Ex vivo T cell activation (Ox40/PDL-1 expression) of PBMCs stimulated with peptide pools derived from 11 German cockroach proteins, including nine official cockroach allergens, plus chitinase and vitellogenin, was determined by flow cytometry. IgE prevalences to chitinase (17%) and hemocyanin (44%) were comparable to values for the other eight allergens that we previously reported (21–57%). Hemocyanin (Bla g 3), was a major allergen  for a sub-group of 15 highly cockroach-sensitized subjects (IgE > 3.5 kUA/L: 53%). Chitinase was officially named as new allergen Bla g 12. Cockroach-specific IgE levels in plasma showed excellent correlation with the sum of 10 allergen-specific IgE . T cell reactivity to 11 proteins was highly variable among subjects, the highest being for vitellogenin, followed by Bla g 3. The main finding was that cockroach allergen-specific IgE and T cell reactivity patterns were unique per subject, and lacked immunodominant allergens and correlation with clinical phenotype/disease severity in the studied cohort. Knowing the subject-specific B/T cell reactivity profiles to a comprehensive panel of cockroach allergens will contribute to diagnosis of cockroach allergy and will be important for planning and assessing allergen immunotherapy outcomes, according to the allergen content in therapeutic cockroach extracts.IgE sensitization to cockroach allergens is associated with development of allergic diseases, such as asthma. To understand the relevance of different cockroach allergens for diagnosis and immunotherapy, a comprehensive analysis of IgE antibody levels and T cell reactivity to an expanded set of cockroach allergens and their relationship to disease was performed in a cohort of USA cockroach sensitized patients. IgE antibody levels to recombinant chitinase and hemocyanin were measured for 23 subjects by custom-made ImmunoCAPs and compared with IgE levels to eight cockroach allergens we previously reported for the same cohort.  Blattella germanica is the most common species associated with allergy. Exposure by inhalation to allergens released by the cockroach into the environment can lead to IgE production (sensitization) in susceptible individuals. However, currently immunotherapy is not used for cockroach allergy and there are no standardized extracts for therapy. To date, no strong immunodominant allergen/s regarding IgE prevalence have been described for German cockroach in a given population .In 2005, Satinover et al. showed that the IgE reactivity to five cockroach allergens  was variable among cockroach allergic patients n = 118), with unique subject sensitization profiles . Bla g 2, with unPeriplaneta americana in Taiwan, resulting in four polymorphisms that are currently listed as Per a 3 in the above mentioned official Allergen Nomenclature database  of cockroach allergic subjects , aiming to compare and identify the most relevant molecules in terms of B and T cell reactivity, and their association to disease. The ultimate goal was to gain insight into immunological responses associated with cockroach allergy for improving diagnosis and treatment, especially in light of the current cockroach immunotherapy trial from the Inner City Asthma Consortium (CRITICAL).A/L) were recruited from San Diego, California; St Louis, Missouri; and New York, New York, according to institutional review board approval   on average. Two negative controls were a non-allergic individual and a subject sensitized to Fel d 1, Bet v 1, and Phl p 5, but not to cockroach (IgE < 0.1 kUA/L using i6 ImmunoCAP). Both were negative for Bla g 3 and Bla g 12.A cohort of 23 subjects sensitized to cockroach (IgE titer ≥ 0.35 kU13-0691) Table 1.13-0691) . All hadPichia pastoris using the pPICZαB vector, by methanol induction. The proteins were chitinase, hemocyanin (Bla g 3.0101) and an N-terminal fragment of vitellogenin named N-vitellogenin (18-322 with 6-His-tag). The rational for expression of the N-terminal vitellogenin is explained below. Their expected MW calculated from the amino acid sequence including the C-terminal 6-His-tag were 58.1 kDa for chitinase , 79.6 kDa for hemocyanin and 36.3 kDa for the N-terminal vitellogenin  (Three German cockroach proteins were expressed in ctively) , 19, 20.Chitinase and Bla g 3 were purified by metal affinity chromatography . N-vitellogenin was purified by metal affinity chromatography followed by size exclusion chromatography (20 mM TrisBase 0.02 M NaCl pH 7.2). A purity of >90% was confirmed for the three molecules on silver-stained SDS-PAGE (data not shown). Purity of chitinase and N-vitellogenin was additionally confirmed by mass spectrometry . Mass spectrometry was performed with a Thermofisher Q Exactive mass spectrometer, equipped with a Vanquish Flex UHPLC liquid chromatography system. Samples were digested with trypsin and reduced with tris(2-carboxyethyl) phospine (TCEP), followed by alkylation with iodoacetamide. The LC-MS/MS analysis was performed using ThermoFisher Proteome Discoverer 2.2 software, employing the Sequest HT search engine.in vivo, typically close to polyserine sequences at an RXXR consensus sequence motif by subtilisin-like endoproteases  from German cockroach was expressed.IgE antibody binding to the full vitellogenin could not be tested because attempts to express the full-length protein from German cockroach (MW I18-N1862) resulted in a fragmented recombinant protein. Proteolytic cleavage of vitellogenin also occurs roteases , 22, learoteases , 24. BasPichia pastoris, comprising the amino acids I18 to S322 plus a C-terminal 6xHis-tag for purification by nickel affinity chromatography. The recombinant N-vitellogenin was >90% pure in silver-stained SDS-PAGE gel and further shown to be 93% pure by mass spectrometry. Residues 1–17 were not included since they are the putative signal peptide according to the SignalP-5.0 server  and Comas et al.  was that: a) an equivalent 40 kDa unit had been isolated from the abdominal fat body tissue of honeybees , b) a res et al. . The expEZ-Link Sulfo-NHS-LC-Biotin  was added to a defined amount of each allergen  at a 10-fold molar excess and incubated for 30 min at room temperature. The biotinylated mix was put over a pre-washed Zeba Desalt Spin Column  two times and the concentration was determined after biotinylation by Advanced Protein Assay .The quantification of biotinylation was carried out by using a Quant Tag™ Biotin Kit . Samples were tested in triplicate against a known biotin standard curve to determine the number of biotins per allergen molecule.Streptavidin ImmunoCAPs  were loaded and incubated on a Phadia 100, with the biotinylated allergen at amounts ranging from 0.5 to 10 µg/CAP. Two different human plasma samples from individuals allergic to the allergen  were selected for optimization experiments. Their IgE levels to the allergen-loaded CAPs were measured in a Phadia 250 following manufacturer’s instructions  to select optimal amount of biotinylated allergen to be loaded to the streptavidin ImmunoCAPs.Biotinylated cockroach allergens Bla g 3 and Bla g 12 were loaded and incubated on streptavidin ImmunoCAPs using the Phadia 100. The ImmunoCAPs were transferred to the Phadia 250, where measurements of allergen-specific IgE antibody binding were performed according to manufacturer’s instructions.2O2. Absorbance was read at 405 nm on a Bio-Tek EL800 Microplate Reader .IgE antibody binding N-vitellogenin was assessed by chimeric enzyme-linked immunosorbent assay. Microtiter plates were coated at 4°C overnight with 10 μg/ml of N-vitellogenin. Plate was blocked for 30 min with 1% bovine serum albumin/phosphate-buffered saline, 0.05% Tween 20, pH 7.4. A 1 h incubation with sera (dilutions 1:2 and 1:10) was performed. Bound IgE was detected using biotin-labeled goat anti-human IgE  at a 1:4,000 dilution (1h incubation), and quantified using a human/mouse chimeric IgE antibody as standard . StreptaA/L, had IgE against N-vitellogenin and served as positive control for the immunoassay. These two plasma were obtained from PlasmaLab International , which operates in full compliance of US Food and Drug Administration. Informed donor’s consent was obtained from each subject before the first donation. None of the patients tested (n = 22) from the cohort had IgE specific for N-vitellogenin.IgE binding to N-vitellogenin was tested in 22 of the cohort patients, and two additional plasma from: 1) a patient sensitized to dust mite but not to cat, dog, and cockroach that was used as an additional negative control, and 2) a patient sensitized to Fel d 1, Can f 1, Bet v 1 and Phl p 5, with cockroach-specific IgE of 2.34 kUPeripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation according to the manufacturer’s instructions  as previously described . Cells wSequences of 11 cockroach proteins  were collected from UniProt. A strategy using 15-mer peptides overlapping by 10 amino acids was selected and peptide sequences generated to get the full coverage of the antigen  for each individual allergen, PHA and PT (positive controls), or DMSO (negative control). Cells were incubated for 24 h. After the incubation, cells were labeled with a cocktail of antibodies  was similar to the average of geometric means of the 10 cockroach allergen-specific IgEs (0.78 kUA/L), and approximately half the maximum value, which was for Bla g 2 (1.36 kUA/L) (A/L). There was a highly significant correlation between cockroach-specific IgE and the sum of 10 allergen-specific IgE levels . With individual sensitization profiles, there was overall a correlation of the number of allergens recognized per subject and their titer with the IgE antibody binding to the cockroach extract.To assess the relevance of hemocyanin (Bla g 3) and chitinase, the IgE antibody levels to both purified molecules were measured and compared with the IgE reactivities to 8 other cockroach allergens that we previously reported for the same cohort (n = 23)  FigureFigureA/LA/L (A/L) were 47.8% (11/23) and 30.4% (7/23), respectively. Based on these data, the chitinase was proven to be an allergen and was submitted to the WHO/IUIS Allergen Nomenclature database, which approved the assignment of this new allergen to group 12. Chitinase is now listed as Bla g 12 in the official database, and this name will be used from now on in this article.The prevalences of IgE sensitization among the 23 subjects tested were 43.5% (10/23) for Bla g 3 and 17.4% (4/23) for chitinase, using a conservative cut-off of 0.35 kUA/L Table 2.A/L  the IgE prevalences were 53.3 and 20.0%, respectively, regardless of cut-off. In subjects with cockroach-specific IgE > 5 kUA/L, the IgE prevalences were 58.3% for Bla g 3 and 16.7% for Bla g 12 (0.35 kUA/L cut-off), and 66.7% and 33.3% (0.1 kUA/L cut-off), respectively. These results indicate that Bla g 3 was a major allergen for this sub-group of highly cockroach-allergic subjects, adding to the other four major allergens   was above the average of IgE prevalences for the 10 allergens tested (38.7 ± 13.0%), and for Bla g 1, Bla g 4, Bla g 5, and Per a 7  (n = 23) Table 2.3.5% was Patterns of IgE reactivity to the 10 cockroach allergens were variable in the cohort. Among 10 (out of 23) subjects that recognized Bla g 3, four  also recognized Bla g 12 Figure 2Bordetella pertussis (PT) vaccine antigens. Good signal was observed in response to the positive control PHA . The evaluations were based on previously described Activation Induced Marker (AIM) assays , 28, utiNo