During April 17–May 5, 2023, 13 monkeypox (mpox) cases were reported to the Chicago Department of Public Health (CDPH) after 2 months during which only a single case had been reported. The cluster was remarkable because it comprised more than 10 cases at a time when sporadic cases or small clusters  were being reported in the United States, and >69% of the persons in this cluster had received 2 doses of JYNNEOS or 1 dose of ACAM2000 vaccine.On May 9, 2023, CDPH issued a health alert,**3 and viral load <200 viral copies/mL). Three (8%) patients experienced concurrent sexually transmitted infections at the time of mpox diagnosis.During March 18–June 12, 2023, 40 laboratory-confirmed mpox cases were identified in Chicago, including 22 (55%), five (13%), and 13 (33%), respectively, among patients who had received 2 doses of JYNNEOS or 1 dose of ACAM2000 vaccine, those who had received 1 vaccine dose of JYNNEOS vaccine, and those who had not received any vaccines for mpox . All casMost vaccinated patients in this cluster received 1 or 2 JYNNEOS vaccine doses during July–August 2022; the timing of vaccination is similar to an mpox cluster in France involving vaccinated persons who acquired mpox >6 months after vaccination3). Preliminary sequencing results from one unvaccinated patient and three patients who had received 2 doses of JYNNEOS or 1 dose of ACAM2000 vaccine indicate that Monkeypox virus (MPXV) among these Chicago patients is consistent with the B.1 variant of clade IIb MPXV, the predominant variant during the 2022–2023 outbreak. Genomic sequences revealed very few point mutations relative to published MPXV genomes, with no changes predicted to cause amino acid changes or increased pathogenicity. In terms of risk factors, patients who received 2 doses of JYNNEOS or 1 dose of ACAM2000 had a median of three sex partners (range = one to 20) during the 3 weeks before symptom onset, compared with 1.5 (range = 0–6) among patients who received 1 dose of JYNNEOS vaccine and unvaccinated patients. Preliminary medical record review indicates that vaccinated patients experienced self-limited illness, managed in outpatient settings. Compared with patients who received 2 doses of JYNNEOS or 1 dose of ACAM2000 vaccine, patients who received 1 dose of JYNNEOS or no vaccines experienced a higher prevalence of lesions affecting the genital (43% versus 6%) and ocular (29% versus none) mucosa. The two hospitalized patients in this cluster had not received any mpox vaccine and had advanced HIV (<200 CD4 cells/mmAlthough the cause of this cluster has not yet been determined, leading hypotheses include a potential high number of sexual exposures in a network with many vaccinated persons, decreased vaccine effectiveness due to waning of humoral immunity, or vaccine mishandling or administration errors. Health departments are encouraged to report vaccination status of mpox patients to CDC for rapid detection of similar clusters among persons who were previously vaccinated. Persons with known or suspected mpox exposures should isolate and seek testing if they develop mpox symptoms, even if they have been vaccinated. Temporary sexual behavior changes, such as limiting the number of new or multiple sex partners and limiting sex in settings where anonymous sexual contact with multiple partners occurs can also help prevent mpox. Persons eligible for vaccination, particularly those with advanced HIV and other immunocompromising conditions, should receive 2 doses of JYNNEOS vaccine. Additional research on the durability of JYNNEOS vaccine–induced immunity is needed.


Medication nonadherence is a major concern for many health care stakeholders. Improving medication adherence in health plan members who have both hypertension and diabetes is essential for the successful management of these chronic diseases, with anticipated outcomes in decreased health care utilization, all-cause mortality and cost.To (a) identify patients who are potentially nonadherent to antidiabetic or antihypertensive agents within 1 managed care organization and (b) determine the relationship of rates of medication nonadherence with 2 mail intervention programs that involved quarterly medication-specific profiles of patients with potential nonadherence sent to primary care physicians (PCPs) and general medication adherence letters sent to patients with potential nonadherence.The study sample consisted of commercial members, Medicare Advantage-Prescription Drug Plan (MA-PD) members and Medicare Prescription Drug Plan (PDP) members who filled prescriptions for antihypertensive and antidiabetic medications and utilized their managed care pharmacy benefit during each measurement quarter (3 months) in the 2-year study period. Nonadherence was defined as a medication possession ratio (MPR) less than 77.0% for 1 or more antihypertensives and/or antidiabetic medications for each standalone calendar quarter. The first intervention, letters to PCPs with patient-specific medication profiles for 2008 Q2, began 6-8 weeks after 2008 Q2 and continued for each standalone calendar quarter through the end of the study period in 2010 Q1 . We assumed that patient care was managed by PCPs for hypertension and diabetes treatment. The medication profile also included antihyperlipidemic medication claims information, but there was no adherence analysis performed for antihyperlipidemic medications. The second intervention, letters sent to potentially nonadherent patients, began 6-8 weeks after 2009 Q1 for patients with MPR less than 77% for 1 or more antidiabetic or antihypertensive medications in 2009 Q1 and continued for each standalone calendar quarter through the end of the study period in 2010 Q1.Because there were 2 different interventions, 2 baseline adherence rates were calculated, for 2008 Q2 for the PCP mailing and for 2009 Q1 for the patient mailing. Compared with the baseline nonadherence rate in 2008 Q2 (35.6%), a small increase in nonadherence was observed in 2008 Q3 (36.4%), following by 6 calendar quarters of lower rates of nonadherence with a 27.7% nonadherence rate in the last measurement period in 2010 Q1. Compared with the nonadherence rate of 30.8% in baseline 2 (2009 Q1), the patient mailings were associated with small increases in nonadherence to 31.4% in 2009 Q2 and 31.1% in 2009 Q3, respectively, followed by lower nonadherence rates in 2009 Q4 (29.2%) and 2010 Q1 (27.7%).A 2-part intervention that involved mailings to PCPs for patients with both diabetic and antihypertensive medications who were potentially nonadherent to at least 1 medication, followed 9 months later by a general mailing sent to these potentially nonadherent patients regarding medication adherence, was associated with apparent improvement. However, the effect of the 2-part intervention on medication nonadherence could not be isolated because of coincident disease management interventions in diabetes and hypertension during the 2-year study period.


Pak1 deficiency in the cardiac atria have not been well explored. In this study, Pak1 cardiac-conditional knock-out (cKO) mice were studied under baseline and adrenergic challenge conditions. Pak1 cKO mice show atrial arrhythmias including atrial fibrillation (AF) in vivo, detected during anaesthetized electrocardiography without evidence of interstitial fibrosis upon Masson's trichrome staining. Optical mapping of left atrial preparations from Pak1 cKO mice revealed a higher incidence of Ca2+ and action potential alternans under isoprenaline challenge and differences in baseline action potential and calcium transient characteristics. Type-2 ryanodine receptor (RyR2) channels from Pak1 cKO hearts had a higher open probability than those from wild-type. Reverse transcription-quantitative polymerase chain reaction and Western blotting indicated that pCamkIIδ and RyR2 are highly phosphorylated at baseline in the atria of Pak1 cKO mice, while the expression of Slc8a2 and Slc8a3 as a Na+–Ca2+ exchanger, controlling the influx of Ca2+ from outside of the cell and efflux of Na+ from the cytoplasm, are augmented. Chromatin immunoprecipitation study showed that pCreb1 interacts with Slc8a2 and Slc8a3. Our study thus demonstrates that deficiency of Pak1 promotes atrial arrhythmogenesis under adrenergic stress, probably through post-translational and transcriptional modifications of key molecules that are critical to Ca2+ homeostasis.P21-activated kinase 1 (Pak1) signalling plays a vital and overall protective role in the heart. However, the phenotypes of This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’. Abnormal ‘phosphorylation states' of Ca2+-handling proteins occurs in several disease conditions including AF  approximately 50 mM) on the trans  side of the bilayer at 21°C. The luminal chamber was voltage-clamped at ground. The free [Ca2+] and pH of the solutions were measured using a Ca2+ electrode  and a Ross-type pH electrode  as previously described i under Ang II stress at single cell level.Our electrophysiological studies in intact animals and isolated atrial independently implicated out mice  that sho2+ alternans recorded in LA in particular when they are acutely exposed to isoprenaline stress in our study suggests the alteration of Ca2+ homeostasis that could be linked to either abnormal SR Ca2+ release or decrease in kinetics of SERCA2A and NCX currents and an increase in diastolic Ca2+ levels in as we found in Pak1 cKO mouse ventricular cardiomyocytes under baseline conditions [2+ reuptake into the SR via SERCA2A inhibition using the antagonist thapsigargin was demonstrated to increase the susceptibility to cellular Ca2+ alternans [et al. [Canditions . Upon islternans . Additio [et al.  have sho [et al. .2+ refilling time in Pak1 cKO ventricular myocytes when compared to WT littermates. This observation along with slow NCX and SERCA2A kinetics, may imply longer cytosolic Ca2+ transients. In fact, ventricular myocyte experimentation using total Pak1 KO mice have reported longer Ca2+ transient recovery time in KO than in WT mice [Pak1 cKO mice under baseline conditions in the present study.We previously  have als WT mice . The two2+ sensitivity of RyR2 channels from PAK1 cKO mice. This increase in RyR2 activity might have resulted from the higher phosphorylation levels at S2808 and S2814 detected in the PAK1 cKO heart preparations since enhanced phosphorylation at these sites has been linked to RyR2 dysregulation and increased RyR2-mediated cytosolic Ca2+ leak [2+ would probably impact on the consistency of local SR Ca2+-release events, providing an explanation of the cellular basis for an increase in arrhythmogenesis and abnormal Ca2+ transient observed in Pak1 cKO mice.Our data on RyR2 single-channel recordings revealed that channels derived from PAK1 cKO hearts exhibit a markedly higher Po than channels from control hearts, suggesting an increase in cytosolic Caa2+ leak –39. The Pak1 cKO mice with alterations of the activity of RyR2 and CamKII and expression of Serca2a and Ncx genes that are essential to Ca2+ homeostasis and electrophysiological stability in the atria. These changes may reflect multiple regulatory mechanisms that PAK1 is involved in Ca2+ handling proteins activity and expression at both post-translational and transcriptional levels. Firstly, in our previous study, we have established the action of PAK1 on the phosphatase PP2A and the resulting balance between kinase and phosphatase activity controlling LTCC and IK activity in sinoatrial node pacemaker cells [Pak1 cKO mouse atrial tissue. Admittedly, such mechanisms require more investigations in the future, while the downregulated SERCA2a and upregulated Slc8a2 and Slc8a3 transcripts in Pak1 cKO atrial tissue may reflect a complicated transcriptional regulation but important role of Pak1 as a regulator of cardiac SERCA2a and Slc8a2 and Slc8a3 through transcriptional mechanisms, which is consistent with our previous finding in the ventricular tissue [Our molecular studies then associated PAK deficiency in er cells ; An impoer cells . As a coer cells . Thus Par tissue .2+ homestasis in the atria through regulation of calcium handling proteins RyR2, Serca2a and NCX via either post-translational and transcriptional mechanisms, thus providing new insights into atrial Ca2+ signalling regulatory mechanism that has an implication in developing new therapeutic strategies for atrial tachyarrhythmias.In conclusion, our study demonstrates for the first time, to our knowledge, a key regulatory role of Pak1 for maintaining electrophysiological stability and Ca


