with low-grade oyster mushroom was successful. Satisfactory extrudates were obtained at a barrel temperature of 140 °C, screw speed range of 100–160 rpm, and oyster mushroom addition at 0%, 7.5%, and 15% via factorial experiments. Single-screw extrusion equipped with a slit die successfully produced expanded oyster mushroom-soy protein extrudates. However, the increase in the oyster mushroom content significantly decreased ( The combined effects of an increasing human population worldwide, awareness on animal protein sustainability, environmental concerns, and human health considerations are expected to increase the global market venue of plant protein-based meat to USD 7.5bn by 2025 . MoreoveExtrusion is a continuous process of mixing, kneading, and shaping that involves both thermal and mechanical energies. It is a versatile cooking technique that can be applied to produce varieties of food such as cereals, meat analogs, and ready-to-eat food. Extrusion is a high-temperature/short-time process that is remarkably adaptable in fulfilling consumer demand for attractive products. The production of various food products is workable by simultaneously adjusting the conditions of feed ingredients and the setting parameters of the extruder. Feed ingredients generally consist of multiple components that will experience different structural transformations under extrusion cooking in the presence of heat, shear force, and water. In the extrusion of proteins, their original states are disrupted (denaturation of protein) and altered  by high temperature, shear, and pressure. However, protein texturization is also dependent on the type of ingredient used, as the ingredients can enhance or inhibit the desired final product quality. Several authors have considered the effects of adding extra ingredients  on the final quality  of meat analogs ,6,9,10. Mushrooms have been used as meat replacers in human diets due to their good source of macronutrients (protein and fiber) and micronutrients , as well as low fat, sodium, and energy contents ,11,12,13Low-grade oyster mushroom can be classified into the no market value category by the Federal Agricultural Marketing Authority, Malaysia . They arPleurotus sajor-caju) were obtained from Ladang Tanaman Cendawan, Universiti Putra Malaysia, Serdang. Soy protein concentrate (SP) and isolated soy protein (ISP) were purchased from Shaanxi Jintai Biological Engineering Co., Ltd., China, and Imaherb Biotech Co., Ltd., China, respectively. Commercial wheat flour produced by Syarikat Faiza Sdn. Bhd., regular single refined rock salt manufactured by Halagel (M) Sdn. Bhd., and cooking palm oil produced by FFM Berhad were purchased from a local shop in Serdang, Malaysia. Additives such as food-grade soy lecithin and sodium metabisulfite were acquired from Evergreen and Engineering Resources, Malaysia. These additives were used to improve the texture of extrudates. Distilled water was obtained from the Packaging and Preservation Laboratory, Universiti Putra Malaysia, Malaysia.Low-grade gray oyster mushrooms  for 1 min. On the basis of the analyses performed according to the Official Methods of AOAC 16th Edition 1995) [95 [19], A base mixture was prepared by mixing 50% of SP and 50% ISP. Additional ingredients such as wheat flour, soy lecithin, sodium metabisulfite, salt, cooking palm oil, distilled water, and oyster mushroom were added on a weight percentage basis of the base mixture. Five feed mixtures were prepared at different percentages of oyster mushroom content: 0% (acts as control), 7.5%, 15%, 25%, and 35%. The formulation ingredient details of the five feed mixtures on a weight basis (wb%) are given in Extrusion cooking was carried out using a laboratory-scale single-screw Brabender 19/20D extruder . The extruder has a grooved barrel with a length–diameter ratio of 20:1, two heating zones, and a heating element at the extruder’s die head. A slit extrusion die with a dimension of 20 × 2 mm (width × height) was attached to the extruder discharge. Different extruder temperatures were set at 80 °C (feeding zone), variable temperature , and 45 °C (die) for the first zone, second zone, and at the die, respectively. A compression screw (compression ratio 2:1) was used to extrude the meat analog. Feed mixtures were fed through a standard stainless steel hopper with a dosing screw rotating at a constant speed of 25 rpm. The extrusion screw speed was set between 110 and 160 rpm. The extrudates discharged at the die were only collected once extrusion reached a steady state as indicated by the consistent values of an extruder’s torque and die temperature (Zone 3) recorded in the extrusion torque–temperature over time profiles. Extrudate samples were left to cool down to room temperature