correlation was observed between the prevalences of IgE sensitization and the frequencies of the T cell response to 10 cockroach allergens at the population level in the cohort tested Table 2,A/L). Moderate to severe asthma alone was found in individuals with low cockroach-specific IgE . Conversely, mild asthma was found associated to relatively high cockroach-specific IgE (8.32 kUA/L), in addition to low levels (0.91 kUA/L).The clinical phenotypes of the subjects in the cohort ranged from mild asthma or allergic rhinitis alone, to more severe forms of these diseases, or their combination Table 1.A/L) had allergic rhinitis and moderate to severe asthma, among the three subjects with the lowest IgE levels (2.25 to 2.79 kUA/L), two had mild asthma and one moderate to severe asthma.No allergen was specifically associated with severity of the disease. For example, four patients with the most severe disease  had the highest levels of IgE to an allergen from either group 2, 7, 9, or none of the 10 tested. The allergen to which more subjects had the highest IgE levels was Bla g 2 (six subjects). Among those, there was not a clear association between Bla g 2-specific IgE levels and severity of the disease: whereas three out of those six with the highest levels of IgE to Bla g 2  for diagnosis and therapy. This is especially relevant for component analyses of clinical immunotherapy trials, given the variability of allergen content in extracts used for treatment, which may impact clinical efficacy , 29. Thein vitro expansion steps to allow their characterization on a global scale. Although this approach allows for greater sensitivity, it may alter the phenotype of responding T cells. We and others previously demonstrated that allergen-specific T cells can be detectable ex vivo using a novel assay strategy with the combination of pools of T cell epitopes. This technique uses the upregulation of the activation markers as a read-out for T cell reactivity (AIM assay)  , and canM assay) , but a lM assay) –35. ThisPichia pastoris has the secretion signal sequence from the Saccharomyces cerevisiae α-factor prepro peptide , suggesting a correct folding of the molecules. Second, the pPICZαB vector used for protein expression in  peptide , 37. P.  folding . In our  hexamer . A surfaAscaris lumbricoides, as it also occurs in Africa  did not correspond to the subject with the highest T cell response (fold activated T cells) to this protein (#14). Conversely, the patients with the highest T cell reactivity to Bla g 3 (subject #13) or Bla g 12 (#5), did not have IgE against these two allergens. Similarly, the subject with the highest T cell response to Bla g 2 (#5) did not have the highest Bla g 2-specific IgE (#18). These results indicate the existence of different individual allergen immunodominance for either B or T cell reactivity. They also indicate that a variability in subjects’ reactivity to extracts used for immunotherapy is expected, according to their allergen composition.At the individual level, a unique profile of IgE sensitization and T cell responses to 10 allergens showed dominance of different allergens per subject. The four patients with IgE specific for chitinase and hemocyanin  were not the ones with the highest cockroach-specific IgE. For subject #12, chitinase dominated the response. However, overall, there was a correlation of the number of allergens recognized and their titer with the IgE antibody binding to the cockroach extract, as reported previously for eight allergens . Unlike in vivo  despite similar number of peptides per pool . Current evidence also shows that allergen T cell dominance varies in other cohorts, as reported for Bla g 5, Bla g 9 and Bla g 11  , than among individuals with rhinitis alone (n = 20) (81% versus 45%). On the other hand, 80% of allergic rhinitis patients had IgE-binding activity to Per a 9, versus only 28.5% of asthmatic patients. These results suggested that sensitization to Per a 2 could be a marker of more severe airway disease . HoweverThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Review boards: La Jolla Institute’s Institutional Review Board (IRB protocol: VD-112-0217), Mount Sinai’s Institutional Review Board (IRB protocol: GCO 13-0691), and Washington University Institutional Review Board (IRB protocol: 201305110). No vulnerable populations were involved. The patients/participants provided their written informed consent to participate in this study.JG expressed and purified the new cockroach allergens and measured IgE reactivity. VS and RdSA planed the experimental T cell work and wrote the T cell section. ASu performed all the T cell focused experimental work. LBB, AB, and PB provided clinical samples. AF provided input for data analysis. AP and ASe designed the study. AP wrote the manuscript. All authors contributed to the article and approved the submitted version.Research reported in this publication was supported by the National Institute of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI077653 (to AP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.AP is an employee of Indoor Biotechnologies, Inc. and the contact principal investigator of the NIH R01 Award that funded the study.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.


NKX2-5 and TBX5 genes encoding cardiac transcription factors. No pathogenic variants were identified in patients with isolated LVNC in the absence of cardiac dysfunction or syndromic features. In conclusion, the diagnostic yield of genetic testing in adult index patients with LVNC is low. Genetic testing is most beneficial in LVNC associated with other cardiac and syndromic features, in which it can facilitate correct diagnoses, and least useful in adults with only isolated LVNC without a family history. Cardiac transcription factors are important in the development of LVNC and should be included in genetic testing panels.The genetic etiology and heritability of left ventricular noncompaction (LVNC) in adults is unclear. This study sought to assess the value of genetic testing in adults with LVNC. Adults diagnosed with LVNC while undergoing screening in the context of a family history of cardiomyopathy were excluded. Clinical data for 35 unrelated patients diagnosed with LVNC at ≥18 years of age were retrospectively analyzed. Left ventricular (LV) dysfunction, electrocardiogram (ECG) abnormalities, cardiac malformations or syndromic features were identified in 25 patients; 10 patients had isolated LVNC in the absence of cardiac dysfunction or syndromic features. Exome sequencing was performed, and analysis using commercial panels targeted 193 nuclear and mitochondrial genes. Nucleotide variants in coding regions or in intron-exon boundaries with predicted impacts on splicing were assessed. Fifty-four rare variants were identified in 35 nuclear genes. Across all 35 LVNC patients, the clinically meaningful genetic diagnostic yield was 9% (3/35), with heterozygous likely pathogenic or pathogenic variants identified in the  Genetic testing is of limited use for diagnosing a rare heart muscle disorder called left ventricular non-compaction (LVNC), a structural abnormality of the lower left chamber of the heart in which the muscular wall appears soft and spongy rather than smooth and compacted. A team led by Christopher Semsarian from the University of Sydney, Australia, carried out a a retrospective analysis of genetic data from 35 patients diagnosed with LVNC but with no family history of the disease. They identified only three likely pathogenic gene variants, all in known heart regulatory genes and all in individuals with symptoms of cardiac dysfunction. The researchers conclude that genetic testing can facilitate a correct diagnosis of LVNC in patients showing signs of heart problems, but is largely unhelpful for those without symptoms and no family history. LVNC is classified as an inherited cardiomyopathy of autosomal dominant inheritance, and clinical screening of first-degree family members is indicated2. Outside such families, LV trabeculation identified in an individual may be an incidental finding or a feature of a syndrome4. Major adverse events include life-threatening arrhythmias, thromboembolism, and cardiac failure.Left ventricular noncompaction (LVNC) is an arrhythmogenic cardiomyopathy characterized by prominent left ventricular (LV) trabeculations with deep intertrabecular recesses and thinning of the compact epicardium. LVNC is increasingly being diagnosed in adults, though the clinical significance of LVNC presenting in adulthood remains unclear, particularly when the diagnosis is made outside the context of an affected familyMYH7, MYBPC3 and TTN, with reported genetic testing yields between 17 and 41%5. The diagnostic genetic yield in these studies may be biased by the inclusion of individuals screened in the context of a family history of cardiomyopathy, for which the genetics are better understood. Genetic testing is typically performed using clinical LVNC gene panels that screen a large number of cardiomyopathy-associated genes. Exome sequencing has the added advantage of facilitating analysis of nuclear and mitochondrial genomes as well as reassessment of data when new gene-disease associations are elucidated. Both nuclear and mitochondrial genome variants have been associated with LVNC6.Identification of a genetic etiology is important because it can aid in correct diagnoses, identify at-risk family members and inform reproductive decisions. Studies of the genetic causes of LVNC have primarily identified variants in cardiomyopathy genes, including In this study, we sought to assess the value of genetic testing in adult index patients diagnosed with LVNC outside the context of family screening and to explore the potential of genetic testing to guide the clinical diagnosis and management of such individuals.This study included all unrelated index patients diagnosed with LVNC in adulthood, defined as >18 years of age, who attended the Genetic Heart Disease Clinic, a cardiac tertiary referral centre at the Royal Prince Alfred Hospital in Sydney, Australia, between 2002 and 2018. Blood used for genetic testing was available for all enroled subjects. The patients were previously diagnosed with LVNC in the context of symptomatic or asymptomatic assessment and were referred for evaluation and management. Patients who attended the clinic for cardiac screening in the context of a significant family history were excluded. Informed consent was obtained, and the Sydney Local Health District Ethics Committee approved the study.8. Past clinical data were retrospectively retrieved from medical records: clinical assessment included physical examination, 12-lead electrocardiogram (ECG), and echocardiogram. LV systolic dysfunction was defined as a systolic left ventricular ejection fraction (LVEF) < 50% on echocardiogram. Cardiac magnetic resonance imaging results were available for the majority of the patients. The patients were also assessed for any syndromic features, including pectus excavatum and upper limb abnormalities.Confirmatory diagnoses were made by experienced cardiologists based on clinical presentation and interpretation of echocardiogram and cardiac magnetic resonance imaging results Fig.  using th10. Ultrarare variants were defined as those with a gnomAD allele count = 0.Exome sequencing of DNA isolated from whole blood with QIAamp DNA Mini Kit  was performed using the Illumina NovaSeq platform. The genetic investigation