Candida albicans is a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously, C. albicans colonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about how C. albicans responds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposed C. albicans to high concentrations (10% vol/vol) of ethanol—a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants of C. albicans isolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug. Candida albicans is an opportunistic pathogen causing severe morbidity and mortality in immunocompromised patients. Candida spp are the second most common cause of nosocomial bloodstream infection in the US, and cause about 70% of all fungal infections  / pellet weight. Equation 2: % DHE = [(A230/518) × dilution factor]/pellet weight. % ergosterol = Equation 1 – Equation 2. The dilution factor is the dilution factor used for diluting the sterol-containing heptane solution with ethanol, 290 = E for ergosterol at 282 nm, and 518 = E for DHE at 230 nm -2H-tetrazolium hydroxide (XTT) (Alfa-Aesar) in PBS with 4 uL of menadione per/100 uL was added to each well. The plates were incubated for one hour at room temperature and then 50 uL of solution from each well was removed and read at 492 nm to determine metabolic activity of each well. The no-drug well averaged between the two duplicates was used to calculate the OD492 that indicates a 50% reduction. The concentration of drug that yielded a reduction in metabolic activity immediately less than 50% of the no-drug well was considered the MIC50. The heat map of the metabolic activity of these wells is shown as the medians of 6 biological replicates for each strain in For the broth-microdilution assay, cells were grown as stated above and 10Supplement 1Supplemental Figure S1: Quantification of small colony variants throughout passaging of four separate populations of cells that were exposed repeatedly to increasing concentrations of ethanol in medium A or repeatedly exposed to water in medium A. Quantification of small colony variants throughout passage numbers of four separate populations of cells that were exposed repeatedly to increasing concentrations of ethanol in media A or repeatedly exposed to water in media A. Green squares were repeatedly exposed to ethanol and black dots were repeatedly exposed to water. Y-axis shows % small colonies relative to total number of colonies. Bars represent the mean values of the populations at that passage number. Small colonies were classified as less than 50% of the size of the normal sized colonies on a plate by eye.Supplement 2Supplemental Figure S2: Enc and Wnc survival in 10% ethanol. Enc and Wnc survival in 10% ethanol. Colonies exposed for 4 hours in 10% ethanol or 10% water and plated on YPD-agar to count final colonies. The percentage of final colonies in 10% ethanol compared to 10% water were calculated and used to calculate a relative survival compared to SC5314 within each experiment (y-axis). Normal-sized colonies from ethanol populations (Enc) or water population (Wnc) were used. One-way ANOVA with Welch’s correction was used for statistical testing with multiple comparisons. Median values with 95% confidence interval are plotted for 8–16 replicate cultures from 2–4 separate experiments for each strain.Supplement 3Supplemental Figure S3: Growth curves and doubling times of Esc strains and normal-sized colony strains in YPD. Isolated colonies were picked and grown for 20 hours to post-exponential phase and seeded into YPD in 96 well plates at an approximate OD600 of 0.05. Cells were grown for 48 hours at 30°C with constant shaking in a Biotek Epoch2 plate reader with OD600 read every 30 minutes. Three biological replicate cultures from each strain were used to graph the growth curves. Growth curves show the mean and standard deviation of each timepoint. The wildtype, background strain: SC5314, a culture from the repeat media-A (with no ethanol exposure) is shown as RepDMEM-2, and a normal sized colony from a repeat ethanol exposure population is shown as RepEtOH-8. Esc6, Esc7, and Esc8 are all shown as well. Timepoints 0–24 hours were used to calculate doubling times by performing a nonlinear regression in GraphPad Prism with the exponential plateau fit to find the rate constant: k. All R2 values were above 0.9331. k values were then used to calculate doubling times with the formula LN(2)/k. These were standardized to the SC5314 doubling times and then plotted in this figure as standardized doubling times.Supplement 4Supplemental Figure S4: Principal component analyses for SC5314 vs Esc6, Esc7, or Esc8 in 10% ethanol from RNA sequencing. Principal component analyses for SC5314 vs Esc6, Esc7, or Esc8 in 10% ethanol each from RNA sequencing. PC1 variance ranges from 81%–93% and PC2 variance ranges from 4%–9%. SC5314 is shown in red dots in each and Esc strains are shown in blue. Each dot represents a separate biological replicate culture that was sequenced.Supplement 5Supplemental Figure S5: Gene Ontology Terms of the Top 20 Processes of Significantly Enriched Transcripts in Esc strains exposed to ethanol compared to SC5314 exposed to ethanol. Gene Ontology Terms of the Top 20 Processes of Significantly Enriched Transcripts in Esc strains exposed to ethanol compared to SC5314 exposed to ethanol. Transcripts that were significantly enriched in Esc strains were used to determine GO term process clustering on Candida Genome Database GO term finder. The top 20 hits from each strain were added to the graph and shown on y-axis. Esc6 bars are shown in blue, Esc7 bars are shown in black, and Esc8 bars are shown in orange. X-axis shows the cluster frequency of each of these processes relative to the total number of genes in the respective sets of enriched transcripts used for the analysis.Supplement 6Supplemental Figure S6: Histogram Fit Curve of Esc6 Allele Depth Ratio. Histogram fit curve of Esc6 allele depth ratio is shown. Esc6 is used as an example of what was done for all of the strains that were sequenced. Binning was performed by calculating the number of genes that had allele depth ratios within bins of 0.05. The bin of 0–0.05 was set to an arbitrary number as this represents homozygous for the reference strain and should not be called in the sequence variant report. Then a trimodal fit was performed in excel and minima values were calculated and used for further determination of heterozygous and homozygous polymorphisms.Supplement 7Supplemental Figure S7: MIC50 values from broth-microdilution experiments of SC5314 and Esc6, Esc7, and Esc8. MIC50 values from broth-microdilution experiments of SC5314 and Esc6, Esc7, and Esc8. Six separate biological replicate experiments were conducted and plotted. MIC50 values were determined for each by measuring metabolic activity (XTT) and determining the well that represented a 50% reduction in metabolic activity. Geometric means with geometric SD are graphed and ordinary One-way ANOVA was performed for statistical significance.Supplement 8Supplemental Figure S8: Voriconazole Disk-Diffusion Images of SC5314 and Esc Strains. SC5314 and Esc strains  were grown to post exponential phase and then seeded onto YPD agar at a concentration of 105 cells per plate. A 1 ug disk of voriconazole was placed in the center of the plate. These plates were incubated at 30°C for 48 hours and then imaged with the same settings and converted to black and white images.Supplement 9Supplemental Figure S9: UPC2 to HSP90 Ratio of SC5314 vs Esc7 in different conditions. UPC2 to HSP90 Ratio of SC5314 vs Esc7 in different conditions. Arbitrary transcript values from RNA-seq data were used to calculate transcript ratios of UPC2 to HSP90 and were plotted (y-axis). Ordinary one-way ANOVA used for statistical significance. NS = not significant. * = p-value of 0.0377.Supplement 10Supplemental Table S1 Transcripts showing significant changes in abundance between SC5314 with ethanol vs water medium by RNASeqSupplement 11Supplemental Table S2 Transcripts showing significant changes in abundance between Esc7 with ethanol vs water medium by RNASeqSupplement 12Supplemental Table S3 Transcripts showing significant changes in abundance between Esc7 with ethanol vs SC5314 with ethanol medium by RNASeqSupplement 13Supplemental Table S4 Transcripts showing significant changes in abundance between Esc7 with water vs SC5314 with water medium by RNASeqSupplement 14Supplemental Table S5 Transcripts showing significant changes in abundance between Esc6 with ethanol vs water medium by RNASeqSupplement 15Supplemental Table S6 Transcripts showing significant changes in abundance between Esc8 with ethanol vs water medium by RNASeqSupplement 16Supplemental Table S7 Transcripts showing significant changes in abundance between Esc6 with ethanol vs SC5314 with ethanol medium by RNASeqSupplement 17Supplemental Table S8 Transcripts showing significant changes in abundance between Esc8 with ethanol vs SC5314 with ethanol medium by RNASeqSupplement 18Supplemental Table S9 Transcripts showing significant changes in abundance between Esc6 with water vs SC5314 with water medium by RNASeqSupplement 19Supplemental Table S10 Transcripts showing significant changes in abundance between Esc8 with water vs SC5314 with water medium by RNASeqSupplement 20Supplemental Table S11 Esc6 Genomic PolymorphismsSupplement 21Supplemental Table S12 Esc7 Genomic PolymorphismsSupplement 22Supplemental Table S13 Esc8 Genomic PolymorphismsSupplement 23Supplemental Table S14: Primers used in this study.


BRCA1 and BRCA2 in metaplastic breast cancer. Their headline rate of 56% for BRCA1 is extremely high, but caution is necessary in translating this to all metaplastic cancers as there is likely a strong testing bias.In this issue Corso et al.  have carBRCA1 or BRCA2. A previous report of exome sequencing of 30 metaplastic breast cancers with paired normal tissue did not report any mutations in BRCA1 or BRCA2 [Metaplastic breast cancers are a heterogenous group of invasive breast cancers which share differentiation toward squamous or mesenchymal-appearing elements . The repor BRCA2 , althougor BRCA2 , 7.BRCA1 and BRCA2 genes, of which 23 (0.4%) were diagnosed with metaplastic breast cancers. They identified 13/23 (56.5%) with a BRCA1 pathogenic variant compared to 11.5%  with other breast cancer types (p < 0.0001). Interestingly one of the BRCA1 variants was found in one of the two cases with HER2 overexpression which is not usually a feature of BRCA1 cancers [BRCA1 driven than those that are oestrogen overexpressed [BRCA1 pathogenic variants in metaplastic breast cancer is about 10-fold less based on the detection rates in all breast cancers tested. This is because population testing studies outside clear founder populations only find around a 1% rate for BRCA1 [BRCA1 variant carriers were <53 years of age at diagnosis and 7/13 had a family history of breast or ovarian cancer with 2 being tested for a known variant in the family. Although the detection rate in this study is extremely high this bias in testing should mean a cautious approach to assessing the likelihood of a BRCA1 pathogenic variant in those with a sporadic metaplastic breast cancer aged >60 years as the detection rates are likely to be very much lower. For instance, the report of exome sequencing came from a cohort of 347 with over 2/3rds of tumours >51years at diagnosis and no BRCA1/2 variants were reported in 30 metaplastic tumours. Although the age range in the 30 cases undergoing exome sequencing was not recorded [BRCA1 variants if they had been seen, but large rearrangements that account for around 20% of BRCA1 pathogenic changes [In this edition of the journal Corso et al.  assessed cancers , 9. Althxpressed . The pred 13/23 5.5% with  changes  would noBRCA1 and BRCA2 Pathogenic Variant carriers and the diagnosis of metaplastic breast cancer should certainly not dissuade clinicians from ordering germline testing if other local criteria are met [BRCA2 in this study should not deter testing in this gene as they have also been reported and indeed shown to respond to PARPi [The Corso et al. report  clearly to PARPi .MLH1 and TP53. The TP53 associated tumour was a spindle cell sarcoma, but interestingly this was also the pathology in two of the BRCA1 carriers and indeed two BRCA1 carriers were noted to have squamous type. It is of note that other genes specifically associated with TNBC were not tested in all the samples and in particular more testing of PALB2, RAD51D, RAD51D, and BARD1 [Two further pathogenic germline variants were identified in nd BARD1 , 11 woul