before being packed in polyethylene Ziplock bags. The extrudates were kept in a freezer at −18 °C until further analysis.A range of suitable extrusion process parameters was selected based on satisfactory evaluation, which meets extrusion and the product’s specific criteria. The evaluation was categorized into two criteria: (1) the observation of the extrusion ability and (2) extrudate’s physical appearances , for each case of extrusion conditions. Thus, the factorial experimental design was applied to conduct a preliminary investigation for this selection purpose. Three extrusion process variables, namely barrel temperature, screw speed, and oyster mushroom percentage, were considered. The first stage was to determine the suitable extruder barrel temperature (compression zone). Feed mixture containing 0% oyster mushroom (control) was extruded at four levels of barrel temperature  and six levels of screw speed . The extruder barrel temperature that exhibited a smooth extrusion flow produced unpuffed extrudates with a smooth surface and fibrous structure. These extrudates were then further applied to the second stage of the preliminary investigation to determine the suitable percentage of oyster mushroom addition. Feed mixtures containing 7.5%, 15%, 25%, and 35% oyster mushroom were extruded at the selected extruder barrel temperature and at the same six levels of screw speed. The obtained extrusion flow behavior and physical appearances of each extrudate were summarized using Microsoft Excel 2016 .Expansion ratio was calculated using Equation (1), in which the dimension of the extrudate (width: mm × height: mm) was divided by the flat sheet die dimension (width × height: 20 × 2 mm). A digital Vernier caliper  was used to measure three replicates of the sample dimension. The average expansion ratio measurement was recorded.Equation (2) was applied to calculate the apparent density of the extrudate. The width, height, and length of the extrudate were measured by using a digital Vernier caliper . An analytical balance  was employed to measure the extrudate’s weight. The apparent density of three replicates was obtained as an average.W = weight of extrudate (kg); L = length of the extrudate (m);The WAI was determined following the procedure of Anderson . A suspeThe moisture content of the extrudate was determined immediately after the extrudate exits the extruder’s die. The MX-50 Moisture Analyzer  was employed to measure the moisture content. Before measurement, 2 g of the extrudate was heated at 105 °C until a constant weight was achieved. The mean of triplicate moisture content measurements was recorded.The TI was determined following the procedure reported by Wu et al. , with soextruder ,23. The Scanning electron microscopy (SEM) analysis was conducted to observe fibrous structures and air cell formations in the extrudates. Extrudates were cut into the dimensions of 1 × 1 × 0.2 cm. For each test extrudate, they were cut in transversal and longitudinal directions for further observations. The test samples were mounted onto aluminum stubs by using double-sided carbon tape. Both sample surfaces of cutting directions were viewed under low-vacuum mode in a JSM-IT100 InTouchScope™ Scanning Electron Microscope  at 40× and 100× magnification with an accelerating voltage of 3 kV for raw extrudates. The low accelerating voltage was applied to minimize the charging effects on the test sample. p ≤ 0.05. The Pearson coefficients (r) of linear correlation among the physical properties of extrudates were also determined. Tukey’s multiple comparison test was used to determine the significant difference at p ≤ 0.05 among a set of mean.A two-factor full factorial design was performed to determine the influences of the extrusion process parameter on the extrudate’s physical properties by using Minitab 17 software . One-way analysis of variance was carried out to determine the level of significance at Extrusion parameters and performance can vary depending on the ingredients used and target extrudate characteristics. Therefore, a satisfactory evaluation can be conducted in preliminary investigations to identify the extrusion process variables  . Table 2p ≤ 0.05; p ≤ 0.05). This result showed that increased oyster mushroom retarded extrudate expansion, possibly because of the presence of high protein, fiber, and fat contents in the feed. In the present study, oyster mushroom was added into the feed mixture because of its rich source of dietary fiber and protein [In this study, single-screw extrusion equipped with a slit die of feed mixture produced expanded oyster mushroom-soy protein extrudates. This finding was consistent with that of Rehrah and Samard, who obtained expanded meat analogs from the extrusion of a