MMP2hi Fibroblasts Regulate CD8+ T Cell Residency and Inflammation via CD100 in Psoriasis.

BACKGROUND: Psoriasis, a T cell-mediated chronic inflammatory skin condition, is characterized by the interaction of T cells with various cell types, forming an inflammatory microenvironment that sustains psoriatic inflammation. The homeostasis of these tissue-resident T cells are supported by fibroblasts, the primary structural cells in the dermis. In psoriasis, there is an increased expression of matrix metalloproteinase 2 (MMP2), mediating the structural alterations of skin tissues and the modulation of inflammation. Additionally, the CD100-PLXNB2 axis is known to enhance psoriasis inflammation via keratinocytes, and CD103 levels are associated with the severity of psoriasis upon relapse. OBJECTIVE: To elucidate the role of fibroblasts and the MMP2/CD100 axis in modulating psoriasis inflammation. METHODS: CD100 expression and function in psoriasis were assessed using immunofluorescence, ELISA, single-cell transcriptome sequencing, cellular interaction analyses, and qRT-PCR. CD8+ T cells from psoriasis patients were isolated using magnetic beads to investigate the regulatory effect of MMP2 on CD100 expression on their membranes. Single-cell transcriptome sequencing, spatial transcriptome sequencing, mimetic timing analysis, immunofluorescence, and flow cytometry were utilized to determine the origin of MMP2 and its impact on CD103+CD8+ T cells. The hypotheses were further validated in vivo using MMP2 and CD100 inhibitors. RESULTS: Soluble CD100 (sCD100) was significantly upregulated in both psoriatic lesions and peripheral blood, amplifying psoriasis inflammation by promoting the production of inflammatory cytokines by keratinocytes, fibroblasts, and endothelial cells through the sCD100-PLXNB2 axis. Fibroblasts with high MMP2 expression (MMP2hi) exacerbate psoriasis symptoms by facilitating CD100 shedding from CD8+ T cell membranes. Additionally, it was demonstrated that fibroblasts enhance the upregulation of the CD8+ T cell residency factor CD103 in co-cultures with CD8+ T cells. Inhibitors targeting MMP2 and CD100 proved effective in reducing inflammation in a model of imiquimod-induced psoriasis. CONCLUSION: Our findings underscore the pivotal role of MMP2hi fibroblasts in the amplification and recurrence of inflammatory responses in psoriasis. These fibroblasts augment psoriasis inflammation through the CD100-PLXNB2 axis by facilitating CD100 shedding on CD8+ T cell membranes and by upregulating CD103, thereby enhancing CD8+ T cell residency.

Salvianolic Acid B Reduces Oxidative Stress to Promote Hair-Growth in Mice, Human Hair Follicles and Dermal Papilla Cells.

Background: Existing research links oxidative stress and inflammation to hair loss. Salvianolic acid B (SAB) is known for its anti-oxidative, anti-inflammatory, and other beneficial pharmacological properties. Objective: To assess the efficacy of SAB in modulating hair growth. Methods: In vivo experiments were conducted using C57BL/6 mice to evaluate the effects of SAB on hair and skin parameters. The study involved ex vivo analysis of human hair follicles (HFs) for hair shaft length and hair growth cycle assessment. In vitro, human dermal papilla cells (hDPCs) were cultured with SAB, and their proliferation, protection against H2O2-induced oxidative damage, and gene/protein expression alterations were examined using various analytical techniques, including Real-Time Cell Analysis (RTCA), DCFH-DA Assay, RNA-seq, and KEGG pathway analysis. Results: SAB treatment in mice significantly improved hair growth and vascularization by day 21. In human HFs, SAB extended hair shaft length and delayed the transition to the catagen phase. SAB-treated hDPCs showed a notable decrease in the expression of oxidation-antioxidation-related genes and proteins, including reduced phosphorylation levels of ERK and p38. Conclusion: The study indicates that SAB promotes hDPC proliferation and offers protection against oxidative stress, highlighting its potential as a therapeutic agent for enhancing hair growth and treating hair loss.

Enhancement of Zyxin Promotes Skin Fibrosis by Regulating FAK/PI3K/AKT and TGF-ß Signaling Pathways via Integrins.