This article has been corrected: The authors found an error in Figure 3B. During assembly of the figure, incorrect images of colonies formed by ACC cell lines in which ASXL1 was silenced by shRNA#1 (KD1) were used. The authors prepared a new Figure 3 using representative images from the original experiments. This correction has no impact on the main conclusion. The authors would like to apologize for any inconvenience caused.Figure 3 is presented below.New


The INO80 complex stood out in a large family of ATP-dependent chromatin remodelers because of its ATPase domain binding and translocating on DNA at the edge of nucleosomes, rather than at two helical turns from the center of DNA that is wrapped around nucleosomes. This unique property of INO80 was thought to account for its singular role in nucleosome placement at gene promoters in a DNA-sequence dependent manner that is crucial for transcription regulation. Now, we uncover INO80 functions differently than previously thought with its ATPase domain translocating on DNA close to the center of nucleosomes, like other remodelers. Our discovery also reveals the physical properties of the first ~36 bp of DNA on the entry side of nucleosomes is the main determinant for the DNA specificity of INO80 rather than the properties of the extranucleosomal DNA. The DNA sequence sensitive step of INO80 is after DNA is displaced from the histone octamer on the entry side of nucleosomes and 20 bp of DNA are moved out the exit side. We find the ATPase domain and Arp5 subunit of INO80 are likely involved in INO80’s DNA specificity and the mechanism of INO80 remodeling is substantially different than originally proposed. Building on this observation, a scan was performed to find where single nucleotide gaps and nicks in nucleosomal DNA interfere with nucleosome movement, given DNA gaps/nicks are known to block DNA translocation of chromatin remodelers5. Gaps from Superhelical Locations (SHL)-2 to -6, where DNA enters nucleosomes, were found to efficiently block nucleosome mobilization by INO80 and helped confirm the idea that the ATPase domain of INO80 translocates at SHL-61. The following year cryo-EM revealed a mixed population of nucleosome bound Chaetomium thermophilum and human INO80 with the ATPase domain bound on nucleosomal DNA at SHL-6 or SHL + 2 positions7. Given the earlier biochemical data, it was assumed that the structure with the ATPase domain at SHL-6 represented the active form of INO80. Interestingly, the structure of INO80’s ATPase domain bound at SHL-6 differs from that observed with Chd1 and Isw1a ATPase domains bound to SHL + 2 9. In the same year, the cryo-EM structure of yeast SWR1 complex-bound nucleosomes revealed its ATPase domain bound to DNA at SHL + 2, quite different than INO80 despite sharing numerous subunits and both belonging to the same sub-family of ATP-dependent chromatin remodelers10. Since then, INO80 is the only ATP-dependent chromatin thought to translocate on DNA near the edge of nucleosomes and this assumption has influenced many subsequent studies on the properties of this complex including its DNA sequence specificity.In 2017, chemical crosslinking and DNA footprinting techniques revealed that the ATPase domain of the INO80 chromatin remodeling complex associates with nucleosomal DNA at the SHL-6 position11. These studies found INO80 alone can correctly position more than 90% of nucleosomes at the + 1-position, next to the transcription start site. INO80 positioning of the + 1 nucleosome is not due to nucleosome spacing, a property that INO80 had been previously shown to exhibit, because the proper positioning of nucleosomes was not dependent on the density of nucleosomes assembled on DNA in these experiments13. These data show INO80 has intrinsic DNA sequence specificity and the DNA near promoters can arrest INO80 from further mobilizing nucleosomes. Examination of the physical properties of INO80 positioned + 1 nucleosomes compared to salt gradient dialysis (SGD) positioned nucleosome revealed linker DNA had more pronounced DNA propeller twist and the H2A-H2B bound DNA had less twist, corresponding to respectively more and less rigid DNA in the INO80 positioned than SGD nucleosomes14. From these experiments, the physical properties of extranucleosomal DNA was thought to restrict INO80 remodeling and is supported by the idea of the ATPase domain translocating on DNA close to where extranucleosomal DNA enters and the Arp8/Arp4/actin module bound to extranucleosomal DNA known to control INO80 nucleosome mobilization activity16. This premise however was not well experimentally validated when the remodeling activity of nucleosomes with rigid and flexible linker DNA showed only minor differences17.The DNA sequence specificity of INO80 was found using yeast genomic DNA reconstituted into chromatin and scanning for ATP-dependent chromatin remodeler(s) that could restore the same nucleosome positioning as observed in vivo 1. The physical properties of DNA displaced from H2A-H2B on the entry side of nucleosomes are critical for the efficient passage of DNA from the entry to the exit sides of nucleosomes. Although previously overlooked, we find that the DNA sequence bound to H2A-H2B is the primary driver for INO80’s DNA sequence specificity and Arp5’s grappler and DNA “gatekeeping” activity are likely involved.Currently, we find that our view of how INO80’s ATPase domain engages nucleosomes is incomplete and we need to revise the models of INO80 remodeling nucleosomes. Our current data support INO80’s ATPase domain translocating on DNA at SHL + 2 not SHL-6 and the ATPase contacts at SHL-6 being important in regulating INO80’s remodeling activity. The DNA gaps shown earlier now appear to not block translocation but instead likely impact the physical properties of DNA being displaced at this region akin to that seen with DNA sequence specificity18. The side of nucleosomes with the 70 bp extranucleosomal DNA is the (−) side in reference to the dyad axis and the (+) side is on the opposite side of the nucleosomes. In 70N5 nucleosomes the (−) and (+) sides are where DNA respectively enter or exit nucleosomes when remodeled by INO80. Ino80, the catalytic subunit of the INO80 complex, is crosslinked to DNA at a new position from nt + 4 to + 18 from the dyad axis that was not previously observed and at nt −58 and − 110/−111, positions that had been previously reported16  ported16 . Peptideported16 . The DNA21. When comparing these models, the site crosslinked at SHL-6, spanning from amino acid 683 to 799, is consistent with both models, making it inconclusive as to which model is more likely correct . However, the region of Ino80 from amino acid 915 to 1080 is only proximal to the correct site in DNA when the ATPase domain is actively engaged at SHL + 2 and not at SHL-6 .We model key segments of yeast Ino80 that were absent in the available structures using Alpha Fold2 and align this structure to those of INO80 and SWR1 bound to nucleosomes to find if the region of Ino80 mapped to SHL + 2 and − 6 are consistent with the ATPase domain actively translocating at SHL + 2 DNA footprinting shows INO80 extensively interacting with DNA similar to that observed by DNA crosslinking and highlights where the ATPase domain of Ino80 translocates on nucleosomal DNA. INO80 protects the upper strand of DNA at the SHL-1 to + 3 and SHL - 5/-6 consistent with where the ATPase domain of Ino80 binds . There i22. DNA initially moves 20 base pairs (bp) on the exit side of nucleosomes then rapidly advances an additional 12 bp for a total movement of 32 bp from the starting point, as evident by the 20 bp step being marginally detected and not increasing over time while DNA moving 32 bp continuously increases over a period of 5 minutes  that binds to the H3-H4 tetramer and then separately switching the flanking 36 bp of DNA (referred to as M2) that bind to the H2A-H2B dimer . There aThe outcomes of these experiments are quite revealing. Altering the flanking DNA (M2) has a more pronounced effect on INO80 nucleosome mobilization compared to changing the orientation of the core DNA (M1). Depending on the orientation of INO80 switching the flanking 36 bp DNA either stimulated or repressed nucleosome movement with 70N5 M2 nucleosomes being remodeled 16-fold less efficiently and the rate of remodeling of 5N70 M2 nucleosomes increasing at least 5-fold as estimated after remodeling for 30 minutes . The difIncorporation of the histone variant H2A.Z in place of H2A does not cause any differences in INO80’s DNA sequence specificity and shows that DNA sequence specificity does not depend on the type of H2A present . Next, we determine if the DNA sequence specificity of INO80 is shared by other ATP-dependent remodelers that sense linker DNA length and space nucleosomes by comparing the properties of ISW2 to INO80. We noticed that while ISW2 did show some DNA sequence-dependent effects, they were generally less pronounced and opposite to what we observed with INO80 and demonstrates the uniqueness of INO80’s DNA specificity .7. The rate of ATP hydrolysis with all three mutant nucleosomes are equivalent to the original 601 nucleosomes and indicates that the reduction of remodeling observed with replacing the 10 bp DNA (M2.2) is uncoupled from ATPase activity, consistent with earlier observations . Arp5 interactions near the acidic pocket appear not to be changed; however, its interaction with H3, close to the SHL2 position on DNA, is reduced when INO80 is bound to M2 nucleosomes . Furthermore, we find a loss of Ies6 crosslinking at residue 80 of H3 but not at residue 109 of H2B. These differences in Arp5 photocrosslinking were not observed at residues 109 of H2B or 56 of H4, confirming the site specificity of Arp5 loss. DNA footprinting and histone crosslinking indicate two changes in INO80 interactions: altered binding of both the ATPase domain and the Arp5/Ies6 module with the histone octamer and nucleosomal DNA that is tied to DNA sequence changes at the H2A-H2B interface.We map DNA movement during remodeling as described earlier using DNA photocrosslinking and cleavage to discern at which stage in remodeling is DNA sequence important. The first obvious difference when remodeling M2 nucleosomes is that DNA movement on the entry and exit sides of nucleosomes are uncoupled from each other with new DNA positions only detected on the exit side . A shortThe question arises as to where does the 20 bp come from to translocate out the exit side if no DNA movement is observed at the entry side of nucleosomes. We find that DNA displacement occurs on the entry side of M2 nucleosome, equivalent to that observed with 601 nucleosomes, as indicated by reduced crosslinking to DNA not coupled to DNA movement . DNA disChaetomium thermophilum of the ATPase domain engaging either SHL-6 or SHL + 2. Based on our findings, SWR1 and INO80 complexes likely engage nucleosomes more similarly than thought and the observed binding of the ATPase domain at SHL + 2 for INO80 is not an artifact of cryo-EM.The model of INO80 engaging and remodeling nucleosomes needs to be substantially revised starting with the ATPase domain’s interaction and translocation on nucleosomal DNA. DNA photocrosslinking and peptide mapping reveals the ATPase domain of Ino80 contacts both SHL-6 and SHL + 2, bridging these two superhelical locations. It becomes evident from DNA footprinting and crosslinking that Ino80 binds simultaneously to both positions as indicated by the extent of the footprint at these positions and the distinct regions of the ATPase domain that are crosslinked. Several approaches are used to decipher at which of these two sites is the cleft region of the ATPase domain bound and therefore translocating on when ATP is added. Both structural modeling of where the ATPase domain is crosslinked to DNA and mapping where DNA translocation occurs when an ATP is analog is added indicates the active cleft region of the ATPase domain is bound and translocates on DNA at SHL + 2 rather than the previously stated SHL-6 position. These findings resolve two previous conundrums as to