protein-based mixture employed with a slit die ,24. As s protein ,13. Rece protein .p ≤ 0.05) when the screw speed and oyster mushroom increased (3) than the extrudate produced with 15% oyster mushroom addition (1272.73 ± 2.94 kg/m3). The apparent density of the extrudates was more likely affected by the increment of screw speed from 100 to 160 rpm than the addition of oyster mushroom. The apparent density of the extrudate made from soy proteins and oyster mushrooms was lower than that of the extrudate added with flavor enhancer as reported by Milani et al. [No significant negative correlation was found between the expansion ratio and apparent density of the extrudates . The extncreased . The datp ≤ 0.05). However, no remarkable changes in the moisture content of extrudates run at the screw speed of 110–150 rpm were observed. A reduction in moisture content was observed for the extrudate produced with 0%, 7.5%, and 15% oyster mushroom at the speed of 160 rpm. These trends showed that the moisture content was negatively affected by screw speed but positively affected by oyster mushroom addition at a significance level of p ≤ 0.05 (p ≤ 0.05) between moisture content and expansion ratio, but no significant positive correlation was observed between moisture content and apparent density (p ≤ 0.05 . A stron density . The lesp ≤ 0.05) with the increase in screw speed and oyster mushroom addition (p ≤ 0.05) with the expansion ratio and apparent density but positively correlated (p ≤ 0.05) with the moisture content and TI of the extrudate  with increasing percentage of oyster mushroom addition (The fibrous structure formation in a meat analog can be expressed by the TI with a dimensionless value of >1. The TI is the ratio of the maximum cutting strength in the vertical (lengthwise strength, extruder ,30. The addition . Extrudaaddition . The screw speed insignificantly affected the TI values of extrudates produced at speeds ranging from 110 to 160 rpm, but TI variation was observed. As shown in The oyster mushroom-soy protein meat analog microstructures in transverse and longitudinal surfaces are presented in A factorial experiment design was successfully used to determine the suitable barrel temperature of 140 °C, screw speed ranging from 110 to 160 rpm, and oyster mushroom addition at 0%, 7.5%, and 15% for the extrusion of unpuffed extrudates with fibrous structure. Addition of oyster mushroom to the soy protein-based meat analog resulted in a significantly reduced expansion ratio and apparent density but increased the moisture content and WAI. In this study, the apparent density of the extrudates was found to be affected more by the increment of screw speed compared with the oyster mushroom content. The TI of the extrudates increased significantly when the oyster mushroom content increased. However, the screw speed only had a minor effect on the TI of the extrudates. The application of single-screw extrusion equipped with a slit die produced expanded protein extrudates with non-uniform air cell distribution. At the addition of 15% oyster mushroom, the extrudate had a smaller air cell and a defined fibrous structure compared with the control extrudate. Overall, the study could be useful for researchers looking for a better understanding of extrusion flow performance and extrudate physical changes, as well as food manufacturers that aim for the development of meat analogs with improved aesthetic properties based on plant-based mixtures and processing.


Translation is a fundamental process in gene expression. Ribosome profiling is a method that enables the study of transcriptome-wide translation. A fundamental, technical challenge in analyzing Ribo-Seq data is identifying the A-site location on ribosome-protected mRNA fragments. Identification of the A-site is essential as it is at this location on the ribosome where a codon is translated into an amino acid. Incorrect assignment of a read to the A-site can lead to lower signal-to-noise ratio and loss of correlations necessary to understand the molecular factors influencing translation. Therefore, an easy-to-use and accurate analysis tool is needed to accurately identify the A-site locations.We present RiboA, a web application that identifies the most accurate A-site location on a ribosome-protected mRNA fragment and generates the A-site read density profiles. It uses an Integer Programming method that reflects the biological fact that the A-site of actively translating ribosomes is generally located between the second codon and stop codon of a transcript, and utilizes a wide range of mRNA fragment sizes in and around the coding sequence (CDS). The web application is containerized with Docker, and it can be easily ported across platforms.https://a-site.vmhost.psu.edu/. The