was primarily limited to 193 genes included in clinically available genetic testing panels offered by Invitae, Blueprint genetics, Ambry, and GeneDx . Mitochondrial variants were compared with the Cambridge reference sequence (CRS). A variant was considered potentially disease causing if it was not haplotype associated and had an overall allele frequency of <0.05% according to frequency data in GenBank, which contains 49,135 full-length human mitochondrial sequences14. MitoTip scores based on frequency, the type of nucleotide change, and nucleotide conservation were taken into consideration as variants in tRNA-encoding genes15. All rare nuclear and mitochondrial variants are reported  guidelinesn = 33, 94%) and female , with a mean age at diagnosis of 43.8 ± 12.3 years (range 21–77 years). Seventy-four percent of the patients (n = 26) were diagnosed following a symptomatic presentation; nine patients were diagnosed incidentally. Patients had LV dilation , asymmetric LV hypertrophy  and atrial septal defects . Extracardiac manifestations included skeletal muscle weakness , pectus excavatum  and upper limb abnormalities . Half of the patients were taking aspirin , and 26% (n = 9) had an in situ cardioverter defibrillator implanted.A total of 35 LVNC index patients were included Table ; of thesNKX2-5 and TBX5 . Cosegregation analysis identified two daughters who were heterozygous for the TBX5 variant, and both were diagnosed with HOS. ECG parameters and echocardiography were within normal limits in one daughter aged 25 years at the time of this study. Echocardiography revealed mild LV dilation, apical hypokinesis and possible LVNC in the other daughter aged 30 years. One granddaughter was found to have upper limb skeletal abnormalities consistent with a clinical diagnosis of HOS.Female index patient BRP1 was diagnosed with LVNC at 49 years of age, and her symptoms included general fatigue, chest tightness, palpitations and presyncope. Clinical examination revealed brachydactyly, ankylosing spondylitis and pectus deformity. ECG and Holter monitor investigations revealed sinus node dysfunction, intermittent sinus pauses and evidence of isorhythmic dissociation. Assessment of family members revealed three generations of triphalangeal thumbs Fig. . Geneticn = 15, 43%) being the most common  among adult index patients who present outside the context of family screening. All causative variants were identified in those patients with overt disease, suggesting that genetic testing is likely to be most beneficial in LVNC associated with cardiac dysfunction or malformations and noncardiac systemic features and least useful in clinically unaffected adults with only isolated LVNC.In recent years, the number of adults diagnosed with LVNC has increased, though the benefit of genetic testing is unclear among patientsMYBPC3 and MYH7 cardiomyopathy genes were not identified5. This difference likely reflects the exclusion of patients presenting in the context of a family history of cardiomyopathy. Interestingly, all three pathogenic or likely pathogenic variants were identified in genes encoding transcription factors. In addition, we identified highly suspicious VUS in a heterogeneous group of genes, suggesting that many different mechanistic pathways may result in a noncompaction phenotype and highlighting the importance of using a broad genetic testing panel.To our knowledge, this is the first study to assess a large number of LVNC genes (193 genes) using a targeted exome sequencing approach in adult index patients. In contrast to previous studies, multiple variants in NKX2-5 and TBX5, and LVNC. A small number of human genetic studies have identified causative variants in NKX2-5, an important regulator of cardiac trabeculation and conduction system development in LVNC18. Analysis of 50 patients carrying NKX2-5 variants showed that AV block is the most consistent phenotype, and patients are at risk of sudden death even in the absence of significant cardiomyopathy18. A recent study has shown that mice with conditional knockout of NKX2-5 develop excessive trabeculation associated with conduction defects and heart failure progression19. Assessment of induced pluripotent stem cell-cardiomyocytes from LVNC patients with the stop-gain variant p. Tyr317Ter in TBX20, encoding a transcription factor, highlights its regulation of PRDM16 and how the loss of PRDM16 regulation can result in LVNC20. Thus, we provide further evidence for the causative role of the PRDM16 loss-of-function variant identified in the present cohort and the important role of the cardiac transcription factor network in the pathogenesis of LVNC.Our findings add to existing evidence that suggests an important association between transcription factor variants, specifically in 3. This issue is exemplified by the classification of the previously mentioned loss-of-function variant in PRDM16 as a VUS. Loss-of-function mutations in PRDM16 have previously been found in patients with LVNC, DCM and sudden cardiac death; however, more evidence is required for these variants to be considered a known disease mechanism, which would facilitate a likely pathogenic or pathogenic classification5. Furthermore, there is increasing evidence supporting the role of ACTC1 variants in the pathogenesis of LVNC, and it is likely that the ACTC1 p. The Ser241Arg variant is the cause of disease in index patient BLM21.Twenty-six percent of clinically affected patients in this study were heterozygous for highly suspicious VUS, which highlights the lack of genotype-phenotype evidence guiding pathogenicity classifications in LVNCDSP deletion is more meaningful in the context of LVNC with dyskinesis and scarring. The classification of loss-of-function variants in TBX5 relies on the identification of upper limb abnormalities consistent with Holt-Oram syndrome (HOS)16. Although there is minimal evidence, namely, in the form of case reports, identifying LVNC in the context of TBX5 variants is mechanistically plausible given the high prevalence of other structural abnormalities, including septal and valvular defects22. Additional studies are required to better understand whether the association of LVNC with TBX5 variants and HOS represents a true mechanistic link or an incidental finding. As the use of genetic testing in LVNC increases, we are seeing the development of stronger gene-disease associations, including that of LVNC and variants in HCN423. We identified three patients with rare missense variants in HCN4 despite the conserved nature of the gene. Of particular interest is variant p. Gly480Ser, which may be located in a hotspot as p. Gly480Arg; it has previously been described in a large family with sinus bradycardia24. However, it is difficult to determine whether variants identified in adult patients are related to increased trabeculation or coexisting cardiac abnormalities; it is possible that hypertrabeculation distracts from the true phenotype. If we can improve phenotype accuracy, it may be feasible to reduce the size of the panel screened, which would have the added benefit of reducing the number of VUSs that are identified.The requirement of a large gene panel relates to the lack of standardized diagnostic criteria, which results in a heterogeneous patient cohort for which variant interpretation is difficult. Indeed, thorough clinical phenotyping can aid variant interpretation, and in many cases, it is the coexisting feature that guides classification. The identification of a MT-TV gene cause of disease in a patient with a mixed LVNC and hypertrophy phenotype. Variants in MT-TV have previously been associated with HCM25. Across the cohort, a small number of rare mitochondrial variants were identified and might be associated with the LVNC phenotype. A previous study has shown that mitochondrial variants may operate in tandem with nuclear variants in the pathophysiological process that underlies LVNC6.A review of the mitochondrial genome, facilitated by our exome sequencing approach, revealed a likely monogenic Our findings have implications for clinical practice. The current body of work highlights that performing genetic testing for all LVNC index patients is likely to result in a low diagnostic yield and that the greatest utility is for individuals with LV dysfunction, ECG abnormalities or syndromic/coexisting clinical features Fig. . For theThe main limitation in studying the genetic etiology of LVNC stems from issues with the diagnostic criteria that result in highly heterogeneous patient cohorts. As such, our small cohort of 35 index cases may not be representative of the general population. Although we identified a potentially causal variant in an additional 26% of patients, only three (9%) carried variants with sufficient evidence to classify as likely pathogenic or pathogenic. By utilizing exome sequencing, it is possible that we missed variants in deep intronic regions, as well as large rearrangements and karyotype abnormalities. Moreover, genetic testing results were not available for all first-degree members in this study, and therefore lack of cosegregation is a limitation of this study. This limitation is particularly highlighted by the lack of variant cosegregation in the BHS and BKS families.Genetic testing applied broadly to adult index patients with LVNC is likely to have a low diagnostic yield. This work suggests that genetic testing is likely to be most beneficial in LVNC associated with other cardiac features, such as LV dysfunction, other cardi and noncardiac syndromic features, and least useful in adults with only isolated LVNC in the absence of cardiac dysfunction and syndromic features. Although the diagnostic yield of genetic testing was low in this study for adult index patients with LVNC who did not present in the context of family screening, genetic testing can aid in facilitating correct diagnoses and appropriate patient management. The results from this study highlight the important role of cardiac transcription factor variants in the development of LVNC. Further research is required to elucidate the pathogenesis of LVNC, which shows significant clinical and genetic heterogeneity, and to confirm our finding that there is minimal benefit of genetic testing in individuals diagnosed with isolated LVNC.Supplementary Data


Their protein products are extracytoplasmic enzymes  and can hydrolyze extracellular cyclic dinucleotides, thus reducing the innate immune responses of infected hosts. This makes CpdB(-like) enzymes potential targets for novel therapeutic strategies in infectious diseases, bringing about the necessity to gain insight into the molecular bases of their catalytic behavior. We have dissected the two-domain structure of Escherichia coli CpdB to study the role of its N-terminal and C-terminal domains (CpdB_Ndom and CpdB_Cdom). The specificity, kinetics and inhibitor sensitivity of point mutants of CpdB, and truncated proteins CpdB_Ndom and CpdB_Cdom were investigated. CpdB_Ndom contains the catalytic site, is inhibited by phosphate but not by adenosine, while CpdB_Cdom is inactive but contains a substrate-binding site that determines substrate specificity and adenosine inhibition of CpdB. Among CpdB substrates, 3′-AMP, cyclic dinucleotides and linear dinucleotides are strongly dependent on the CpdB_Cdom binding site for activity, as the isolated CpdB_Ndom showed much-diminished activity on them. In contrast, 2′,3′-cyclic mononucleotides and bis-4-nitrophenylphosphate were actively hydrolyzed by CpdB_Ndom, indicating that they are rather independent of the CpdB_Cdom binding site.CpdB is a 3′-nucleotidase/2′3′-cyclic nucleotide phosphodiesterase, active also with reasonable efficiency on cyclic dinucleotides like c-di-AMP  and c-di-GMP . These are regulators of bacterial physiology, but are also pathogen-associated molecular patterns recognized by STING to induce IFN-β response in infected hosts. The  On the other hand, substrate saturation datasets obtained in the presence of three different inhibitor concentrations (one of them null), were used to find the best fitting kinetic equation out of six possibilities, i.e., complete or partial, competitive, mixed or uncompetitive inhibition [Vmax, KM and Ki were left to fluctuate. The precision of fit was estimated with a χ2 parameter, with lower values indicating better fits [Ki values.The procedures followed to derive kinetic parameters lsewhere . Datasethibition ,48. Eachter fits . In each


Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely ‘non-canonical’ substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both  PLK2EE motif ,46. In cEE motif . Consiste arrays  and a vae arrays ,38. Consposition , as wellrylation . Justifirylation ,38. WhilN-terminal to the site of substrate phosphorylation . Data were processed using the Boltzmann equation to generate sigmoidal denaturation curves, and average Tsoftware .in vitro peptide-based enzyme assays were carried out using the Caliper LabChip EZ Reader platform, which monitors phosphorylation-induced changes in the mobility of a fluorescently labelled PLK4 peptide substrate P motifs on multiple cellular proteins. Notably, protein phosphorylation in this broad [pS/pT]P consensus is markedly down-regulated in human cells exposed to centrinone, and PLK4 is able to phosphorylate residues with a Pro at +1 in recombinant proteins (but not derived synthetic peptides) that includes two new PLK4 substrates, PTPN12 and NMYC. In the future, the biological effects of these phosphorylation events will be explored further, in the context of catalytic output of the tyrosine phosphatase PTPN12 and the potential for PLK4 integration into the Aurora A/FBX7/NMYC signalling network. It is anticipated that when assessed alongside complementary PLK4 chemical genetic, depletion or elimination strategies, our proteomic datasets will help define further physiological PLK4 substrates across the cell cycle. These findings will also allow the extent to which a [pS/pT]P phosphorylation consensus represents a direct, indirect or substrate-dependent PLK4 modification in cells, and thus permit the biological roles of PLK4 to be examined in more detail.All data needed to evaluate the conclusions made are available in the main or supplementary sections of the paper. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository  with the


Leymus mollis (Pooidae), that had not previously been examined. Rhizome tissue had strong ice-structuring and ice recrystallization inhibiting (IRI) activities, indicating the probable presence of IBPs. The gene sequence of an IBP was obtained. The sequence encoded a 118-amino acid IRI domain composed of eight repeats and that was 80% identical to the IRI domain of the IBP of perennial ryegrassLolium perenne. The predicted 3D structure of the IRI domain had eight beta-roll coils like those inL. perenne IBPSeveral cold-hardy grasses have been shown to have ice-binding proteins (IBPs) that protect against freeze-thaw injury. Here, we looked for IBP activity in an Alaskan coastal grass, Lolium perenne  tolerance .Within the grass family (Poaceae), the subfamily Pooideae includes many cold-adapted grasses including wheat, barley and forage grasses. These grasses have developed a large family of ice-binding proteins (IBPs) that protect the plants from freezing damage , Alaska (71.3°N) on 6 October 2019, and stored at -80°C. The grass was shipped frozen to University of Nevada Las Vegas for analysis, but accidentally rose to ~10°C for about one day during shipment.Festuca sp.) at the University of Nevada Las Vegas (52 mg) was similarly homogenized and used as a control. Ice-structuring activity was observed by examining the growth of an ice seed crystal with well-defined icec- anda-axes submerged in the grass extract supernatant, as described previously  were aligned. Regions with high identities at the 5’ and 3’ ends were selected for primer sequences to obtain an amplicon that covered as much of the gene as possible. Primers for 18S ribosomal RNA were selected from conserved regions in the 18S sequences fromDupontia fisheri,L. perenne andD. antarctica  that flanked a region of high variability. At that time, we suspected the Utqiaġvik grass sample wasD. fisheri and did not use theL. mollis 18S sequence. PCR was carried out with an Eppendorf Mastercycler Personal thermal cycler, with 3 minutes initial denaturation at 95°C, followed by 35 cycles of 30 seconds denaturation at 95°C, 30 seconds annealing at 59°C and 40 seconds extension at 72°C, followed by 3 minutes final extension at 72°C using Promega GoTaq polymerase . PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide and observed and photographed with a UVP transilluminator. IBP and 18S bands of the expected sizes were obtained  according to the manufacturer’s instructions. To make IBP primers, the nucleotide sequences of IBPs fromng data ), cleaneLeymus IBP was predicted withSWISS-MODEL (Lolium perenne IRI domain (Protein Data Bank accession no.3ULT) (Leymus IRI domain based on the template. We selected the one with the highest sequence identity (76.5%), which also was the only one that had eight orderly coils like those in theL. perenne template. The free energy of this structure was then minimized (from -42.56 to -52.58 MJ mol-1) with theYASARA Energy Minimization Server  most closely matched the sequence ofLeymus mollis (GenBank accession no.EF581964) (98.3% identity). Among the Pooidae,L. mollis is a member of the Triticodae, which includes wheat (Triticum) and barley (Hordeum), whileLolium andDeschampsia are members of the Poodae. The grass was confirmed asL. mollis from photos of the remnant spikes and leaves by Matthew Carlson (University of Alaska Anchorage) and Carolyn Parker, University of Alaska Fairbanks .The obtained 18S rRNA sequence  encoded 260 a.a., which corresponds to all but the first 25 a.a. of theL. perenne IBP sequence. The sequence included the stop codon and contained no introns. Although the sequence was close to the sequence ofL. perenne (65% in the region of overlap), it most closely matched an IBP-like protein fromTriticum aestivum (QBE94480) , in agreement withL. mollis’s classification as a member of the Triticodae. When only the IRI domains were compared, theLeymus IRI domain was also closer to theTriticum IRI domain than it was to theLolium IRI domain. The ice-binding activities ofTritium spp. IBPs have not yet been reported.Primers based on the IBPs of other pooid grasses succeeded in amplifying a sequence that encoded the C-terminal part of the IBP gene ofL. mollis, like that ofL. perenne, has eight repeats, each consisting of 14 or 15 residues . InLeymus, the average spacing was calculated as 4.77 Å. The regular array of hydrophilic residues is thought to order water molecules that act as a glue between the IBP and the ice  activities. The collection site of the grass, a gravelly beach on the Chukchi Sea, is an extreme habitat subject to numerous environmental stresses.L. mollis appears to be better adapted to high latitudes thanL. perenne, which does not grow well north of 60°N .Data are available under the terms of theFigshare: Table S1. Number of days with subzero air temperatures during the growing season at Utqiaġvik, AK.https://doi.org/10.6084/m9.figshare.12765983.v1 .Data are available under the terms of the In the present paper Sformo and Raymond present data on an ice binding and recrystallisation inhibiting protein from the Alaskan grass Leymus mollis, which lives in areas where it is exposed to several kinds of stresses including low temperatures.L. mollis was closely matched to theTriticum aestivum IRI domain.The authors have observed ice structuring, recrystallisation inhibition activity in aqueous homogenates of the rhizome tissue. They have sequenced DNA from similar homogenates using primers based upon the conserved sequences from other IBP containing grasses. The obtained DNA sequences were translated into the amino acid sequences presumably involved in the ice recrystallisation inhibition and ice structuring. The amino acid sequence of the IRI domain ofLolium perenne. It appears that the tree dimensional structure of the ice binding domain ofL. mollis IBP quite resembles that ofL. perenne.The 3D structure of the ice binding domain was predicted using the obtained sequence and the structure of the IRI domain inThe paper is an interesting comparative account of the ice related properties of a grass found at high latitudes - again indicating that ice structuring and recrystallisation inhibition is very important in plants adapted to cold climates.I miss an extension of the discussion of the comparative aspects of cold tolerance and adaptive aspects of the findings in the paper.Figure 1 could be explained a bit better as some readers may not understand the differences between the A and the B panel very well.Figure 2 is not very clear and also this figure would benefit from a bit more explanation in the text.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Cold tolerance of ectothermic animals, antifreeze proteins, cryptobiosisI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. •    I miss an extension of the discussion of the comparative aspects of cold tolerance and adaptive aspects of the findings in the paper.We revised the last paragraph to give some perspective to the cold tolerance of L. mollis and how it probably differs from that of L. perenne.•    Figure 1 could be explained a bit better as some readers may not understand the differences between the A and the B panel very well.We added some sentences to better explain the difference between Leymus and Festuca.•    Figure 2 is not very clear and also this figure would benefit from a bit more explanation in the text.We added new photos to show RI of Festuca. It more clearly shows that Leymus has much stronger RI than Festuca. This brief report outlines a description of the sequence and structure of ice-binding protein in Leymus mollis from the Beaufort Coast in Arctic Alaska, and compares the findings to IBPs of other more temperate grass species. The paper seems pretty straightforward, however, I do not have the expertise to comment on the amino acid sequence, protein modeling, and comparative approach to other species. The paper was well-written, the figures were informative, and I believe it warrants indexing. While this is a brief note, I do think that additional interpretation of results and providing some brief explanations related to mechanisms of freeze tolerance in light of their findings would make the manuscript substantially more valuable. I was not able to gauge if the similarities and differences between Leymus and Lolium/Festuca are likely to have physiological/ecological bearing on their respective environmental contexts. .here. I provide additional comments and edits embedded as notes in a PDF - please see the file I appreciate the opportunity to read this paper and I sincerely hope that my comments are useful in generating a more robust paper.