Skin fibrosis is a common pathological manifestation in systemic sclerosis (SSc), keloid, and localized scleroderma (LS) characterized by fibroblast activation and excessive extracellular matrix (ECM) deposition. However, few effective drugs are available to treat skin fibrosis due to its unclear mechanisms. In our study, we reanalyzed skin RNA-sequencing data of Caucasian, African, and Hispanic SSc patients from the Gene Expression Omnibus (GEO) database. We found that the focal adhesion pathway was up-regulated and Zyxin appeared to be the primary focal adhesion protein involved in skin fibrosis, and we further verified its expression in Chinese skin tissues of several fibrotic diseases, including SSc, keloid, and LS. Moreover, we found Zyxin inhibition could significantly alleviate skin fibrosis using Zyxin knock-down and knock-out mice, nude mouse model and skin explants of human keloid. Double immunofluorescence staining showed that Zyxin was highly expressed in fibroblasts. Further analysis revealed pro-fibrotic gene expression and collagen production increased in Zyxin over-expressed fibroblasts, and decreased in Zyxin interfered SSc fibroblasts. In addition, transcriptome and cell culture analyses revealed Zyxin inhibition could effectively attenuate skin fibrosis by regulating the FAK/PI3K/AKT and TGF-ß signaling pathways via integrins. These results suggest Zyxin appears a potential new therapeutic target for skin fibrosis.

GRB2 serves as a viable target against skin fibrosis in systemic sclerosis by regulating endothelial cell apoptosis.

BACKGROUND: Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune system dysfunction, along with tissue fibrosis. Our previous study found GRB2 was downregulated by salvianolic acid B, a small molecule drug that attenuated skin fibrosis of SSc. OBJECTIVES: Here we aim to investigate the role of GRB2 in SSc. METHODS: The microarray data of SSc skin biopsies in Caucasians were obtained from the Gene Expression Omnibus (GEO) database. The expression of GRB2 was further detected in Chinese SSc and healthy controls. Bleomycin (BLM)-induced skin fibrosis mice were used to explore how GRB2 downregulation affected fibrosis. The apoptosis of EA.hy926 endothelial cells was induced by H2O2 and apoptosis ratio was measured by flow cytometric. Transcriptome and phosphoproteomic analyses were performed to explore the regulated pathway. RESULTS: The expression of GRB2 was significantly enhanced in SSc patient skin, 1.51-fold in Caucasians and 1.40-fold in Chinese. Double immunofluorescence staining showed the endothelial cells of SSc patient's skin highly expressed GRB2. The in vivo study revealed that GRB2 knockdown alleviated skin fibrosis and apoptosis of endothelial cells in BLM mouse skin. The in vitro study showed that GRB2 downregulation inhibited the apoptosis of EA.hy926 and protected them from H2O2-induced hyperpermeability. Moreover, transcriptome and phosphoproteomic analysis suggested the focal adhesion pathway was enriched in GRB2 siRNA transfected endothelial cells. CONCLUSIONS: Our results demonstrated GRB2 highly expressed in endothelial cells of SSc skin, and inhibiting GRB2 could effectively attenuate BLM-induced skin fibrosis and endothelial cell apoptosis. GRB2 is expected to be a new therapeutic target for SSc.

Risks of central lymph node metastasis in papillary thyroid carcinoma with or without multifocality in at least one lobe: A multi-center analysis.

PURPOSES: To quantitatively predict central lymph node metastasis (CLNM) risks by comparing the clinicopathological features of different multifocal manifestations in papillary thyroid carcinomas (PTC) patients. METHODS: A total of 998 PTC patients from three medical centers were retrospectively analyzed. RESULTS: PTC patients with multifocal lesions in at least one thyroid lobe (MF group) yielded significantly higher CLNM rates than those with unifocal lesions in one or both lobes (UF group). Multifocality in at least one lobe rather than bilateral presence was confirmed to be an independent risk factor of CLNM for PTC patients. Four (age, gender, maximum tumor diameter, and thyroid capsular invasion (TCI)) and three (age, gender, and TCI) factors were proven to be independent risk factors of CLNM for patients within UF and MF groups, respectively. Predictive nomograms were established for these patients based on their respective high-risk factors. The accuracy and validity of these newly-created models were verified using C-index and calibration curves. Patients within UF and MF groups possessing significantly different CLNM risks based on individualized nomogram risk scores were further classified into different subgroups. A detailed CLNM risk stratification flow chart covering all PTC patients was then established. CONCLUSION: A meticulous evaluating system that quantifiesCLNM risk for PTC patients with unifocal lesions in one or both lobes and multifocal lesions in at least one lobe was established.

Dominant-negative CK2alpha induces potent effects on circadian rhythmicity.