code is publicly available at https://github.com/obrien-lab/aip_web_docker under the MIT license.The Integer Programming method that RiboA utilizes is the most accurate in identifying the A-site on Ribo-Seq mRNA fragments compared to other methods. RiboA makes it easier for the community to use this method via a user-friendly and portable web application. In addition, RiboA supports reproducible analyses by tracking all the input datasets and parameters, and it provides enhanced visualization to facilitate scientific exploration. RiboA is available as a web service at  Regulation of the translation process influences steady-state protein levels in cells. Hence, it is important to understand translation to determine its role in gene expression. The development of ribosome profiling (Ribo-Seq), a high-throughput Next-Generation Sequencing (NGS) method, has greatly advanced the transcriptome-wide study of translation –3. The AS. cerevisiae [The A-site location is identified by an offset value, which is the number of nucleotides separating the start of the A-site codon from the 5′-end of the ribosome-protected mRNA fragment. For example, in one study , the A-srevisiae –6 and morevisiae , 8. Thisrevisiae  and scikrevisiae , and R previsiae , riboWalrevisiae  and Riborevisiae .Recently, a novel method was created that utilizes Integer Programming . This meTherefore, we developed RiboA, a user-friendly web application that employs this Integer Programming method. RiboA identifies the most accurate A-site location on a ribosome-protected mRNA fragment and generates the A-site read density profiles. In addition, RiboA tracks all the input datasets and parameters, and hence supports reproducible analyses. It also provides enhanced visualization to facilitate scientific exploration. RiboA is containerized with Docker , and it S. cerevisiae [E. coli [The homepage of RiboA introduces the Integer Programming method for identifying A-site locations and includes a tutorial and a video on how to use RiboA. While login is not required and anonymous users can submit jobs, they are limited to using the existing input datasets hosted on the server. These include a published dataset for revisiae , a poole[E. coli –20, and [E. coli . On the ∆, for fragments of a particular size (S) and frame (F) that map onto gene i by maximizing the total number of reads between the second codon and stop codon of a transcript T, subject to constraints 0 ≤ ∆ ≤ S and ∆ mod 3 = 0. Sometimes the top two highest scores T and T can be very close, where ∆′ and ∆″ are the two corresponding offset values. To avoid bias caused by small sample size, genes who on average have less than one read per codon are filtered out. To further improve the robustness of the offset table, we implemented two additional thresholds to identify unique offsets. First, at least 70% of genes should exhibit the most probable offset and at least ten genes should be present in each dataset. This threshold can be lowered to a value above 50% to generate more uniquely identified offsets. However, a lower threshold may lead to less accurate results and it is up to the researcher’s discretion. Secondly, the average number of reads in the second, third and fourth codon is at least five times the number of reads in the first codon. Three of the thresholds can be customized when the user submits a RiboA offset job (see below). Note that if the A-site location cannot be uniquely determined the top two offset values will be included in the generated offset table, and we recommend users disregard reads with uncertain A-site offsets.The workflow diagram of using RiboA is illustrated in Fig. S. cerevisiae and E. coli. If the user chooses to upload a GFF file, RiboA will convert it to the CDS annotation file in the backend. The format of the CDS annotation file is specific to the alignment mode, e.g. genome or transcriptome. Upon a user’s selection of the alignment mode, the corresponding instruction for the annotation format is shown on the webpage. We have provided examples for both the GFF files and the TAB-based annotation files on the “Upload Data” page of the website. A number of parameters can be customized, such as the range of fragment size measured in nucleotides and the number of nucleotides beyond the CDS region of a gene which are to be avoided to overlap with another gene, the minimum average number of reads per codon for filtering genes, the minimum percentage of genes with the most probable offset for assigning a unique offset, and the minimum ratio between the average reads in the second, third and fourth codon and the reads in the first codon also for assigning a unique offset. These parameters help to improve the robustness of the