why the ATPase domains of SWR1 and INO80 complexes engage nucleosomes so differently when they are paralogs and share many of the same subunits and the observation in the original cryo-EM structures of human and 27More evidence for the ATPase domain of Ino80 actively translocating on DNA at SHL + 2 comes from the DNA sequence specificity of INO80 providing an effective way to capture early stages in INO80 remodeling. When INO80 nucleosome remodeling is dramatically slowed due to changes in the DNA sequence, we see DNA only be moved to new positions on the exit side of 70N5 M2 nucleosomes. Although no new DNA positions are observed on the entry side of nucleosomes, DNA is displaced where H2A-H2B binds at the same rate and extent as DNA translocation on the exit side. These data suggest DNA displacement near where the ATPase domain binds SHL-6 is likely coupled to DNA translocation at SHL + 2 and not due to DNA translocation at SHL-6 as originally proposed Next, DNA translocation is arrested after moving 20 bp on the exit side and uncouples the normally fast transition from moving 20 to 32 bp observed with the preferred DNA substrate (70N5 601 nucleosomes). These results indicate that the movement of 32 bp of DNA on the exit side occurs after DNA is displaced from the histone octamer on the entry side and this transition is the stage at which DNA sequence influences the rate of nucleosomes being moved by INO80 .33. In contrast to these other remodelers, we find that the contacts of the ATPase domain at SHL-6 is crucial for INO80’s DNA sequence specificity and likely vital for INO80’s positioning of + 1 nucleosomes near transcription start sites in vivo, while with these other remodelers only modest effects have been conferred on remodeling33.There are several clues as to how DNA sequence might alter INO80’s binding to nucleosomes and thereby effect its ability to mobilize nucleosomes based on DNA footprinting and site-directed histone crosslinking. Binding of the ATPase domain at SHL-6 is altered by DNA sequence as seen with a loss of INO80 protection at SHL-5 on one strand of DNA and on the other strand at SHL-6 along with DNA lifting off from nt −65 to −67. The ATPase domain still binds to part of DNA at this region and is not completely lost consistent with some type of conformation change. The importance of the ATPase domain binding at SHL-6 for regulating INO80’s DNA sequence specificity is validated by replacement of a 10 bp A/T rich DNA centered at SHL-6 with a G/C rich DNA decreasing the rate of nucleosome movement by a factor of 4–5. These data show the interactions of the ATPase domain at SHL-6 is an important part of the DNA sequence sensing ability of INO80. The ATPase domain of other remodelers including the SWR1 complex are known to have a secondary binding site at SHL6 while also primarily binding to SHL2, as observed here with INO807. The observation with M2 nucleosomes of DNA translocation at SHL + 2 aberrantly moving pass the dyad axis till the SHL-2 position is consistent with the loss of Arp5 contacts at SHL-2 and Arp5 restraining DNA movement between the two sides of nucleosomes.There appears to be more factors involved in the DNA sequence dependence of INO80 because replacement of the 10 bp of DNA at SHL-6 does not show the same extent of inhibition as seen by swapping the 36 bps of DNA bound to the H2A-H2B dimer. It appears likely the Arp5 subunit of INO80 is involved and we observe like the ATPase domain of Ino80 that Arp5 contacts are also DNA sequence dependent. The DNA binding domain of Arp5 binds differently to nucleosomal DNA when the sequence is changed as seen with a loss of protection at SHL-2 on both DNA strands. Second, we observe by site-directed histone crosslinking a loss of Arp5 crosslinking to residue 80 of histone H3 in M2 nucleosomes compared to 601. These changes indicate Arp5 interactions with nucleosomal DNA are perturbed in a DNA-sequence dependent manner rather than through a complete loss of their binding. The involvement of Arp5 is consistent with its previously suggested role as a “gatekeeper” that regulates DNA movement from one side to the other of nucleosomes1. We had previously interpreted DNA gaps in the SHL-2 to −6 region that interfere with INO80 mobilizing nucleosomes to be due to blocking DNA translocation1. Based on the current data, we realize this is incorrect because INO80 is not translocating on DNA at this region and the gaps instead are likely tied to the strong DNA dependence observed at this same location. Together these data suggest the physical properties of the DNA being displaced from the histone octamer, including flexibility/rigidity, are crucial for regulating the rate of nucleosome mobilization by INO80 which are impacted by DNA sequence or breaks in the DNA strand. Studies from Phillip Korber, Karl-Peter Hopfner and Sebastian Eustermann have shown that INO80 with yeast chromatin reconstituted in vitro preferentially stops mobilizing nucleosomes when encountering DNA that is highly enriched with a less negative propeller twist from SHL-3 to SHL-6, indicative of DNA flexibility at this region being important for the specificity of INO80 to position nucleosomes14. These data are consistent with our observations and further highlight the importance of DNA physical properties in this region. More work is needed to better understand how DNA shape effects this important step in INO80 remodeling.In summary, our data indicates the DNA sequence specificity of INO80 resides principally at the H2A-H2B region proximal to where linker DNA enters nucleosomes, the ATPase domain of Ino80 and the Arp5 subunit are involved, and the sequence impacts the ability of DNA to pass from the linker proximal to distal sides of the nucleosome. It seems that not only is DNA sequence important, but also the integrity of the DNA strand in this region of the nucleosomes based on earlier studies34.This revised understanding of INO80’s mechanism raises questions about whether hexasomes are the correct substrate for INO80 and if hexasomes remodeled by INO80 would exhibit the same DNA sequence specificity as nucleosomes, given the importance of the DNA bound to the proximal H2A-H2B dimer that is typically absent in the hexasome experimentsSaccharomyces cerevisiae INO80 and ISW2 complexes were purified by immunoaffinity purification with 2 - FLAG (DYKDDDDK) epitopes attached at the C-terminus of the catalytic subunit. Yeast was grown in 15L YPD cultures , until the OD600 reached 5–6. Cells were harvested, washed with ice-cold water followed by ice-cold H-0.3 buffer  containing protease inhibitors . The cell pellet was collected and passed through a syringe to make yeast spaghetti, which were frozen in liquid nitrogen. The spaghetti was ground into fine powder by cryogenic grinding using (Spex freezer mill 6870). The yeast powder was suspended with ice-cold H-0.3 buffer with protease inhibitors, and nuclear proteins were extracted (S-100 extract) by ultracentrifugation at 100,000 g for 1 h using a Ti-55.2 rotor in an ultra-centrifuge (Thermo Fisher) at 4°C. The supernatant containing the soluble protein fraction from ultracentrifugation was incubated overnight at 4°C with the Anti-FLAG M2 agarose beads (Sigma Aldrich) (10 μl beads per ml S-100 extract) equilibrated with buffer H-0.3 with protease inhibitors. The resin was washed several times with buffer H-0.5 followed by H-0.1  with protease inhibitors. FLAG-tagged protein complexes were eluted from the resin with 1 mg/mL solution of 3X-FLAG peptide in buffer H-0.1 containing PMSF only (no other protease inhibitor). Complex purity and integrity was determined by analyzing samples on 4–20% gradient SDS–polyacrylamide gels and staining with coomassie brilliant blue R-250 and SYPRO Ruby protein stain.Native wild-type The M1 and M2 601 DNA constructs were synthesized by IDT-DNA. The M1 construct was generated by swapping and taking the reverse complement of the middle 36 bp DNA fragments, whereas the M2 construct was generated by swapping and taking the reverse complement of the outer 36 bp DNA fragments. The 601 DNA sequence mutant M2.1, M2.2, and M2.3 constructs were generated by site directed mutagenesis using the NEB SDM Kit by swapping the nucleotide regions with the opposite M2 corresponding nucleotide regions.32P]-labeled DNA at 37°C by rapid salt-dilution with 3–5 μg of recombinant Xenopus laevis histone octamers . The DNA and histone octamer was serially diluted from 2 M to 280 mM NaCl in steps at 37°C. Reconstituted nucleosomes were examined on a native 4% polyacrylamide gel (35.36 acrylamide: 1 bisacrylamide) (PAGE) and the 32P signal was captured on phosphorimaging .Mononucleosomes were reconstituted with 1.7 μg of PCR-generated 70N5 and 5N70 DNA (70 and 5 bp of flanking DNA on either side of 145 bp of 601 nucleosome position sequence DNA) and 100 fmol of 5 ´-labeled Ino80-FLAG markers of known molecular weights prepared by in vitro coupled transcription and translation using TnT® T7 Quick Coupled Transcription/Translation System (Promega) as described before 1. Data images in figures are representative of ≥ 3 experiments.Photoaffinity-labeled INO80 complex (after digestion of DNA and label transfer) was denatured with 0.4% SDS and heating at 90°C for 3 minutes, followed by buffer exchange using Amicon Ultra filters to remove SDS and FLAG peptides. C-terminal FLAG-tagged INO80 was purified by immobilization on ANTI-FLAG1. Briefly, Protease (ArgC) digested Ino80 fragments were resolved in 4–20% Tris-glycine SDS-polyacrylamide gels and transferred onto PVDF membranes using Bio-Rad Trans-Blot® electrophoretic transfer cell. The membranes were blocked with 5% fat-free milk in TBST , overnight at 4°C, washed with TBST, and incubated with mouse monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody  diluted 1:1000 for 1 hour at room temperature. The blots were washed with TBST, and developed with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher) visualization using an Image Quant LAS 4000 . .Western Blots were performed as described before 36. dUMP and dCMP analogs coupled with p-azidophenacyl bromide were incorporated along with [α-32P] dGTP/dATP, in tandem, at specific positions. Photo-reactive DNA was reconstituted into nucleosomes and bound with saturating amounts of INO80 at 30°C for 30 min. The extent of enzyme binding was assessed on 4% native polyacrylamide gels. In all experiments > 90% of nucleosomes were bound by the enzyme. After binding, INO80 was crosslinked to DNA by UV irradiation , and DNA was digested with DNaseI and S1 nuclease for transfer of the radioactive label to the crosslinked protein(s)36. Protein subunits were separated on 4–20% SDS polyacrylamide gels and radiolabeled subunit(s) were visualized by phosphorimaging. Data images in figures are representative of ≥ 3 experiments.Site-specific photo-reactive DNA probes with 601-nucleosome positioning sequence for 70N5 nucleosomes were synthesized by enzymatic incorporation of modified nucleotides into double stranded DNAXenopus laevis histones with amino acids replaced by cysteine at specific positions  were expressed, purified, and reconstituted into octamers with other histones as discussed above. Homogeneous 70N5 601/M2 nucleosomes were reconstituted using the respective 601-nucleosome positioning sequence containing Cy5 label at one end. The PEAS [N-((2-pyridyldithio)ethyl)-4-azidosalicylamide] (Thermo Fisher) -conjugated nucleosomes were iodinated with [125I] (Perkin Elmer). Detailed protocol had been discussed and standardized earlier 16. Nucleosomes with photoreactive octamers were bound to saturating amounts of INO80 at 30° C and were irradiated directly under UV  for crosslinking. After crosslinking, samples were treated with 100 mM DTT and incubated at 37° C for 30 minutes to transfer [125I]-radiolabel to the crosslinked proteins. Samples were resolved on 4–20% SDS-polyacrylamide gels, dried and viewed by phosphor-imaging following 14–16 hours of exposure to identify the radiolabeled subunits. Data images in figures are representative of 3 individual experiments..Recombinant