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Plant ecology and evolution - emphasis on Arctic and Boreal systemsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for many good comments. Revisions have been made to address all of them. here.Thank you for many helpful comments. We revised the manuscript to address each of them. Our response to your comments can be viewed in the PDF file Leymus mollis) collected in Alaska and have observed the presence of an ice-binding protein (IBP) based on ice crystal faceting and the inhibition of ice recrystallization. From another extract they prepared DNA and used primers based on conserved regions in other grass IBP transcripts to amplify theL. mollis gene, which fortunately lacks introns. From the sequence of this DNA they were able to provide the amino acid sequence of the IBP domain involved in ice recrystallization. They used this to model the three-dimensional structure of the domain, which looks very much like that ofLollium perenne. Another set of primers was used to amplify a diagnostic section of the 18S ribosomal RNA gene whose sequence identified the species.Sformo and Raymond have prepared aqueous extracts of a hardy coastal grass  extracted and analyzed. This report is also a good illustration of the use of molecular modeling to avoid solving a structure that would not differ much from the template. Indeed, the main features ofL. perenne andD. antarctica are compared. It is not helpful to say their sequences “are very similar”. In the last paragraph of the Introduction theL. mollis IBP sequence is again said to be “very similar” to those of the other two grasses. Please provide % identity and % similarity.The Abstract should include the percent identity of the IRI domain between the two grass species. This type of information should also be provided in the Introduction where the sequences of the IBPs ofFigure 1A shows ice structuring. However, since panel B also shows ice crystals it is important to point out to the average reader that the distinction lies in the faceting seen in A where flat, angular surfaces are the result of IBP binding to ice. The ice in B is smoother and rounded.Figure 2 is a rather poor presentation of ice recrystallization. One must look closely at the ice control images to see the grains are larger after 21 h. The methods say the ice recrystallization was done in water. From our experience the inclusion of 150 mM NaCl makes a huge difference to the visibility of the ice grains. The other suggestion is to take the starting picture at zero time with the ice held colder than – 3 °C. We see substantial recrystallization happening in the first hour, even at -6 °C, and will typically hold the ice wafer at -10 °C until the starting picture is captured, and then raise the temperature for more rapid recrystallization. I recommend that this analysis be repeated and improved.L. perenne IBP as a template. Then, the one with the ‘highest sequence identity’ was selected. Surely the sequence identity is the same for all models because they are based on the same template. We would typically compare models by root mean square deviation (RMSD) – how the atoms deviate from the model on average. Is this what is meant here?In Methods, under ‘Structure prediction’, it is stated that three structures were proposed by SWISS MODEL operating withL. mollis open reading frame was incomplete, it would be useful to report the sequence upstream of the ice-binding domain. Presumably this is a series of leucine-rich repeats similar to those seen in other grass IBPs?Although the sequence of theIn reference to ice crystal axes the a- and c- should be italicized.Replace the contraction "didn’t" with "did not". Suggestions for improvement:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Protein biochemistry, structural biology, ice-binding proteinsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Percent identities added to Abstract. Identities were removed from Introduction to make a revision requested by reviewer 2.Fig. 1A: The sentence was revised to point out that the ice is smooth and rounded with no evidence of facet development.Fig. 2: The experiment requires much preparation and is difficult to repeat now that one of the authors is back in Alaska. However, we think there is a clear difference in recrystallization between the two samples when the figure is viewed at full scale.Swiss-Model: Swiss Model tries different regions of the the sequence to model, e.g., residues 131-247 in one model and residues 144-228 in another. Each gives a slightly different model. We selected the one that had the highest a.a. sequence identity.N-terminal region of IBP: For this, we would need a consensus sequence of the 5’ UTR in other grasses (for use as a forward primer) but we were unable to find such a sequence. Percent identity was added to Abstract and the Results. Identities were removed from Introduction to make a revision requested by reviewer 2.Fig. 1A: The sentence was revised to point out that the ice is smooth and rounded with no evidence of facet development.Fig. 2: We added a new control, Festuca (lawn grass) extract, which shows more clearly the greater RI activity of Leymus.Swiss-Model: Swiss Model tries different regions of the sequence to model, e.g., residues 131-247 in one model and residues 144-228 in another. Each gives a slightly different model. We selected the one that had the highest a.a. sequence identity.N-terminal region of IBP: For this, we would need a consensus sequence of the 5’ UTR in other grasses (for use as a forward primer) but we were unable to find such a sequence.


The protein kinase enzyme family plays a pivotal role in almost every aspect of cellular function, including cellular metabolism, division, proliferation, transcription, movement, and survival. Protein kinase A (PKA), whose activation is triggered by cyclic adenosine monophosphate (cAMP), is widely distributed in various systems and tissues throughout the body and highly related to pathogenesis and progression of various kinds of diseases. The inhibition of PKA activation is essential for the study of PKA functions. Protein kinase inhibitor peptide (PKI) is a potent, heat-stable, and specific PKA inhibitor. It has been demonstrated that PKI can block PKA-mediated phosphorylase activation. Since then, researchers have a lot of knowledge about PKI. PKI is considered to be the most effective and specific method to inhibit PKA and is widely used in related research. In this review, we will first introduce the knowledge on the activation of PKA and mechanisms related on the inhibitory effects of PKI on PKA. Then, we will compare PKI-mediated PKA inhibition vs. several popular methods of PKA inhibition. The protein kinase enzyme family plays a pivotal role in almost every aspect of cellular function, including cellular metabolism, division, proliferation, transcription, movement, and survival . ProteinProtein kinase A (PKA) was the first protein kinase to be discovered. It was identified in skeletal muscle by Edmond H. Fisher and Edwin G. Krebs when they found that glycogen phosphorylase was activated following its phosphorylation by protein kinase, which was later termed “phosphorylase kinase” and now “protein kinase A” . Almost As a prototypical serine/threonine kinase, PKA is distributed in all systems and tissues throughout the body and related to pathogenesis and progression of various diseases. Numerous studies have focused on the function, regulation, and pathological roles of this signaling pathway. Manipulation of this signaling pathway has been explored to treat several diseases such as cardiovascular diseases, Alzheimer disease, Parkinson’s disease, ischemia, and diabetes. .The discovery of cAMP-mediated activation of PKA led to the “Second Messenger Hypothesis” in cellular signaling. It is described as that extracellular ligands, such as a certain kind of hormones like glucagon and epinephrine acted on membrane receptors and cause the generation of intracellular second messengers like cAMP, which in turn activated a kind of protein kinase and lead to the activation of cellular processes like glycogenolysis . cAMP isPKA is inactive in the absence of the triggering cAMP. Like most protein kinases, PKA was found in an inactive state under basal conditions and activated by a variety of ligand-induced regulatory mechanisms . The und2+ channels to the membrane in a supermolecular complex to coordinate the regulation of the channel  domain . Small sPKIα consists of 75 amino acids, of which amino acids 1–25 are the PKA inhibition domain (Ki = 0.2 nM) and the NES is within residues 37–46 (LALKLAGLDI). This NES contains several hydrophobic amino acids and is one of the strongest NES identified . Normallvia two mechanisms: direct inhibition of PKA and the regulation of intracellular localization of PKA. The inhibition of PKA activation relies on the pseudosubstrate amino acid sequence in PKI located at the N-terminus, which is similar to that in the RI subunit of PKA. PKI binds to the catalytic subunit of PKA with high affinity via the pseudosubstrate sequence but cannot be phosphorylated, leading to the inhibition of PKA activity ethyl]-5-isoquinoline sulfonamide, is a cell permeable, relatively PKA-specific derivative from its predecessor, H8, a PKA, and protein kinase G (PKG) inhibitor. It is by far the most widely used PKI inhibitor in research related to PKA . This is50 of H89, the concentration at which a compound inhibits 50% of a given activity, varies according to the ATP concentration, which makes it hard for researchers to get the effective dose of H89 in the treatment of cells. Secondly and more importantly, H89 is a nonspecific inhibitor with effects on ion channels, receptors, Ca2+-ATPase, small GTPase RhoA, and other kinases such as ROCK1 and PKB  or many non-specific effects off the target PKA. Consequently, more effective and specific inhibitors for PKA are required for the study of PKA.50, thus PKI may provide a more effective and specific approach in the inhibition of PKA. Since the PKA inhibition domain has been identified, several synthetic peptide forms of PKI have been developed and widely applied for the study of PKA function. So far, synthetic peptide analogs of PKI include PKI-(6-22)-amide (Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH2), PKI-(14-24)-amide (Gly-Arg-ThrGly-Arg-Arg-Asn-Ala-Ile-His-Asp-NH2), andPKI-(5-24)-amide .As described above, PKI is an endogenous molecule that specifically inhibits PKA activity with a low ICAnabas testudineus  or RNA interference (RNAi). Since siRNA was reported in 1998 , it has PKA has been extensively studied, and it is an important regulator of various cellular processes and disorders in almost all systems. Recently, methods taking advantage of PKI have proven to be more effective and specific in the inhibition of PKA than chemical PKA inhibitors and widely used in studies regarding biological roles of PKA. However, PKI can inhibit PKG at high concentrations and thus should be used with caution. More research on PKI ought to be conducted to improve its specificity.CL, PK, and XZ were in charge of searching all the relative papers and writing this manuscript. JZ was in charge of drawing the picture. XC guided the organization and drafting of this manuscript. All authors contributed to the article and approved the submitted 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.