Circadian clocks organize the precise timing of cellular and behavioral events. In Drosophila, circadian clocks consist of negative feedback loops in which the clock component PERIOD (PER) represses its own transcription. PER phosphorylation is a critical step in timing the onset and termination of this feedback. The protein kinase CK2 has been linked to circadian timing, but the importance of this contribution is unclear; it is not certain where and when CK2 acts to regulate circadian rhythms. To determine its temporal and spatial functions, a dominant negative mutant of the catalytic alpha subunit, CK2alpha(Tik), was targeted to circadian neurons. Behaviorally, CK2alpha(Tik) induces severe period lengthening (approximately 33 h), greater than nearly all known circadian mutant alleles, and abolishes detectable free-running behavioral rhythmicity at high levels of expression. CK2alpha(Tik), when targeted to a subset of pacemaker neurons, generates period splitting, resulting in flies exhibiting both long and near 24-h periods. These behavioral effects are evident even when CK2alpha(Tik) expression is induced only during adulthood, implicating an acute role for CK2alpha function in circadian rhythms. CK2alpha(Tik) expression results in reduced PER phosphorylation, delayed nuclear entry, and dampened cycling with elevated trough levels of PER. Heightened trough levels of per transcript accompany increased protein levels, suggesting that CK2alpha(Tik) disturbs negative feedback of PER on its own transcription. Taken together, these in vivo data implicate a central role of CK2alpha function in timing PER negative feedback in adult circadian neurons.

The ion channel narrow abdomen is critical for neural output of the Drosophila circadian pacemaker.

Circadian clocks consist of transcriptional feedback loops housed in interdependent pacemaker neurons. Yet little is known about the neuronal output components essential for rhythmic behavior. Drosophila mutants of a putative ion channel, narrow abdomen (na), exhibit poor circadian rhythms and suppressed daylight activity. We find that NA is expressed in pacemaker neurons and induced expression within circadian neurons is sufficient to rescue these mutant phenotypes. Selective na rescue in distinct pacemaker neurons influences rhythmicity and timing of behavior. Oscillations of the clock protein PERIOD are intact in na mutants, indicating an output role. Pore residues are required for robust rescue consistent with NA action as an ion channel. In na mutants, expression of potassium currents and the key neuropeptide PDF are elevated, the latter consistent with reduced release. These data implicate NA and the pacemaker neural network in controlling phase and rhythmicity.

A G protein-coupled receptor, groom-of-PDF, is required for PDF neuron action in circadian behavior.

The neuropeptide Pigment-Dispersing Factor (PDF) plays a critical role in mediating circadian control of behavior in Drosophila. Here we identify mutants (groom-of-PDF; gop) that display phase-advanced evening activity and poor free-running rhythmicity, phenocopying pdf mutants. In gop mutants, a spontaneous retrotransposon disrupts a coding exon of a G protein-coupled receptor, CG13758. Disruption of the receptor is accompanied by phase-advanced oscillations of the core clock protein PERIOD. Moreover, effects on circadian timing induced by perturbation of PDF neurons require gop. Yet PDF oscillations themselves remain robust in gop mutants, suggesting that GOP acts downstream of PDF. gop is expressed most strongly in the dorsal brain in regions that lie in proximity to PDF-containing nerve terminals. Taken together, these studies implicate GOP as a PDF receptor in Drosophila.

TIMELESS is an important mediator of CK2 effects on circadian clock function in vivo.

Circadian oscillations in clock components are central to generation of self-sustained 24-h periodicity. In the Drosophila molecular clock, accumulation, phosphorylation, and degradation of PERIOD (PER) and TIMELESS (TIM) proteins govern period length. Yet little is known about the kinases that phosphorylate TIM in vivo. It has been shown previously that the protein kinase CK2 phosphorylates TIM in vitro. Here, we identify a role for CK2 in TIM regulation in vivo. Induction of a dominant-negative CK2alpha, CK2alpha(Tik) (Tik), increases TIM protein and tim transcript levels, reduces oscillation amplitude, and results in persistent cytoplasmic TIM localization. Exposure to light and subsequent TIM degradation results in an increase in the fraction of the transcriptional repressor PER that is nuclear and suppression of per and tim RNA levels. TIM protein, but not tim transcript, levels are elevated in Tik mutants in a per(01) background. In contrast, Tik effects on PER are undetectable in a tim(01) background, suggesting that TIM is required for CK2 effects on PER. To identify potential CK2 target sites, we assayed TIM phosphorylation rhythms in a deletion mutant that removes a conserved serine-rich domain and found that TIM protein does not show robust rhythmic changes in mobility by Western blotting, a hallmark of rhythmic phosphorylation. The period lengthening effects in Tik heterozygotes are reduced in a tim(UL) mutant that disrupts a putative CK2 phosphorylation site. Together, these data indicate that TIM is an important mediator of CK2 effects on circadian rhythms.