GRAIN WIDTH AND WEIGHT 2  exhibit abnormally wide grains (UBIQUITIN SPECIFIC PROTEASE 15 (OsUBP15) promotes grain size and weight  proteasome pathway. The Ub-proteasome pathway participates in a wide range of physiological processes, including plant reproduction  was identified in an EMS-generated mutant population; smg3-1 displayed shorter panicles and reduced grain number per panicle; the reduced grain size was found to be due to defects in cell expansion  displayed similar defects, confirming the role of SMG3 as a positive regulator of grain size.A small grain mutant  revealed that SMG3 is localized to the endoplasmic reticulum where it colocalized with the ER-marker HDEL. At the whole plant level, the staining patterns of the pSMG3::SMG3-GUS showed that the SMG3 gene is expressed in the developing panicles and the roots. pathway . SMG3 inDGS1 gene, Li and colleagues generated four alleles in the ZH11 and KNK rice varieties , which displayed defects in grain size. Interestingly, the double mutant smg3-2 dgs1-1 showed a greater reduction in grain size than either single mutant alone. On the other hand, overexpression of DGS1 in a ZH11 background resulted in longer rice grains. The defects in the dgs1 mutant alleles resembled those of the smg3 mutant. Interestingly, both DGS1 and SMG3 genes were expressed in developing panicles. At the subcellular level, analysis of the GFP-DGS1 line indicates localization to the ER. To test if DGS1 is a functional E3 ligase, the authors expressed the protein in E. coli to conduct a biochemical analysis. Since a full DGS1 could not be expressed in E. coli they used a version lacking the transmembrane domain (DGS1ΔTM) to perform a ubiquitination assay. This analysis indicated that DGS1 can autoubiquitinate; and that mutations in the RING domain in DGS1 abolished the activity.To identify other components in the ERAD pathway, the authors tested protein interactions between SMG3 and other known regulators of grain development in rice using yeast two-hybrid assays. The analysis indicated that SMG3 physically interacts with DECREASED GRAIN SIZE1 (DGS1), which has been previously characterized in the control of grain size regulation . The SMGsmg3-1 and dgs1 mutants were less sensitive to brassinosteroids. Additionally, the smg3 and dgs1 mutants had changes in gene expression consistent with changes in BR function . In contrast, the expression of BR-signaling genes BZR1 and GSK2 was decreased. Furthermore, in vitro experiments demonstrated the interaction between BRI1 and DGS1, also found in planta with split luciferase assays. This interaction is critical since DGS1 was identified as a functional E3 ubiquitin ligase and can ubiquitinate BRI1.A previous study of the SMG3 homolog in Arabidopsis (UBC32) indicated that this protein participates in the degradation of the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1), so authors tested the relationship between SMG3, DGS1, and BR signaling in rice. They found that the dgs and smg3 mutants, suggesting that SMG3 and DGS1 affect the abundance and hence the function of the BRI1 receptor.To explore the effect of DGS1 on BRI1 function, the authors used an anti-BRI1 antibody to measure the accumulation of the BR receptor in different genetic backgrounds. The BRI1 protein levels were increased in the In summary, this work provides evidence for the participation of ER-associated degradation in plant hormone signaling in the control of reproductive development. Some interesting phenomena are still to be discovered regarding the involvement of SMG3 in the responses to abiotic stresses and its possible participation in root development.


This article has been corrected: The authors found that they incorrectly submitted two images: Figure 2I (tumors from the NC and FBXO28 groups), showing upregulation of FBXO28 in BxPC-3 cells in a mouse tumorigenesis experiment, and Figure 5G (FBXO28+SMARCC2 48h panel), showing results obtained with transfected BxPC-3 cells in a wound healing experiment. The authors stated that these corrections were prepared using images from the original experiments and do not change the results or conclusions of this paper. The authors apologize for this error.Figure 2 and 5 are presented below.Corrected


Recent quality initiatives require that the routine annual therapeutic drug-monitoring (TDM) parameters for the high-risk medication digoxin include a measure of renal function and a serum potassium level but not a serum digoxin concentration (SDC) measurement. Several studies have shown that the majority of the SDCs obtained in hospital settings provide little clinically actionable information.To evaluate the appropriateness and utility of SDCs ordered in a medical group practice setting by categorizing the reason the SDC was ordered and identifying action taken in response to the result.The descriptive study was conducted as a retrospective, electronic medical record (EMR) review of 90 primary care patients with continuous prescriptions for digoxin current on their medication profile with no gaps in therapy for at least 2 years prior to an SDC result entered into the EMR between January 1, 2009, and September 30, 2009. The reason the SDC was ordered was abstracted independently by 2 reviewers, who then assigned it to 1 of 8 predefined indication categories based on previously published criteria and practice guidelines. A third reviewer resolved inter-reviewer discrepancy (n?=?1).A total of 90 patients with at least 1 SDC met inclusion criteria. Routine monitoring was the most frequent SDC order indication category with 35 patients (38.9%), 17 (48.6%) of whom did not have the recommended monitoring measures of potassium or renal function drawn concurrently. Patients were included in other categories as follows: confirmation of signs/symptoms of toxicity 30 (33.3%); assessment of factors altering pharmacokinetics 5 (5.6%); assessment of dosage change 5 (5.6%); assessment of drug interaction 3 (3.3%); assessment of clinical response 3 (3.3%); assessment of adherence 1 (1.1%); and other 2 (2.2%). Across all categories, a total of 19 (21.1%) of SDC results were outside the therapeutic range of 0.5 nanograms (ng) per mL and 2.0 ng per mL, 18 of which were below 0.5 ng per mL, with none of the subtherapeutic levels leading to a change in digoxin therapy. Only 1 patient (1.1%) had therapy changed in response to an elevated abnormal SDC result of 2.1 ng per mL and was in the routine monitoring category.The majority of SDC results obtained in our medical group setting did not lead to clinical action, such as dose adjustment or drug discontinuation. SDCs were commonly measured as part of routine monitoring, which is considered an inappropriate indication, and often without being accompanied by better markers for digoxin toxicity such as serum potassium levels and measures of renal function as recommended by drug-monitoring quality initiatives. Provider education is needed regarding the most indicative digoxin TDM parameters to obtain in order to satisfy quality initiatives.


Following the publication of this article , concernThe P21 western blot panel in Fig 4A appears to have no detectable signal or background, nor does the panel present a positive control to confirm a successful blot.The Ad5/F35-siAPE1 panel in Fig 8A appears to partially overlap with the Ad5/F35-siAPE1+IR panel in Fig 8a and the Ad5/F35-siAPE1+IR panel in Fig 9a despite representing different experimental conditions.The Ad5/F35-siAPE1 panel in Fig 9A appears to partially overlap with the Ad5/F35-EGFP+IR panel when rotated despite representing different experimental conditions.The authors did not provide a response to the concern regarding the P21 western blot in Fig 4A nor was any underlying data provided to resolve this issue.The authors stated that the concerns related to Figs 8 and 9 were unintentional errors made during preparation of the figures but stated they were unable to source the original underlying data for both figures. The PLOS Editors remain concerned about the reliability of these figures.PLOS ONE Editors retract this article.In light of the concerns affecting multiple figure panels that question the reliability and validity of these data, the All authors either did not respond directly to the final editorial decision or could not be reached.


To measure the impact of clinical pharmacists in primary care practices who closely monitor patients older than 80 years after initiation of new medications.  The study was an uncontrolled pilot trial performed at a group-model health maintenance organization in the Pacific Northwest between August and December 1999. Forty-eight patients who were older than 80 years and were prescribed at least one new medication in their primary care clinic were called at home 3 to 6 days after starting a new medication and asked questions focusing on compliance and potential adverse drug events.  More than 20% of patients (10 of 48) had a clinically important change made as a result of the pharmacist telephone monitoring; 42% of patients (20 of 48) either experienced an undesired medication effect (14 of 48) or an inadequate effect (6 of 48). Pharmacists spent an average 11.3 minutes at an estimated cost of $6.40 per patient.  A simple, inexpensive pharmacist-based program to screen for medication problems after initiation of new medicines may improve the care to a population older than 80 years.


After this article  was publSpecifically:The LRRK2 KO β-actin panel in Fig 4A appears similar to the LRRK2 WT p38 panel in Fig 9A.The authors stated that the above concern was due to errors made during preparation of the figures and stated that the panel in Fig 9A was correct. The authors provided the underlying panel and accompanying quantification data for Fig 4A  along wiIn reviewing this matter, PLOS noted that the primary data were not provided with the published article  contraryPLOS ONE Editors issue this Expression of Concern.In light of the above concern pertaining to data availability, the S1 File(PPTX)Click here for additional data file.S2 File(PZFX)Click here for additional data file.S3 File(PPTX)Click here for additional data file.S4 File(PZFX)Click here for additional data file.