Fifty years ago, it was speculated that the marine-based West Antarctic Ice Sheet is vulnerable to warming and may have melted in the past. Testing this hypothesis has proved challenging due to the difficulty of developing in situ records of ice sheet and environmental change spanning warm periods. We present a multiproxy record that implies loss of the West Antarctic Ice Sheet during the Last Interglacial , associated with ocean warming and the release of greenhouse gas methane from marine sediments. Our ice sheet modeling predicts that Antarctica may have contributed several meters to global sea level at this time, suggesting that this ice sheet lies close to a “tipping point” under projected warming. The future response of the Antarctic ice sheet to rising temperatures remains highly uncertain. A useful period for assessing the sensitivity of Antarctica to warming is the Last Interglacial (LIG) (129 to 116 ky), which experienced warmer polar temperatures and higher global mean sea level (GMSL) (+6 to 9 m) relative to present day. LIG sea level cannot be fully explained by Greenland Ice Sheet melt (∼2 m), ocean thermal expansion, and melting mountain glaciers (∼1 m), suggesting substantial Antarctic mass loss was initiated by warming of Southern Ocean waters, resulting from a weakening Atlantic meridional overturning circulation in response to North Atlantic surface freshening. Here, we report a blue-ice record of ice sheet and environmental change from the Weddell Sea Embayment at the periphery of the marine-based West Antarctic Ice Sheet (WAIS), which is underlain by major methane hydrate reserves. Constrained by a widespread volcanic horizon and supported by ancient microbial DNA analyses, we provide evidence for substantial mass loss across the Weddell Sea Embayment during the LIG, most likely driven by ocean warming and associated with destabilization of subglacial hydrates. Ice sheet modeling supports this interpretation and suggests that millennial-scale warming of the Southern Ocean could have triggered a multimeter rise in global sea levels. Our data indicate that Antarctica is highly vulnerable to projected increases in ocean temperatures and may drive ice–climate feedbacks that further amplify warming. The projected contribution of the Antarctic ice sheet to 21st-century global mean sea level (GMSL) ranges from negligible . Valuabl4105SI Appendix, Fig. S5) constrained by analysis of trace gases and geochemically identified volcanic layers exposed across the transect, which have been Bayesian age modeled against the recently compiled continuous 156-ky global greenhouse gas time series  ]. Today, precipitation at the site is delivered via storms originating from the South Atlantic or Weddell Sea , exposed in Horseshoe Valley   on the Aand N2O)    and closand N2O) , equivaldell Sea . Crucialdell Sea .SI Appendix, Fig. S7), implying proximal warmer conditions and reduced sea ice extent , is correlated to a volcanic ash from the penultimate deglaciation (Termination II) referred to as Tephra B in marine sediments on the West Antarctic continental margin , allowing the isolation and stagnation of ice in Horseshoe Valley over multiple millennia. Furthermore, glaciological investigations assessing the impact of ice shelf loss on glaciers along the Antarctic Peninsula provide important insights into the preservation of ice, albeit on a smaller scale. The 2002 Larsen B ice shelf collapse led to many of the tributary glaciers abruptly changing from a convex to a concave profile (cross-section) . Radio-echo sounding surveys across the WSE have identified a large subglacial basin comprising landforms reflecting restricted, dynamic, marine-proximal alpine glaciation, with hanging tributary valleys feeding an overdeepened Ellsworth Trough . The ext37section) , with resection) . These ssection) .SI Appendix, Fig. S2) and the subsequent inferred highly variable climate and/or sea ice extent across the wider WSE  imply the preservation of ice from MIS 6/5 (Termination II) and 5/4 transitions in Horseshoe Valley. Our data provide evidence for substantial mass loss across the WSE during the LIG.The ice at Patriot Hills therefore appears to preserve a record of glacier flow in Horseshoe Valley up to the moment when the Filcher–Ronne Ice Shelf collapsed, after which the sequence remained isolated due to regional ice flow reconfiguration for multiple millennia; a situation that persisted until the ice surface had risen sufficiently to enable the regional ice flow to recover sometime during late MIS 5. We cannot, however, discount the possibility that there were one or more cycles of ice mass gain and loss through MIS 5. The presence of a discrete older ice unit along the flanks of the Ellsworth Mountains   may have significantly reduced North Atlantic Deep Water (NADW) formation and shut down the Atlantic meridional overturning circulation (AMOC) , resulti the LIG , a proxy the LIG I. The un the LIG  afforded the LIG , 45, 46  the LIG . The sub the LIG , 48, 49  the LIG C. Recent the LIG , 14, 20  the LIG B. Of con the LIG .Methyloversatilis microbes dominated the detectable microbial diversity (∼130 ky) . Methyloversatilis was only found in high abundance in this sample (with trace amounts identified at ∼22 ky). Crucially, Methyloversatilis are facultative methylotrophs and live on single and multicarbon sources (4 and active methane oxidation by Methyloversatilis or other methanotrophic taxa in marine sediments or in the water column during the end of Termination II (SI Appendix). More work is needed to explore the potential for microbial methane utilization in this unique environment.With Southern Ocean warming and concurrent ice sheet retreat, the large methane reservoirs in Antarctic sedimentary basins  could have become vulnerable to release  and may  sources , consistThe inferred substantial mass loss across the WSE implies a major role for ocean warming during Termination II and the LIG. To provide a framework for interpreting ice sheet dynamics around the Patriot Hills and across Antarctica, we present a series of temperature sensitivity experiments using the Parallel Ice Sheet Model, version 0.6.3  2). We . We 2). SI Appendix, Fig. S17). Ongoing slower ice loss subsequently occurs around the margins of East Antarctica, producing a sustained contribution to sea level rise. Even for relatively cool ocean-forced runs, we find the shelves collapse quickly between the 200-y intervals . Indeed, during the warmer ocean model runs, the shelves disappear too quickly to observe the relevant processes on the timescale covered by the snapshots. For instance, under the scenario of 2 °C linear warming, the ice shelves disappear within 600 y of forcing (when temperatures reached between +0.4 and +0.8 °C). Other modeling studies using a range of different setups have reported similar rapid losses of the ice shelves during the onset of the LIG (For the 2 °C warmer than present day ocean temperature scenario (comparable to reconstructed estimates) , 23, wit the LIG , 25. Our the LIG , 25.SI Appendix, Fig. S19).Recent work has suggested that the Ellsworth Mountains would have experienced a relatively large positive isostatic adjustment (>200 m) accompanying the loss of the WAIS , 25, 32,56Previous work has highlighted the sensitivities of the Ronne–Filchner and Ross ice shelves to warming under a range of model setups , 18. RecSI Appendix, Fig. S17).The Patriot Hills record is consistent with basin-scale mass loss early in the LIG , 32 as aThe evidence for substantial mass loss from Antarctica in the early LIG has important implications for the future , 62. Our63−1) compound glacier system situated within an overdeepened catchment that coalesces with the Institute Ice Stream at the periphery of the WSE . Airborne radio-echo sounding surveys across the Ellsworth Mountains have revealed several wide (up to 34 km across) and long (260 km) subglacial troughs containing ice up to 2,620 m thick (−1 (cf. the main trunk of the Institute Ice Stream reaches speeds up to 415 m⋅a−1). This is in large part due to the configuration of the Horseshoe Valley Trough where the ice thickness measures in excess of 2,000 m at the head of the valley and reduces to ∼1,400 m downstream; toward the mouth of the valley, a subglacial ridge is found at ∼200 m below sea level with the ice thickness some 750 m thick   a long record of ice to accumulate, and 2) the isolation and preservation of ice during periods of regional and Antarctic-wide mass loss.The Patriot Hills BIA  is a slow flowing , al, al−1) cFig. S3) . The new−2⋅y−1   , 37, 38.Fig. S4)  set at z18O stable isotope values, ancient DNA (notably the detection of Methyloversatilis microbes in the sample form −340 m in the Patriot Hills record), and ice sheet modeling are consistent with early offshore warming in the south Atlantic and substantial ice mass loss in the early LIG  and more exposure of katabatic-enhancing nunataks, resulting in increased wind scour , 37. Whi37arly LIG , 62, pre2), methane (CH4) and nitrous oxides (N2O)—and volcanic tephra horizons. The trace gas measurements provide a range of possible age solutions against the recently published 156-ky smoothed global time series for these gas species . A Kovacs 9-cm-diameter ice corer was used to collect ice for gas and taken from >3-m depth to minimize modern air contamination and/or alteration . Shards were extracted by centrifugation of the melted ice samples and put onto a glass slide for electron microprobe analysis. The slides were ground and polished using silica carbide paper and decreasing grades of diamond suspension to expose fresh sections of glass. Single-grain analyses of 10 oxides were performed on a Cameca SX-100 electron microprobe at the Tephrochronology Analytical Unit, University of Edinburgh. See SI Appendix for operating conditions . The shards from −340 m are trachytic in composition and exhibit a tightly clustered population . Both were compared to published tephras from across Antarctica (The presence of visible tephra layers (volcanic ash horizons) provides additional chronological control for the Patriot Hills BIA. Here, we report two new tephras from Patriot Hills at 10 and −340 m, both observed as ∼4-cm units of dispersed shards  strongly support the correlation of Patriot Hills −340-m tephra horizon and the marine West Antarctic Tephra B  has been proposed to correlate to the tephra at Dome Fuji 1,785.14 m, but the correlation has until now remained only tentative in the absence of any reported geochemistry from the latter . Here, wnditions . Accuracnditions  glass weTephra B , 85, andhttps://c14.arch.ox.ac.uk/)  , exceeding the recommended rejection Agreement Index threshold of 60%  in the software package OxCal, version 4.2.4 ( and S4) , 89. Usi and S4) , 91. “Ca and S4) . Taking d of 60% , 33, 34 18O isotopic measurements were performed between 1- and 3-m resolution at James Cook