Clonal dissemination of the multi-drug resistant Salmonella enterica serovar Braenderup, but not the serovar Bareilly, of prevalent serogroup C1 Salmonella from Taiwan.

BACKGROUND: Nontyphoidal Salmonella is the main cause of human salmonellosis. In order to study the prevalent serogroups and serovars of clinical isolates in Taiwan, 8931 Salmonellae isolates were collected from 19 medical centers and district hospitals throughout the country from 2004 to 2007. The pulsed-field eletrophoresis types (PFGE) and antibiotic resistance profiles of Salmonella enterica serovars Bareilly (S. Bareilly) and Braenderup (S. Braenderup) were compared, and multi-drug resistance (MDR) plasmids were characterized. RESULTS: Over 95% of human salmonellosis in Taiwan was caused by five Salmonella serogroups: B, C1, C2-C3, D1, and E1. S. Typhymurium, S. Enteritidis, S. Stanley and S. Newport were the four most prevalent serovars, accounting for about 64% of isolates. While only one or two major serovars from four of the most prevalent serogroups were represented, four predominant serovars were found in serogroup C1 Salmonellae. The prevalence was decreasing for S. Choleraeuis and S. Braenderup, and S. Virchow and increasing for S. Bareilly. S. Braenderup mainly caused gastroenteritis in children; in contrast, S. Bareiley infected children and elderly people. Both serovars differed by XbaI-PFGE patterns. Almost all S. Bareilly isolates were susceptible to antibiotics of interest, while all lacked plasmids and belonged to one clone. Two distinct major clones in S. Braenderup were cluster A, mainly including MDR isolates with large MDR plasmid from North Taiwan, and cluster B, mainly containing susceptible isolates without R plasmid from South Taiwan. In cluster A, there were two types of conjugative R plasmids with sizes ranging from 75 to 130 kb. Type 1 plasmids consisted of replicons F1A/F1B, blaTEM, IS26, and a class 1 integron with the genes dfrA12-orfF-aadA2-qacEDelta1-sulI. Type 2 plasmids belonged to incompatibility group IncI, contained tnpA-blaCMY-2-blc-sugE genetic structures and lacked both IS26 and class 1 integrons. Although type 2 plasmids showed higher conjugation capability, type 1 plasmids were the predominant plasmid. CONCLUSIONS: Serogroups B, C1, C2-C3, D1, and E1 of Salmonella caused over 95% of human salmonellosis. Two prevalent serovars within serogroup C1, S. Bareilly and cluster B of S. Braenderup, were clonal and drug-susceptible. However, cluster A of S. Braenderup was MDR and probably derived from susceptible isolates by acquiring one of two distinct conjugative R plasmids.

Drosophila CK2 regulates lateral-inhibition during eye and bristle development.

Lateral inhibition is critical for cell fate determination and involves the functions of Notch (N) and its effectors, the Enhancer of Split Complex, E(spl)C repressors. Although E(spl) proteins mediate the repressive effects of N in diverse contexts, the role of phosphorylation was unclear. The studies we describe implicate a common role for the highly conserved Ser/Thr protein kinase CK2 during eye and bristle development. Compromising the functions of the catalytic (alpha) subunit of CK2 elicits a rough eye and defects in the interommatidial bristles (IOBs). These phenotypes are exacerbated by mutations in CK2 and suppressed by an increase in the dosage of this protein kinase. The appearance of the rough eye correlates, in time and space, to the specification and refinement of the 'founding' R8 photoreceptor. Consistent with this observation, compromising CK2 elicits supernumerary R8's at the posterior margin of the morphogenetic furrow (MF), a phenotype characteristic of loss of E(spl)C and impaired lateral inhibition. We also show that compromising CK2 elicits ectopic and split bristles. The former reflects the specification of excess bristle SOPs, while the latter suggests roles during asymmetric divisions that drive morphogenesis of this sensory organ. In addition, these phenotypes are exacerbated by mutations in CK2 or E(spl), indicating genetic interactions between these two loci. Given the centrality of E(spl) to the repressive effects of N, our studies suggest conserved roles for this protein kinase during lateral inhibition. Candidates for this regulation are the E(spl) repressors, the terminal effectors of this pathway.