Dynamic chromatin accessibility regulates stem cell fate determination and tissue homeostasis via controlling gene expression. As a histone‐modifying enzyme that predominantly mediates methylation of lysine 27 in histone H3 (H3K27me1/2/3), Polycomb repressive complex 2 (PRC2) plays the canonical role in targeting developmental regulators during stem cell differentiation and transformation. Embryonic ectoderm development (EED), the core scaffold subunit of PRC2 and as an H3K27me3‐recognizing protein, has been broadly implicated with PRC2 stabilization and allosterically stimulated PRC2. Accumulating evidences from experimental data indicate that EED‐associating epigenetic modifications are indispensable for stem cell maintenance and differentiation into specific cell lineages. In this review, we discuss the most updated advances to summarize the structural architecture of EED and its contributions and underlying mechanisms to mediating lineage differentiation of different stem cells during epigenetic modification to expand our understanding of PRC2. Polycomb repressive complex 2 (PRC2) is composed of four core subunits – embryonic ectoderm development (EED), enhancer of zeste homologue 2, suppressor of Zeste 12, and RB‐binding protein 4/7. PRC2 catalyses methylation of H3K27, an enzymatic activity necessary for PRC2‐mediated epigenetic gene silencing during stem cell differentiation and transformation. PRC2 absent EED is incapable of recognizing binding sites to promote deposition of H3K27me3, results in a significant upregulation of genes that are normally associated with H3K27me marks. The nucleosome represents the smallest repeated structural unit of eukaryotic chromatin, consisting of DNA wrapping around histones,Drosophila as repressors of Hox genes and are gradually found in other species, such as human, mouse, Xenopus, and so on.Polycomb group (PcG) proteins are key epigenetic regulators that mediate heritable transcriptional silencing by modifying chromatin states and participating in the establishment and maintenance of cell fates during multicellular development.A vast number of studies report that the core subunits of the PRC2 complex show spatiotemporal specific expression patterns, indicating that they may have distinct functions.This review provides a comprehensive overview of EED in regulating stem cell fate and the underlying regulatory mechanisms during various organ morphogenesis, refining the framework for understanding the functions and mechanisms of PRC2‐mediated regulation of gene expression.2As a WD‐40 repeat family protein, EED contains seven WD40‐repeat motifs at its C terminus preceded by an extended N‐terminal segment.3EZH2 adopts an autoinhibitory conformation through crystal structures of the inactive isolated catalytic domain, suggesting that structural rearrangement of EZH2 is likely required for PRC2 activation.The PRC2 crystal structures provide fundamental insights into the interaction with each subunit, substrate recognition, and allosteric activation of its enzymatic activity. Moreover, PRC2 is more active on di‐nucleosomes and higher‐order chromatin structures than on mononucleosomes or histone tails,PRC2 recruitment is modulated by many different factors, including the interaction of PRC2 subunits with DNA and histones, PRC1‐mediated H2AK119ub1, RNA and other histone modifications.4Mammalian heterochromatin contains great repressive chromatin domains, including H3K9me2/3‐modified constitutive heterochromatin and H3K27me2/3‐decorated facultative heterochromatin. The two categories of histone modifications are catalysed by Suv39h1/2 and PRC2, respectively.5For pluripotent stem cells to expand and differentiate into one or more specialized and committed cell types in the body, critical cell fate decisions and lineage commitment must be made during growth and development. As differentiating stem cells undergo the cascade of lineage decisions on branching points through epigenetic‐threshold modulation, specific genes must be switched on and genes associated with alternative lineages need to be repressed in a dynamic and timely manner. Next, we will dissect the dynamic roles of EED during ontogenesis to further insight into the PRC2 and its role in the specificity and diversity of lineage specification Figure .5.1−/− embryos result in a genome‐wide decrease in H3K27me1, H3K27me2 and H3K27me3.ESCs possess the ability to differentiate into all the cell types of tissues and organs.5.2The primordial germ cells (PGCs) are precursors of the oocyte and sperm, which transmit significant epigenetic information to the offspring.5.3During the development of the nervous system, the PRC2 complex plays a vital role in maintaining the self‐renewal and proliferation capacities of neural stem/progenitor cells (NSPCs).5.4The development and function of the cardiovascular system are vulnerable to epigenetic insults, one of which is epigenetic repressors PRC2.5.5Intestinal stem cells (ISCs), located at the bottom of the crypts, are a cell population self‐renew extensively and differentiates into all cell types within crypts and villi.5.6The skin is the first barrier that protects mammals against external insults and dehydration. Epidermal lineages derive from a single layer of multipotent skin progenitors named basal cells, which attach to underlying basement membranes separating the epidermis from the dermis.5.7The bone marrow (BM) contains haematopoietic and mesenchymal stem cells.5.8Bone formation involves two major distinct mechanisms: intramembranous and endochondral ossification.6In this study, we comprehensively and systematically reviewed the research advances on EED/PRC2 function regulating ontogenesis Table . PRC2 coLiuyan Huang conceived, wrote, revised the manuscript and made the figure and table. Fanyuan Yu and Feifei Li revised the manuscript. Ling Ye, Chenglin Wang and Fanyuan Yu reviewed, revised and edited the manuscript. All authors read and approved the final manuscript.This work was supported by National Natural Science Foundation of China 81873708 (Chenglin Wang), 82201045 (Fanyuan Yu), Sichuan Province Science and Technology Program 2022JDRC0130 (Fanyuan Yu) and 2022ZYD0055 (Fanyuan Yu) and Young Elite Scientist Sponsorship Program by CAST 2022QNRC001 (Fanyuan Yu).The authors declare no potential conflicts of interest.


Point 5 of the section ‘Available diagnostic tools and their limitations’ incorrectly states that the BIOLINE Onchocerciasis IgG4 ELISA (Abbott) is discontinued. As of July 2023, the ELISA kit is still available, catalog number 61EK11.


A new study by Longo, Roy et al. has solved the structure of the RAD51C‐XRCC3 (CX3) heterodimer with a bound ATP analog, identifying two main structural interfaces and defining separable replication fork stability roles. One function relates to the ability of RAD51C to bind and assemble CX3 on nascent DNA, with an impact on the ability of forks to restart upon replication stress. The other relates to effective CX3 heterodimer formation, required for 5′ RAD51 filament capping, with effects on RAD51 filament disassembly, fork protection and influencing the persistence of reversed forks. A new study by Longo, Roy et al. has solved the structure of the RAD51C‐XRCC3 (CX3) heterodimer with bound ATP analog, identifying two main structural interfaces. Studies of cancer mutations of previously unknown significance in the two interfaces define separable replication fork stability roles required for fork protection and restart. Comprehensive analyses of breast and ovarian cancer pedigrees and patients with Fanconi anemia, a rare disorder characterized by hematological and developmental defects, pinpointed RAD51C as a cancer predisposition gene . This fiFunctional inquiries on RAD51C have been going on for decades. This is due to its role in interacting and assisting RAD51's recombinase function , centralTwo main complexes of RAD51 paralogs are known, with RAD51C being common to both . In one Alvinella pompejana (ap), an extreme metazoan living in deep‐sea waters. Leaving out a small, resolution limiting N‐terminal domain (NTD) of RAD51C and using a non‐hydrolyzable ATP mimic bound in the active sites of both RAD51C and XRCC3, the authors solved high‐resolution crystal structures of the CX3 heterodimer. This revealed extensive contacts between the RAD51C C‐terminal domain (CTD) and XRCC3 NTD and CTD, with these subdomains connected by a linker polymerization motif (PM) of XRCC3. Although similar to RAD51 structures in terms of ATP binding [Important insights on these topics came recently through the teamwork of several groups, combining structure elucidation of the CX3 complex with functional studies of specific cancer variants . Ingenui binding , the CX3Relying on the 3D representation of the CX3 heterodimer, the authors noticed that the cancer and patient mutations can be largely segregated in the two distinct interaction regions, CTDi and NTDi , 2, 14. in situ protein interaction with nascent DNA replication forks (SIRF). Besides this separation of function, the authors attempted to predict hCX3‐RAD51 complexes using AlphaFold2. In this way, they realized that XRCC3, but not RAD51C, interacts with RAD51 [What is the nature of the persisting replication stress? A126 is located on the P‐loop required to bind the ATP phosphate and is capable of disrupting the CX3 interface. Indeed, while the G125V mutation greatly impacts the structure and causes loss of interaction with XRCC3 as observed by two‐hybrid, the A126T mutation reduces the interaction with XRCC3 upon replication stress, as measured by proximity ligation assays or by co‐immunoprecipitation. While the G264S mutation does not destabilize the interaction with XRCC3, it greatly reduced the interaction of RAD51C with nascent DNA at stalled forks as revealed by Thus, CX3 plays important hitherto unappreciated roles during DNA replication stress, using different interfaces to facilitate fork restart or to protect forks and resolve emerging reversed forks. This work creates the foundation to study the multiple CX3 functions relevant to cancer biology at the nexus with replication stress response.The authors declare no conflict of interest.