University using diffusion sampling–cavity ring-down spectrometry  . Reported overall analytical precision on long-term ice core standards is <0.32‰ for δD and <0.13 for δ18O values. All isotopic values are expressed relative to the Vienna Standard Mean Ocean Water 2 (VSMOW2). The isotopic datasets generated in this study are available at the publicly accessible National Oceanic and Atmospheric Administration (NOAA) Paleoclimatology Database (δD and δD 16139) . This syDatabase  and are BIAs offer the opportunity to process large-volume samples of continental Antarctic ice in the field , creating the prospect of generating sufficient microbial concentrations to permit detailed genetic biodiversity surveys , 52 Fig. To obtaSI Appendix for detailed methodology and analysis). DNA from all ice samples as well as extensive sampling and laboratory controls were extracted using two methods to maximize species recovery, and 16S ribosomal RNA libraries were amplified in triplicate using published, universal bacterial and archaeal 16S ribosomal RNA (rRNA) primers. After DNA sequencing, all individually indexed 16S rRNA libraries were de-multiplexed, quality filtered, and imported into QIIME, version 1.8.0. Microbial taxa were identified by comparing sequences to the Geengenes, version 13, reference database and binning sequences with 97% similar to known species into operational taxonomic units using closed reference clustering in UCLUST. Sampling and laboratory contaminants were then filtered from ice samples, and an average of 30.8% of the reads for each sample were retained . Retained sequences were then pooled, and the resulting taxa present in each sample were explored as a proportion of the total filtered DNA sequencing reads. Alpha and beta diversity was explored in QIIME, and importantly, no statistically significant differences in diversity were detected across the samples. Ancient DNA sample data are available upon request. While the current sample numbers limit resolution, our study highlights the untapped potential of BIA genetic data to exploit cryosphere microbial communities to investigate glaciological and environmental change  and intended to capture more accurately the weaker mantle of West Antarctica, where the majority of mass loss takes place. In the experiments presented here, we chose not to implement the subgrid scale interpolated ice shelf basal melt component of this scheme  , 11, 23 s scheme . Calvings scheme , 100. Ous scheme , 102. ClSI Appendix, Fig. S18). With a surface ocean warming of 2 °C, our simulations suggest isolation and stagnation of ice in Horseshoe Valley and the loss of the Bungenstock Ice Rise within 400 y of warming  .Reconstructed summer SST anomalies relative to present day (the 1998 World Ocean Atlas) . A limitl years)  and 6 anWe caution that, for the LIG, subsurface ocean warming is poorly constrained. While recent work has suggested that sea surface warming may propagate to depths important for ice shelves (including embayments) within a few decades , 104, prSupplementary FileSupplementary File


The role of sine oculis homeobox 4 (SIX4) has been found in some malignant tumors. However, there have been few studies on the function of SIX4 in esophageal squamous cell carcinoma (ESCC). This study aimed to explore the regulatory mechanism of SIX4 in ESCC.RT‐qPCR and Western blot analysis were used to measure mRNA and protein expression. The function of SIX4 was investigated using CCK‐8, colony formation, flow cytometry, wound healing and transwell assays. A mouse xenograft tumor assay was designed to perform in vivo experiments.SIX4 was upregulated in ESCC and indicated poor clinical outcomes in ESCC patients. Functionally, knockdown of SIX4 inhibited cell proliferation and induced apoptosis in ESCC. In addition, the silencing of SIX4 inhibited cell migration, invasion and EMT in ESCC. More importantly, upregulation of SIX4 could activate the PI3K/AKT pathway in ESCC cells and promote tumor growth in vivo.Upregulation of SIX4 indicates poor clinical outcomes in ESCC patients and promotes tumor growth and cell metastasis in ESCC. 1. SIX4 was upregulated in ESCC and indicated poor clinical outcomes in ESCC patients. 2. SIX4 promotes tumor growth and cell metastasis in ESCC. 3. Upregulation of SIX4 could activate the PI3K/AKT pathway in ESCC cells. Esophageal cancer is a relatively common tumor of the digestive tract. It is a malignant tumor that occurs in the epithelial tissue of the esophagus, accounting for 2% of all malignant tumors.The sine oculis homeobox (SIX) protein family has been reported to play an important role in the development of vertebrate tissues and organs.The epithelial‐mesenchymal transition (EMT) process has become an important mechanism for the distant metastasis of epithelial malignant tumors, including ESCC.Therefore, the abnormal expression and function of SIX4 was investigated in ESCC. At the same time, the effect of SIX4 on EMT and PI3K/AKT signaling pathway in ESCC was also elucidated.http://gepia.cancer-pku.cn/) is a newly developed interactive web server for analyzing RNA sequencing expression data from the 9736 tumor samples and 8587 normal samples of The Cancer Genome Atlas (TCGA). The screening conditions were: (i) datasets selection: esophageal carcinoma (ESCA); (ii) gene: SIX4; and (iii) expression DIY: boxplot, Stage plot.GEPIA  and ESCC cell lines  were purchased from the type Culture Collection of Chinese Academy of Sciences . BEBM medium (Het‐1A), Ham's F12 medium (KYSE150) or RPMI‐1640 (KYSE450) medium supplemented with 10% fetal bovine serum (FBS) was used to incubate the cells at 37°C with 5% COThe SIX4‐specific siRNA (si‐SIX4) was used to downregulate SIX4 expression, and a nonsilencing siRNA (si‐NC) oligonucleotide was used as a negative control (GenePharma). The cDNA encoding SIX4 was subcloned into the vector pcDNA3.1 . The empty pcDNA3.1 vector (pcDNA‐NC) was used as a control. Next, they were transfected into KYSE450 cells using Lipofectamine 2000 (Invitrogen), respectively.‐△△Ct method, with GAPDH used as an internal control.The isolation of total RNA was performed using TRIZOL reagent (Invitrogen). SIX4 was reverse transcribed by using PrimeScript RT Master Mix (Takara). RT‐qPCR assay was performed using real‐time PCR mixture assays (Takara) based on the manufacturer's instructions. The relative quantification of SIX4 was performed using the 2Protein samples were lysed using RIPA buffer (Beyotime). Next, proteins were separated using 10% SDS‐PAGE and blocked with 5% nonfat milk. Protein samples were then transferred to PVDF membranes. The protein sample was incubated with Bax , Bcl‐2 , E‐cadherin , N‐cadherin , vimentin , SIX4  and GAPDH  primary antibodies (Abcam) overnight at 4°C. After washing, the protein samples were incubated with secondary antibodies  for 1 h. Finally, protein bands were assessed using the ECL kit (Beyotime).2, respectively. Cell counting kit‐8 (CCK‐8) (Dojindo) was then used to incubate the cells for 4 h. The absorbance at 450 nm was observed using a microplate reader (Molecular Devices).Transfected KYSE450 cells were incubated for 24, 48, 72 and 96 h at 37°C in a humidified atmosphere with 5% COThe transfected cells were seeded into six‐well plates (400 cells/well) and incubated for 7–14 days. The culture was terminated after the colonies could be seen by the naked eye. Next, the cells were fixed in methanol and stained with 0.1% crystal purple staining solution for 30 min. Finally, colony imaging was counted.The transfected cells were digested by ethylenediaminetetraacetic acid (EDTA)‐free trypsin and collected in a centrifugal tube. Next, the cells were mixed with 5 μl Annexin V‐FITC and PI. The cells were then incubated at room temperature for 5 min in the dark. Finally, a flow cytometer was used to detect cell apoptosis.3 cells/well) prepared on a glass petri dish were incubated overnight at 37°C to allow the cells to adhere to the plate. A straight scratch was then made with a plastic pipette tip after cell samples reached a confluence of 80%. The cells were then rinsed in PBS to remove the separated cells. Finally, the wound was imaged at 0 and 24 h using a light microscope (Olympus).The transfected KYSE450 cells  was added to the lower chamber. After 24 h, the moving cells were stained with 0.1% crystal violet. The cells were calculated and visualized under light microscopy.2/2. After four weeks, the nude mice were sacrificed, and the tumors were collected and weighed.Ten 6‐week‐old male SCID mice were obtained from the National Laboratory Animal Center . This study was approved by the Institutional Animal Care and Use Committee of Yantaishan Hospital. The KYSE150 cells containing pcDNA‐NC or pcDNA‐SIX4 were injected into the right flank of the mice. Next, tumor volume was monitored every four days. The tumor volume was then calculated by length × widtht‐test and one‐way ANOVA in SPSS 17.0 or Graphpad Prism 6 software. p‐values < 0.05 were considered statistically significant.Data are shown as mean ± SD. Experimental data were analyzed using Student's p < 0.05, Figure p > 0.05, Figure p < 0.01, Figure p < 0.05, Table First, the GEPIA database showed that SIX4 was upregulated in ESCA tissues compared to normal tissues (p < 0.01, Figure p < 0.01, Figure p < 0.01, Figure p < 0.05, Figure p < 0.01, Figure p < 0.05, Figure Next, the expression level of SIX4 was detected in ESCC cells. Compared with normal esophageal cells Het‐1A, upregulation of SIX4 was identified in ESCC cell lines KYSE150 and KYSE450 (p < 0.05, Figure p < 0.01, Figure Next, the effect of SIX4 on cell metastasis was investigated in ESCC. Wound healing assay showed that silencing of SIX4 significantly decreased the velocity of cell movements. The percentage of wound closure at 24 h was decreased from 62.3% in the si‐NC group to 12.1% in the si‐SIX4 group (p < 0.01, Figure p < 0.01, Figure Finally, KYSE450 cells with SIX4 vector were subcutaneously injected into nude mice. Compared with the control group, the tumor volume was increased in mice with SIX4 vector Figure . Tumor gRecently, the important roles of SIX4 have been found in various physiological processes. For example, Six4 can regulate male sex determination and mouse gonadal development.Consistent with our results, SIX4 expression has been reported to be increased in ESCC. Abnormal expression of SIX4 in ESCC was correlated with clinical prognosis.As we all know, EMT is an important process in the distance metastasis of cancer cells.In conclusion, for the first time, this study revealed that SIX4 is upregulated in ESCC and indicates poor clinical outcomes in ESCC patients. Functionally, downregulation of SIX4 inhibits cell proliferation and metastasis in ESCC. Moreover, upregulation of SIX4 can promote ESCC tumor growth in vivo. This study will extend the understanding of SIX4 role in ESCC tumorigenesis.The authors declare that they have no competing interests.