method. Optionally, users can also upload a filter file to include or exclude genes from the analysis.On the offset job submission page, users need to provide the input datasets and parameters Fig. b. The inS. cerevisiae that was used in our previous study [A profile job takes in a custom offset table for specified read lengths and reading frames and generates A-site read density profiles mapped to genes. A default offset table is provided for us study . Other ihttps://redis.io) and a celery (https://docs.celeryproject.org) task queue to orchestrate jobs asynchronously. Upon job submission, users are immediately redirected to the job’s reporting page that will show that the job is in the status PENDING or RUNNING; while in the background, the job is appended to a queue and multiple workers take on jobs from the queue sequentially. Once the job finishes, an email notification with the link to the reporting page is sent to the user. If the job finishes successfully, the reporting page provides the output files to download and visualizes some of the important results. The input datasets and parameters are also tracked and shown on the reporting page so that the analysis can be easily reproduced. If the job fails, users can review the log file for causes.Depending on the input file size, the job may take over one hour to run for a 3 GB SAM file with 5 million alignments. Thus, we utilize a redis message broker (https://www.djangoproject.com) web service, (2) a nginx (https://www.nginx.com) web server, (3) a redis message broker, (4) a celery asynchronous job queue, and (5) a PostgreSQL (https://www.postgresql.org) database. Each service resides in its own Docker container and the five containers are connected with Docker Compose. Containerization makes it easy to port the application across platforms and deploy it to a cloud. In addition, users can simply set up a local environment and run it as a standalone application on their own machines.The web application is containerized with Docker. It consists of five services: (1) a Django  the A-site reads per nucleotide, (2) the A-site reads per nucleotide mapped to Frame 0 by applying the transformation that for reads in frame 1 and 2 the offset is reduced by 1 and 2, respectively, (3) the A-site reads per codon. Each A-site read density profiles generated from RiboA is contained in a tab file . The only exception is the Hussmann method where the difference is not statistically significant.To verify that RiboA generates the most accurate A-site offsets, we examined the ribosome density at the strongest stalling PPX motifs assigned by RiboA in comparison with other A-site methods. To date, this is the best approach to compare A-site methods because these stalling motifs have been identified by both Ribo-Seq and biochemical studies, and the A-site location is known to be at the codon encoding the third residue of the motif –25. We f dataset  . Therefore, RiboA is the most accurate tool in identifying A-site locations.We then examined the stalling motif PPE in an mESCs dataset  Fig. 2bb2b. We d.  where ine.g. 15 nt and 18 nt, perform relatively well in some of the cases, and have previously proven useful in studying translation properties. However, RiboA gives more accurate offset in most cases, and hence better signal-to-noise ratio in Ribo-Seq analysis.We note that the heuristic constant offsets, RiboA assumes the ribosomes are undergoing steady-state translation, and it can only be applied to steady-state ribosome profiling data. RiboA is not appropriate for datasets from non-steady-state experiments, such as the ribosome run-off experiments where initiation is blocked by antibiotics, such as harringtonine treatment.RiboA is a web application that identifies A-site locations and generates read density profiles. We have shown that RiboA is the most accurate in identifying A-site on Ribo-Seq mRNA fragments compared to other tools. In addition, by monitoring all the input datasets and parameters, RiboA supports reproducible computation. And the interactive visualization it presents can facilitate the scientific exploration. As a user-friendly web application, the use of RiboA requires zero programming skills. The containerization has further increased its portability. Detailed tutorials on how to use RiboA and on how to setup the container have been provided both on the RiboA’s home page and the GitHub repository. By making this tool easier to use, we hope RiboA will find widespread use by the community.Project name: RiboA.Project home page: A-site.vmhost.psu.edu.Operating system(s): Platform independent.Programming language: Python, PostgreSQL, HTML, JavaScript, CSS.Other requirements: Web browser.License: MIT license.Any restrictions to use by non-academics: None.