In vivo circadian function of casein kinase 2 phosphorylation sites in Drosophila PERIOD.

Phosphorylation plays a key role in the precise timing of circadian clocks. Daily rhythms of phosphorylation of the Drosophila circadian clock component PERIOD (PER) were first described more than a decade ago, yet little is known about their phosphorylation sites and their function in circadian behavior. Here we show that serines 151 and 153 in PER are required for robust in vitro phosphorylation by the casein kinase 2 (CK2) holoenzyme, a cytoplasmic kinase shown to be involved in circadian rhythms. Mutation of these sites in transgenic flies results in significant period lengthening of behavioral rhythms, altered PER rhythms, and delayed PER nuclear localization in circadian pacemaker neurons. In many respects, mutation of these phosphorylation sites phenocopies mutation of the catalytic subunit of CK2. We propose that CK2 phosphorylation at these sites triggers PER nuclear localization.

Prospective diagnostic accuracy evaluation and clinical utilization of a modified assay for platelet-associated immunoglobulin in thrombocytopenic and nonthrombocytopenic dogs.

BACKGROUND: No diagnostic tests reliably distinguish primary immune-mediated thrombocytopenia (pIMT) from other causes of thrombocytopenia. OBJECTIVES: The purpose of the study was to evaluate diagnostic sensitivity and specificity using modified direct and indirect platelet-associated immunoglobulin (PAIg) assays and reticulated platelets (RP) by flow cytometry for the classification of thrombocytopenic dogs and differentiating pIMT. METHODS: Platelets were isolated from plasma samples of thrombocytopenic dogs and nonthrombocytopenic healthy and ill dogs. For direct PAIg, they were analyzed by flow cytometry after incubation with anti-human amylase fluorescein isothiocyanate (FITC, negative control), anti-canine IgG-FITC, anti-canine IgM-FITC, and anti-human CD61-conjugated fluorochrome (AF647). For indirect PAIg, platelets from normothrombocytic dogs were incubated with thrombocytopenic dog plasma and analyzed similar to direct PAIg. RP percentages were determined based on forward light scatter vs thiazole orange fluorescence. RESULTS: Seventy-five thrombocytopenic dogs, 16 nonthrombocytopenic ill dogs, and 24 healthy dogs were evaluated. Diagnostic sensitivity and specificity utilizing direct IgG was 29.4% and 75.9%, respectively; when combining direct/indirect assays (IgG/IgM), it was 76.5% and 65.5%, respectively, for distinguishing pIMT. For RP, no significant difference between pIMT and sIMT was noted. RP > 8% with positive PAIg had a sensitivity of 94% and specificity of 27.6% for distinguishing pIMT. There was a significant difference in platelet concentration and CD61% staining between control and pIMT. CONCLUSIONS: The combined modified assays resulted in fair diagnostic sensitivity and specificity for the diagnosis of pIMT. The modification of the immunoglobulin assays improved diagnostic accuracy; however, a single panel to accurately classify thrombocytopenia remains elusive.

Diagnostic utility of CD4%:CD8 low% T-lymphocyte ratio to differentiate feline immunodeficiency virus (FIV)-infected from FIV-vaccinated cats.

Antibody testing based on individual risk assessments is recommended to determine feline immunodeficiency virus (FIV) status, but ELISA and Western blot tests cannot distinguish between anti-FIV antibodies produced in response to natural infection and those produced in response to FIV vaccination. The aim of this cross-sectional study was to test the hypothesis that FIV-infected cats could be differentiated from FIV-vaccinated uninfected cats using lymphocyte subset results, specifically the CD4%:CD8(low)% T-lymphocyte ratio. Comparisons of the CD4%:CD8(low)% T-lymphocyte ratio were made among the following four groups: Group 1 - FIV-infected cats (n=61; FIV-antibody positive by ELISA and FIV PCR positive); Group 2 - FIV-uninfected cats (n=96; FIV-antibody negative by ELISA); Group 3 - FIV-vaccinated uninfected cats (n=31; FIV-antibody negative by ELISA before being vaccinated against FIV, after which they tested FIV ELISA positive); and Group 4 - FIV-uninfected but under chronic/active antigenic stimulation (n=16; FIV-antibody negative by ELISA; all had active clinical signs of either upper respiratory tract disease or gingival disease for ≥ 21 days). The median CD4%:CD8(low)% T-lymphocyte ratio was lower in Group 1 (1.39) than in each of the other three groups (Group 2 - 9.77, Group 3 - 9.72, Group 4 - 5.64; P<0.05). The CD4%:CD8(low)% T-lymphocyte ratio was also the most effective discriminator between FIV-infected cats and the other three groups, and areas under ROC curves ranged from 0.91 (compared with Group 4) to 0.96 (compared with Group 3). CD4%:CD8(low)% shows promise as an effective test to differentiate between FIV-infected cats and FIV-vaccinated uninfected cats.