Protein phosphorylation is essential for regulating cellular activities by modifying substrates at specific residues, which frequently interact with proteins containing phosphoprotein-binding domains (PPBDs) to propagate the phosphorylation signaling into downstream pathways. Although massive phosphorylation sites (p-sites) have been reported, most of their interacting PPBDs are unknown. Here, we collected 4458 known PPBD-specific binding p-sites (PBSs), considerably improved our previously developed group-based prediction system (GPS) algorithm, and implemented a deep learning plus transfer learning strategy for model training. Then, we developed a new online service named GPS-PBS, which can hierarchically predict PBSs of 122 single PPBD clusters belonging to two groups and 16 families. By comparison, GPS-PBS achieved a highly competitive accuracy against other existing tools. Using GPS-PBS, we predicted 371,018 mammalian p-sites that potentially interact with at least one PPBD, and revealed that various PPBD-containing proteins (PPCPs) and protein kinases (PKs) can simultaneously regulate the same p-sites to orchestrate important pathways, such as the PI3K-Akt signaling pathway. Taken together, we anticipate GPS-PBS can be a great help for further dissecting phosphorylation signaling networks. In eukaryotes, protein phosphorylation is by far the most important and widespread post-translational modification that mainly occurs on specific serine (S), threonine (T) or tyrosine (Y) residues in protein substrates, and orchestrates a variety of biological processes including signaling transduction, cell cycle/proliferation, autophagy and metabolism ,2,3,4. IIn 1987, the Tony Pawson group discovered the first PPBD named Src homology 2 (SH2) domain that could bind to a broad range of pY-containing proteins ,12. SubsDue to data accumulation, several databases were developed to maintain known PMIs and PBSs ,18,19,20In contrast to labor-intensive and time-consuming experiments, computational prediction of PBSs from protein sequences is an alternative approach to efficiently prioritize highly potential candidates for further experimental consideration. To date, there were six tools developed for predicting PBSs, including Scansite ,23, SMALIn this work, we manually collected 4458 experimentally identified PBSs in 950 PPBD-binding proteins (PPBPs) that interact with 268 PPCPs from 12 eukaryotic species . We clasFrom PubMed, we initially used multiple keyword combinations, such as “((phosphorylation) AND site) AND bind”, “(recognize) AND phosphorylation site” and “((phosphorylation) AND site) AND protein interaction domain”, to search experimentally identified PBSs by carefully checking abstracts or full texts of the scientific literature published before January 1, 2018. Additionally, we further obtained 2888 unambiguous PBSs from PepCyber:P~PEP , whereasm, n) as a S/T/Y residue flanked by m residues upstream and n residues downstream. As previously described /2 = 231 unique abS scores (abS = baS). These scores reflect the position-weighted similarity of amino acids between the given PBP item and all positive PBP entries. Thus, PVT represents a PBP item into a 231-dimensional vector, as below:PVT. Given the final In this study, we considerably improved our previous GPS algorithm that contained three parts, including a basic scoring strategy, a position weight determination (PWD) method, and a peptide-to-vector transformation (PVT) approach  item. The five hidden layers were mainly used for feature extraction and representation. A rectified linear unit (ReLU) activation function was adopted to activate the outcome of a neuron, and defined as below:x was the weighted sum of a neuron. The output layer contains two sigmoid neurons to calculate a score for a given PBP item y, defined as below:P(y) value denotes the probability score of a PBP item to be a real PBS. For training a general model to predict PBSs recognized by pS/pT- or pY-interacting PPBDs, a framework of seven-layer deep neural networks (DNNs) was implemented, containing one input layer, five fully connected (hidden) layers and one output layer. Each layer consisted of a number of computational units called neurons. To avoid over-fitting, which frequently occurs in deep learning algorithms, the dropout method was used by randomly dropping nodes from the five hidden layers if the A lab computer with an Intel(R) CoreTM i7-6700K@ 4.00 GHz central processing unit (CPU), 32 GB of RAM, and a NVIDIA GeForce GTX 960 core were used for training the two general models for predicting pS/pT- and pY-interacting PPBDs, respectively. The training process was implemented in the Keras 2.1.5 library with the tensorflow 1.10.0 backend. Training parameters including dropout ratio, degree of momentum, learning rate, mini-batch size, number of nodes, and strength of parameter regularization were simultaneously optimized to reach an optimal AUC value from the 10-fold cross-validations.K, the number of its interacting PBSs was counted as Km. For a PK family L, the number of its substrate p-sites was counted as Ln. The number of DRPs for K and L on the same p-sites was counted as x. Then, the GPS-PBS predictions were not changed, whereas the prediction results of GPS 5.0 in 638,909 p-sites were randomly permutated. The number of p-sites modified by L remained to be Ln, whereas the DRPs for K and L was re-counted as x’. Such a permutation test was repeated 10,000 times, and the results were modeled in a Gaussian distribution. The p-value was calculated based on the proportion of x’ ≥ x.Here, we defined a doubly regulated p-site (DRP) as a p-site that was predicted to be phosphorylated by at least one PK and also interact with at least one PPBD, at the family level. For a PPBD family To estimate the FPR values, we randomly retrieved 10,000 PBP items from Swiss-Prot protein sequences downloaded from UniProt to construct a near-negative data set. Then, GPS-PBS was used for a prediction. For each PPBD family or single PPBD, the theoretical FPR value was calculated as the predicted number against the 10,000 PBP items. Such a process was repeated 20 times, and the average value was determined as the final FPR. Again, the FPR values were separately estimated for pS/pT- and pY-interacting PPBDs. The high, medium and low thresholds were adopted with FPRs of 2%, 6%, and 10% for pS/pT-interacting PPBDs and 4%, 9%, and 15% for PPBDs in the pY group, respectively. We also implemented an “All” option to output all predictions for one or multiple selected predictors. GPS-PBS was extensively tested on various web browsers including Internet Explorer, Mozilla Firefox, and Google Chrome to ensure its usability. The full procedure of this study was shown in For the prediction of PBSs specifically recognized various PPCPs, here we improved our GPS algorithm ,29,30 toHomo sapiens, Mus musculus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Rattus norvegicus, Arabidopsis thaliana and other species . The top three mostly enriched GO biological processes were selected and visualized for each family. For the pY group, we observed that PPBPs of the SH2 and PTB families were significantly enriched in tyrosine phosphorylation-associated processes, such as peptidyl-tyrosine phosphorylation (GO:0018108), transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169) and peptidyl-tyrosine autophosphorylation (GO:0038083). The results were highly consistent with experimental studies, which demonstrated that PPBPs played a critical role in reading the dynamic signals of pY phosphorylation networks [Using the 776 known human PPBPs belonging to seven families , we condnetworks .For the pS/pT group, the enrichment GO biological processes such as protein phosphorylation (GO:0006468), positive regulation of GTPase activity (GO:0043547) and peptidyl-serine phosphorylation GO:0018105) for the 14-3-3 family demonstrated that their PPBPs were highly involved in regulating pS/pT phosphorylation events. Further analyses of BRCT, FHA, PBD and WW families in the pS/pT group indicated that their PPBPs were highly involved in regulating various types of DNA-associated processes such as positive regulation of transcription, DNA-templated (GO:0045893), DNA damage checkpoint (GO:0000077) and sister chromatid cohesion (GO: 0007062) D. These 8105 for Previously, we developed the GPS 2.0 algorithm for the prediction of kinase-specific p-sites . Later, In this work, we hypothesized that the amino acid residues in each position around PBSs might be generally and differentially important for the recognition of PPBDs. For developing GPS-PBS, both the scoring strategy and PWD were reserved, and we also added a new method named PVT A. In them, n) items, as well as m and n values, were present for each PPDB family or single PPBD cluster. The ratio of negative: positive PBP items ranged from 4.3 (LCP2_SH2) to 204.3 (MDC1_BRCT), indicating the imbalance of positive and negative data in the benchmark data set , which contained 1,616,804 experimentally identified p-sites in 209,326 proteins collected from 68 eukaryotic species . From EPUsing GPS-PBS, we here performed a large-scale prediction of PBSs to annotate potential PPBDs for the mammalian p-sites. The high threshold of GPS-PBS was chosen with false positive rate (FPR) values of 2% and 4% for the PPBDs in the pS/pT and pY groups, respectively. From the results, we found that only 171,825 (22.44%) potential PBSs were predicted at the family level, whereas up to 325,913 (42.56%) p-sites were computationally annotated to potentially interact with at least one PPBD using the single PPBD cluster-based predictors A. In totp-value < 10−10) and identified 124 pairs of significant associations between PPBD and PK families in regulating the same p-sites , an enrichment analysis was performed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations [In dynamic phosphorylation networks, both PKs and PPCPs are important regulators, and the identification of significant associations between the two types of regulators will be helpful for better understanding the mechanisms of phosphorylation. Using GPS 5.0 with the high threshold , we pred p-sites . In our otations  for 1060Catalyzed by writers named PKs, p-sites in phosphoproteins frequently exert their functional effects through following recognition by PPBDs to interact with PPCPs, which act as readers and disseminate phosphorylation signals into downstream pathways for regulating cellular activities ,6,7. AltH. sapiens, M. musculus, and R. norvegicus. Only 313 (7.0%) known PBSs were curated from other species. Thus, although GPS-PBS was designed for a more general purpose, it could be expected that the prediction of PBSs in other species would achieve a lower accuracy, beyond the three mammalians. Additionally, only the sequence similarity of members in different PPBD families or single PPBD clusters were taken into account for model building, and the sequence diversity of individually PPBDs were not directly considered. Thus, the prediction accuracy might be lower for less studied PPBDs or PPBDs without known PBSs.In this work, we compiled a high-quality benchmark data set containing 4458 PBSs , consideUsing GPS-PBS, we conducted a large-scale annotation of potential PPBDs for 765,779 known mammalian p-sites obtained from EPSD , and idem and n values of PBP items will be tested later. Moreover, the BLOSUM62 matrix was adopted to measure the peptide similarity of PBP items, and we will test other types of amino acid substitution matrices for performance improvement. The computational models in GPS-PBS will be updated if the GPS algorithm was updated. Besides GPS, other algorithms will be tested and integrated into GPS-PBS if the accuracy can be improved. We anticipate that GPS-PBS can be a useful tool for further exploration of dynamic phosphorylation networks.In the future, we will continuously maintain GPS-PBS by curating more experimentally identified PBSs if new data becomes available. In GPS-PBS, we adopted PBP for model training, and different combinations of