Among the genes located in this region, PRRT2 codes for a member of the synaptic SNARE complex that allows the release of synaptic vesicles. PRRT2 is a candidate gene for ASD since homozygote mutations are associated with intellectual disability and heterozygote mutations cause benign infantile seizures, paroxysmal dyskinesia, or hemiplegic migraine. Here, we explored the contribution of PRRT2 mutations in ASD by screening its coding part in a large sample of 1578 individuals including 431 individuals with ASD, 186 controls and 961 individuals from the human genome Diversity Panel. We detected 24 nonsynonymous variants, 1 frameshift (A217PfsX8) and 1 in-frame deletion of 6 bp (p.A361_P362del). The frameshift mutation was observed in a control with no history of neurological or psychiatric disorders. The p.A361_P362del was observed in two individuals with autism from sub-Saharan African origin. Overall, the frequency of PRRT2 deleterious variants was not different between individuals with ASD and controls. Remarkably, PRRT2 displays a highly significant excess of nonsynonymous (pN) vs synonymous (pS) mutations in Asia (pN/pS = 4.85) and Europe (pN/pS = 1.62) compared with Africa . We also showed that whole genome amplification performed through rolling cycle amplification could artificially introduce the A217PfsX8 mutation indicating that this technology should not be performed prior to PRRT2 mutation screening. In summary, our results do not support a role for PRRT2 coding sequence variants in ASD, but provide an ascertainment of its genetic variability in worldwide populations that should help researchers and clinicians to better investigate the role of PRRT2 in human diseases.Inherited and  NLGN3/4X, SHANK2/3, NRXN1 and CNTNAP2 were shown to alter synaptic function and increase the risk for ASD de novo or inherited submicroscopic microdeletion/duplication at 16p11.2 have been associated with a variety of developmental/neuropsychiatric disorders including ASD, intellectual disability (ID), schizophrenia, or bipolar disorders, but also with body mass index −7; deletion OR:38.7 and 2.3×10−13) et al. (2009) screened for rare variations in 8 candidate genes selected for their function and expression in the brain . None of them showed a significant association with ASD at the exception of SEZ6L2 for which a suggestive association was detected PRRT2 gene PRRT2 codes for a membrane protein that interacts with SNAP25, a protein from the synaptic vesicles release machinery (the SNARE complex). PRRT2 is a compelling candidate gene for ASD and other psychaitric diseases since a homozygote frameshift mutation (A217PfsX8) was shown to segregate with non-syndromic ID in one consanguineous family with 9 affected individuals PRRT2 mutations (including the recurrent frameshift A217PfsX8) were identified in individuals and families with benign infantile seizures (BIS), paroxysmal kinesigenic dyskinesia (PKD), hemiplegic migraine (HM), or episodic ataxia, variably associated (e.g. the ICCA syndrome that associates BIS with PKD) PRRT2 in a sample of 961 individuals from worldwide populations.Autism Spectrum Disorders (ASD) are characterized by impairments in reciprocal social communication, and repetitive, stereotyped and ritualistic behaviors PRRT2 was performed in individuals with ASD recruited by the PARIS  study at specialized clinical neuropsychiatric centers disposed in France and Sweden  from blood leukocytes or B-lymphoblastoid cell lines, and was extracted with phenol-chloroform , S2. Allproducts . The 3 cPRRT2 variants in individuals and controls was determined by a two-sided Fisher's exact test on a two-by-two contingency table. PolyPhen-2 (http://psort.hgc.jp) and PSORT (http://genetics.bwh.harvard.edu/pph2) were used to predict the functional impact of amino acid substitutions and trans-membrane segments, respectively. To estimate the selective pressure on PRRT2 in different populations (pN/pS), the number of synonymous SNPs or nonsynonymous SNPs was divided by the total number of synonymous or nonsynonymous positions in PRRT2. The number of synonymous and nonsynonymous sites of PRRT2 were calculated by DnaSP v5 The significance of differences in PRRT2 in a large cohort of 1578 individuals including 431 individuals with ASD, 186 controls and 961 individuals from the HGDP We sequenced all coding exons of PRRT2 mutation screening or in the 1000 genomes (>2000 controls). The genomes of these two individuals were investigated using the Illumina 1M Duo SNP array. Using Identity by state (IBS) analysis, we showed that these two individuals were not relatives . In two individuals with ASD from sub-Saharan African origin, a 6 bp in-frame deletion (p.A361_P362del) was identified. The variant was transmitted by unaffected mothers and never observed in any individuals from our study and from other elatives , but werelatives . This PRof PRRT2 . In siliIn the control sample, we identified one individual carrying the frameshift mutation A217PfsX8 already described as causing autosomal dominant ICCA, BIS, PKD, HM or epilepsy) PRRT2 variants and trace the possible origin of them. For example, the predicted damaging P140A is a relatively frequent polymorphism present in Asia (7%), Oceania (5%), in Native American (7%), but more rare in Europe . The A214P variant was observed in Asian  and Native American populations. The P216L was observed in North Africa , Europe (<1%) and Asia (<1%). Interestingly, we observed a higher ratio of nonsynonymous vs synonymous variants in Asia compared with Africa. In Asia, we found only 1 synonymous variant (E127E) carried by a single individual  and 15 nonsynonymous variants  carried by 78 individuals . In contrast, in Africa, we found 3 synonymous variants  carried by 7 individuals  and only 1 nonsynonymous variant (P45S) carried by 2 individuals .We used a panel of DNA from individuals originated from worldwide populations  and S5. vs Africa EVS P = 0.67; Europe HGDP vs Europe EVS P = 1). In order to test whether this difference in selective pressure was specific to PRRT2, we used the data from EVS and extended our pN/pS calculation to all genes included in the 16p11.2 deletion  to synonymous polymorphism (pS) in each population sample . Usuallydeletion . We obsePRRT2 mutation screening should not be performed on DNA previously amplified through RCA.During the screening of the recurrent frameshift mutation ‘A217PfsX8’, we used whole genome amplified DNA for three individuals with ASD. Strikingly, we observed the A217PfsX8 mutation in these three samples for which the DNA was amplified using rolling circle amplification protocol  . We obtaKCTD13 gene are located within the deletion and can be considered possible driver(s) as well PRRT2 was indeed a very compelling candidate. Homozygous PRRT2 mutations were associated with clinical features also observed in a subset of individuals with ASD such as intellectual disability and epilepsy. PRRT2 encodes a putative membrane protein, localized at the synaptic membrane and interacting with SNAP25, a member of the SNARE protein complex involved in the release of synaptic vesicles. Our study was designed to identify new coding variants of PRRT2 in a relatively large sample of individuals with ASD and to ascertain the genetic variability of the gene in worldwide populations.Both deletions and duplications at 16p11.2 increase the risk for ASD, but the genes and the mechanisms involved remain largely unknown. Whereas several other strong candidate genes including the PRRT2 mutation enrichment in individuals with ASD compared with controls. None of the patients tested were deleted or duplicated for the 16p11.2 locus. In addition, we did not find causative PRRT2 mutations previously associated with neurological diseases such as PDK, ICCA, epilepsy or migraine in our cohort of patients. We observed a significant enrichment of maternally transmitted mutations in the individuals with ASD (9/10 transmitted by the mother). However, this apparent disequilibrium might have occurred by chance since to our knowledge, no transmission disequilibrium was reported for PRRT2 mutations in other neurological diseases. Taken together, these results indicate that mutations in PRRT2 are not a frequent cause of autism. Our study cannot however exclude that, in individuals with 16p11.2 deletions, haploinsufficiency of PRRT2 could increase the risk of ASD.We could not find any deleterious Secondly, we detected the presence of the deleterious truncating mutation A217PfsX8 in a control . This man had no psychiatric diseases and, to his knowledge, no history of any psychiatric disorders in his first and second degree relatives (parents and grand parents). This individual had no diagnostic for migraine and epilepsy, but was unavailable for further clinical explorations in order to detect mild signs of neurological problems. Similar incomplete penetrance of the A217PfsX8 mutation was observed in carriers from families with ICCA syndrome PRRT2 in worldwide populations. We identified 21 novel nonsynonymous variants including 17 never reported before and 4 already listed in the nucleotide variant database , whereas the pN/pS ratio in Asia and Europe is higher than expected and consistent with positive Darwinian selection PRRT2 seems to be the gene with the higher difference of selective pressure between Europe and Africa, but addition samples should be screened to ascertain if this difference is specific to PRRT2 or is also true for other genes within the deletion. Considering this heterogeneous pattern of selective pressure, it would be interesting to see if there is a higher prevalence of PRRT2 related disease in Asia and Europe compared to Africa. It would also be interesting to explore the genetic diversity of the binding partners of PRRT2 in order to decipher whether this lower selective pressure is restricted to PRRT2 or to other members of the same pathway.Thirdly, we ascertained the genetic variability of database . Despitein vivo and in vitro. For this reason, we strongly suggest that PRRT2 mutation screening should be restricted to native DNA and validate using Sanger sequencing. Interestingly, in the last release of the Exome Variant Server database, at the same location of the A217PfsX8 mutation , 10% of the PRRT2 allele contains an additional C  and 10% have a deletion of a C , but with very low average sample read depth (N = 10). Given the very high penetrance of the A217PfsX8 mutation in PRRT2-related diseases, it is impossible that 20% of the PRRT2 alleles carry a frame shift mutation at this position. Especially, since we found the A217PfsX8 mutation in only 1 individual out of 1990 (Allelic frequency = 0.025%). This high frequency of A217PfsX8 mutation in EVS could be related to the DNA amplification step required for whole exome sequencing. Studying the mechanism leading to the A217PfsX8 mutation was beyond the scope of our study, but our observation that a DNA amplification step through RCA could introduce this additional C/G might help in understanding the mechanism leading to A217PfsX8 mutation.Finally, we showed that whole genome amplification using multiple displacement amplification technology such as GenomiPhi and Repli-G could lead to the appearance of the A217PfsX8 mutation. The genomic region surrounding the mutation is made of 4 guanines and 9 cytosines and seems therefore to be at risk for mutation both PRRT2 mutations do not play a major role in the susceptibility to ASD and confirm that truncating mutations of PRRT2 are not fully penetrant. We also provide an ascertainment of the genetic diversity of PRRT2 in worldwide populations as well as important indication on the pitfalls for the mutation screening. All together, these results should help researchers and clinicians to better investigate the role of PRRT2 in human diseases.In summary, our study indicates that Figure S1PRRT2 in Exome Variant server. The nucleotide positions are according to PRRT2 from NCBI37/hg19. The average coverage of 10×, 20× and 40× are indicated in red, yellow and green respectively.Sequence coverage of PRRT2 in Exome Variants Serveur. Total number of sample sequenced (in blue) and average read depth (purple) for (TIF)Click here for additional data file.Figure S2Multiple dimension scale (MDS) of the genetic distances between individuals from the Human Genome Diversity Pannel. Insert. MDS restricted to the African populations including the two individuals with ASD carrying the in frame deletion p.A361_P362del (triangles).(TIF)Click here for additional data file.Table S1Cohorts used in this study.(DOCX)Click here for additional data file.Table S2Ethnicity of the cohorts.(DOCX)Click here for additional data file.Table S3PRRT2 primers.(DOCX)Click here for additional data file.Table S4PRRT2 nonsynonymous variants identified in the HGDP.(DOCX)Click here for additional data file.Table S5PRRT2 synonymous variants identified in the HGDP.(DOCX)Click here for additional data file.Table S6PRRT2 coding variants present in dbSNP, Exome variant Server (EVS) and the 1000 genome project (1KG).(DOCX)Click here for additional data file.Table S7Identity by state (IBS) values for the 2 individuals with ASD carrying the mutation p.A361_P362del compared with the IBS from different African populations.(DOCX)Click here for additional data file.Table S8Evolutionary analysis of all genes located within the 16p11.2 deletion.(DOCX)Click here for additional data file.