Fluorescence flow cytometry methodology to exclude platelet aggregate interference when measuring feline CD4 and CD8 lymphocyte counts.

Changes in individual feline lymphocyte subsets over the course of infection, immune-mediated disease, or treatment can be used clinically to monitor disease progression. However, interference by platelet aggregates is a common problem when measuring feline lymphocyte subtype counts using flow cytometry in whole blood specimens. In this study, buffer was used to lyse red blood cells so that lymphocytes could be isolated, and then a gate containing a highly purified population of lymphocytes was characterized and fixed using fluorescence flow cytometry analysis. After tagging platelets with anti-CD61AF647 antibody to reduce aggregate interference, lymphocyte subtypes were measured using simultaneous 3-color channels with fluorescent anti-CD markers. When CD61AF647 exclusion of platelet aggregates was used, CD4%, CD8%, CD8low% and CD4:CD8 counts increased significantly (all specimens, n=66, P<0.001; >20% CD61 in the fixed gate, n=21, P<0.01). The methodology showed robust stability and precision over 3 days (n=10 specimens), yielding average day-to-day coefficients of variation (CVs) of 2.15%, 5.01%, 7.33%, 7.77% and 9.35% for white blood cell (WBC) counts, lymphocyte counts, CD4 lymphocyte counts, CD8 lymphocyte counts and CD4:CD8, respectively.

Effects of Vitreoscilla hemoglobin on the 2,4-dinitrotoluene (2,4-DNT) dioxygenase activity of Burkholderia and on 2,4-DNT degradation in two-phase bioreactors.

Expression of vgb, encoding Vitreoscilla hemoglobin (VHb), in Burkholderia strain YV1 was previously shown to improve cell growth and enhance 2,4-dinitrotoluene (2,4-DNT) degradation compared with control strain DNT, especially under hypoxic conditions. In the work reported here, the ratio of 2,4-DNT degraded to oxygen uptake was approximately 5-fold larger for strain YV1 than for strain DNT. The addition of purified VHb to cytosolic fractions of strain DNT increased 2,4-DNT degradation 1.5-fold, compared with 1.1-fold for control bovine Hb, but increased the 2,4-DNT degradation 2.7-fold when added to partially purified 2,4-DNT dioxygenase, compared with 1.3-fold for bovine Hb. This suggests a direct transfer of oxygen from VHb to the oxygenase. In a bioreactor at high 2,4-DNT concentration (using 100 ml oleyl alcohol containing 2 g 2,4-DNT as the second phase) with 1.5 l culture, both strains could remove 0.8 g 2,4-DNT by 120 h; and, under the same conditions in a fed-batch reactor, the degradation increased to 1 g for strain YV1 but not for strain DNT.

A role for casein kinase 2alpha in the Drosophila circadian clock.

Circadian clocks drive rhythmic behaviour in animals and are regulated by transcriptional feedback loops. For example, the Drosophila proteins Clock (Clk) and Cycle (Cyc) activate transcription of period (per) and timeless (tim). Per and Tim then associate, translocate to the nucleus, and repress the activity of Clk and Cyc. However, post-translational modifications are also critical to proper timing. Per and Tim undergo rhythmic changes in phosphorylation, and evidence supports roles for two kinases in this process: Doubletime (Dbt) phosphorylates Per, whereas Shaggy (Sgg) phosphorylates Tim. Yet Sgg and Dbt often require a phosphoserine in their target site, and analysis of Per phosphorylation in dbt mutants suggests a role for other kinases. Here we show that the catalytic subunit of Drosophila casein kinase 2 (CK2alpha) is expressed predominantly in the cytoplasm of key circadian pacemaker neurons. CK2alpha mutant flies show lengthened circadian period, decreased CK2 activity, and delayed nuclear entry of Per. These effects are probably direct, as CK2alpha specifically phosphorylates Per in vitro. We propose that CK2 is an evolutionary link between the divergent circadian systems of animals, plants and fungi.