The NLSs of GRK5 and 6 bind DNA in vitro, whilst the NLS of GRK4 does not. Using mutants of GRK5 we identify the regions of GRK5 required for DNA-binding in vitro and nuclear localization in cells. The DNA-binding ability of GRK5 requires both the NLS and an N-terminal calmodulin (CaM)-binding site. A functional nuclear export sequence (NES), required for CaM-dependent nuclear export of the kinase, is also identified. Based on our observations we propose a model to explain how nuclear localization of GRK5 may be regulated. Notably, the nuclear localization of GRK5 and 6 is differentially regulated. These results suggest subfamily specific nuclear functions for the GRK4 subfamily members. Identification of GRK specific small molecule inhibitors of nuclear localization and/or function for the GRK4 subfamily may thus be an achievable goal.G protein-coupled receptor kinases (GRKs) act to desensitize G protein-coupled receptors (GPCRs). In addition to this role at the plasma membrane, a nuclear function for GRK5, a member of the GRK4 subfamily of GRKs, has been reported. GRK5 phosphorylates and promotes the nuclear export of the histone deacetylase, HDAC5. Here we demonstrate that the possession of a nuclear localization sequence (NLS) is a common feature of GRK4 subfamily members . However, the location of the NLS and the ability of these GRKs to bind DNA  The G protein-coupled receptor kinase (GRK) family of serine/threonine kinases were originally identified based on their ability to phosphorylate agonist occupied G protein-coupled receptors (GPCRs). GRK-mediated GPCR phosphorylation leads to recruitment of an arrestin family member, Arrestins 1–4, to the phosphorylated receptor. Arrestin binding sterically inhibits signaling via heterotrimeric G proteins, targets the receptor for internalization and initiates G protein-independent, arrestin-dependent, signal transduction The seven members of the GRK family are grouped into three subfamilies; the GRK1 (GRK1 and 7), GRK2 (GRK2 and 3) and GRK4  subfamilies Notably, expressing the N-terminus of GRK5 (GRK5-NT) has been shown to lead to accumulation and stabilization of the IκBα/NF-kB complex in the nucleus and inhibition of NF-κB activity in vivo and whether they represent the only means by which GRK5 promotes hypertrophy is not known. Similarly, we have very little information regarding how TAC promotes nuclear localization of GRK5 and whether the ability of GRK5 to bind DNA is required for GRK5-dependent hypertrophy. In this study we report the identification of an NES sequence in GRK5 and describe a variety of GRK5 mutants that differ in their ability to localize to the nucleus and/or their ability to bind DNA. These mutant constructs provide insights into the mechanisms regulating nuclear localization of GRK5 and provide useful tools for future dissection of the role of GRK5 in vivo.GRK5-mediated HDAC5 phosphorylation and the ability of GRK5 to regulate IκBα function represent two mechanisms whereby cardiac overexpression of GRK5 could cause pathological cardiac hypertrophy in response to pressure overload in mice. However, the extent to which either of these mechanisms are actually responsible for GRK5-dependent cardiac hypertrophy GRK5 is upregulated in the left ventricle in dilated cardiomyopathy and left ventricular overload disease in vitro suggests subfamily specific nuclear functions for the GRK4 subfamily of GRKs.In this study we also demonstrate that possession of an NLS is a common feature of the GRK4 subfamily of GRKs. However, the location and DNA-binding properties of the NLS differs between subfamily members. Furthermore although binding to calmodulin (CaM) promotes nuclear export of GRK5, nuclear localization of GRK6A is unaffected by this treatment. Thus even though both kinases bind CaM, and have NLSs with similar locations and DNA-binding properties their nuclear export is differentially regulated. Although a nuclear function for GRK5 has been identified, a role for GRK4 and GRK6 in the nucleus remains to be elucidated. The observation that the nuclear export of GRK5 and GRK6A are differentially regulated and that GRK4 fails to bind DNA in vivo. The identification of NLSs in other GRK4 subfamily members further indicates that investigating potential nuclear functions for these enzymes may also be a fruitful exercise.To summarize, the aim of the experiments detailed in this study were, through analysis of mutant GRK5 constructs, to gain more information on the regulatory mechanisms controlling its nuclear localization. Nuclear functions for GRK5 are only just beginning to be characterized. Our study identifies a mutant of GRK5 that reveals that nuclear localization and the ability to bind DNA are dissociable properties of the kinase. This mutant will be a useful tool for probing the functional relevance of the DNA-binding ability of GRK5 All cells were maintained in Dulbecco's modified Eagle medium (GIBCO) containing 10% fetal calf serum (Sigma) and penicillin and streptomycin  at 37°C, 5% CO2.GCG TGT GCA GCT GCT ACC GCC ATG AAC GGC GGG GAC CC TAC GAG ACC AAG GAT GCC  – 3′; GRK5 anti-sense primer, 5′ – G GTC CCC GCC GTT CAT GGC GGT AGC AG′TGCC  ACA CGC GGC ATC CTT GGT CTC GTA GGC – 3′. GRK2 sense primer, 5′ – CAGCC AGC GCA GCT GCT GAT GCC ATG AAC GGC GGG GAC CC ACA CCG GAC AAG  – 3′; GRK2 anti-sense primer, 5′ – G GTC CCC GCC GTT CAT GGC ATC AGC AG′TGCC GGC CTT GTC CGG TGT GTG GCT  – 3′. Nucleotides in bold encode the mutated amino acids.The plasmids for pRK5-GRK5 GCA GCT GCT ATA AAG CGC GAA GAG GTG GAG CGG CC TTC CAG CAG AGG  – 3′; anti-sense primer, 5′ – G CCGAGC AGC TGC CCT CTG CTG GAA GGG CG CTC CAC CTC TTC GCG CTT TAT  – 3′. Nucleotides in bold encode the mutated amino acids.pCMV5-GRK6A/B/C (human) were generous gifts from Dr Mario Tiberi  GCT AGA ATA GCT GCT AGG AAA GGT GAA GCT ATG GC– 3′; anti-sense primer, 5′ - GC AGC AGC TAT TCT AGC TTT TTG TAG CTT TTT GCA GGCCAT AGC TTC ACC TTT CCT  – 3′. The location of the NLS in the splice variants of GRK4 is as follows; α and γ, residues 219–227, lysines 221, 224 and 225 mutated to alanine; β and δ, 187–195, lysine residues 189, 192 and 193 mutated to alanine. Integrity of mutant constructs was confirmed by DNA sequencing.pRK5-GRK4α/β/γ/δ (human) were generous gifts from Richard Premont,  2 at 37°C, 5% CO2. Cells were subsequently immunolabelled with a mouse anti-GRK4-6 antibody  for 1 hr at room temperature. Confocal images were taken at room temperature using a Bio-Rad MRC 1024 laser scanning confocal system with Nikon Plan Apo 60x oil immersion lens and Optiphot 2 microscope using Bio-Rad Lasersharp 2000 software to acquire the images. The confocal plane shown represents a plane through the center of the nucleus as assessed by Hoechst staining. Images shown in a single figure were taken using the same microscope settings to ensure that cells with similar levels of GRK expression were compared within an experiment. Images were optimized for contrast in Adobe Photoshop, but no further manipulations were made. To quantify ionophore-dependent nuclear export at least 50 appropriately transfected cells were counted and GRK distribution scored as nuclear/cytosolic. Cells were deemed to show a cytosolic distribution if no transfected protein was detected in the nucleus. At least three separate transfections were scored per experiment.HEp2 cells (ATTC) were transfected by electroporation as described previously 2, 0.1% Triton X-100, 3 mM dithiothreitol, 0.1 M NaCl, 0.05 mM EDTA) for 1 hr at 4°C. Following incubation, the resin was washed and the amount of GRK retained on the resin determined by Western blot analysis. Films were quantified using a densitometer (Bio-Rad). In each experiment a control and experimental value were compared and significance determined using a t test. For competition assays, purified GRK6A (150 ng) was preincubated with the stated amounts of DNA  or RNA  for 30 min on ice before the DNA-binding assays were performed.As described previously GRK6A was expressed in, and purified from, baculovirus-infected SF9 cells as previously described 388RKEKVKRE395) The GRK4 subfamily contains three kinases, GRK4, GRK5 and GRK6. Four splice variants of human GRK4  and three of GRK6  have been identified. GRK5 contains an NLS located between amino acids 388 and 395  (in vitro suggests distinct nuclear functions for these enzymes.GRK5 binds DNA  its NLS . To deted bound) . GRK6A, d bound) , suggestd bound) . The amoin vitro DNA binding assay using purified GRK6A. Approximately 23 ± 1.7% of the purified GRK6A (150 ng) incubated with native DNA-cellulose was retained by the resin  promotes nuclear export of GRK5  but not the cellulose support  for GRK4 distinct from that of GRKs 5 and 6. The NLS of GRK4 is similar in sequence and location to predicted putative NLSs in GRK1 and GRK7 All members of the GRK4 subfamily contain functional NLSs, however, the NLS sequence of GRKs 5 and 6 differ from that of GRK4 in sequence, location and DNA-binding properties. Thus the NLS of GRKs 5 and 6, but not 4, bind DNA 2+/CaM appears to be responsible for exposing the NES in the cytosolic pool of transfected wild type GRK5 since GRK5NTPB, which lacks the N-terminal CaM binding site, is almost entirely nuclear. The observation that both GRK5ΔNES and GRK5NTPB are not exported from the nucleus in a Ca2+/CaM-dependent fashion further supports this contention since it demonstrates that CaM-dependent nuclear export is both NES and CaM-dependent. A role for CaM in mediating nuclear export has also been proposed for Ca2+/CaM dependent protein kinase I-α where CaM-binding promotes binding of the kinase to the CRM1 nuclear export complex Analysis of the subcellular localization and DNA-binding properties of the seven GRK5 mutants used in this study suggests a potential model whereby GRK5 nuclear localization may be regulated . The wilin vitro  distribution of the kinase and that when the palmitoylation sites on GRK6A are mutated nuclear GRK6A can be detected in vitro. Given the complexity of regulatory mechanisms controlling the nuclear localization of GRK5 it is not beyond the realms of possibility that both sequences may participate in regulating the nuclear localization of GRK6, potentially, in a cell type specific fashion. That the nuclear export of GRK5 and 6 is differentially regulated may suggest a common nuclear function that is differentially modulated following activation of specific receptor subsets, or alternatively, that GRK5 and 6 perform distinct nuclear functions. Additionally, it suggests that it may be possible to design subfamily specific small molecule regulators of the nuclear functions of GRK5 and 6A by selectively blocking their nuclear import/export.Despite having NLSs with similar locations and DNA-binding properties and both being CaM-binding proteins, the nuclear export of GRK5 is Cain vitroThe crystal structure of GRK6A has been solved and suggests that this kinase may exist as a dimer in vivo model of pressure overload disease. Specifically the role GRK5 kinase activity, nuclear localization and DNA-binding ability plays in promoting pathological hypertrophy in response to TAC may reveal additional nuclear functions for this kinase. A more detailed understanding of the molecular features of GRK5 required to promote pathological hypertrophy may lead to the identification of regions on GRK5 or target molecules amenable to regulation via small molecule inhibitors.This study highlights both a structural diversity between the GRK2 and GRK4 subfamilies of GRKs and a potential functional diversity within the GRK4 subfamily. A nuclear function for GRK5, phosphorylation and inhibition of the MEF2 repressor HDAC5, has been identified. Presumably GRK6 and GRK4 also have nuclear functions. What these are and whether they vary in a subtype, or splice variant, specific fashion remains to be determined. In this study we identify a functional NES in GRK5 and describe a number of mutants of this kinase that vary in their ability to localize to the nucleus or to bind DNA. Analysis of the hypertrophic response following TAC in transgenic mice with cardiac specific overexpression of these mutants is anticipated to reveal more information on the role of GRK5 in this