A rapid host-protein test for differentiating bacterial from viral infection: Apollo diagnostic accuracy study.

Objectives: To determine the diagnostic accuracy of a rapid host-protein test for differentiating bacterial from viral infections in patients who presented to the emergency department (ED) or urgent care center (UCC). Methods: This was a prospective multicenter, blinded study. MeMed BV (MMBV), a test based on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), interferon gamma-inducible protein-10 (IP-10), and C-reactive protein (CRP), was measured using a rapid measurement platform. Patients were enrolled from 9 EDs and 3 UCCs in the United States and Israel. Patients >3 months of age presenting with fever and clinical suspicion of acute infection were considered eligible. MMBV results were not provided to the treating clinician. MMBV results (bacterial/viral/equivocal) were compared against a reference standard method for classification of infection etiology determined by expert panel adjudication. Experts were blinded to MMBV results. They were provided with comprehensive patient data, including laboratory, microbiological, radiological and follow-up. Results: Of 563 adults and children enrolled, 476 comprised the study population (314 adults, 162 children). The predominant clinical syndrome was respiratory tract infection (60.5% upper, 11.3% lower). MMBV demonstrated sensitivity of 90.0% (95% confidence interval [CI]: 80.3-99.7), specificity of 92.8% (90.0%-95.5%), and negative predictive value of 98.8% (96.8%-99.6%) for bacterial infections. Only 7.2% of cases yielded equivocal MMBV scores. Area under the curve for MMBV was 0.95 (0.90-0.99). Conclusions: MMBV had a high sensitivity and specificity relative to reference standard for differentiating bacterial from viral infections. Future implementation of MMBV for patients with suspected acute infections could potentially aid with appropriate antibiotic decision-making.

Associations of High-Sensitive Cardiac Troponin T in Healthy Newborns and Prolonged Second Stage of Labor, Neonatal and Maternal Factors: A Prospective Study.

INTRODUCTION: High-sensitivity cardiac troponin T (hs-cTnT) is not used routinely as a diagnostic biomarker in newborns. The high precision of hs-cTnT assays increases the ability to determine small differences in cTnT over time and to detect troponin T elevation; thus, we believe that hs-cTnT assays might improve clinical care. We explored the plausible association between hs-cTnT levels (ng/L) in healthy newborns and prolonged second stage of labor, neonatal, and maternal factors. METHODS: A prospective study was performed among healthy newborns in the Obstetrics and Gynecology Department at Hillel Yaffe Medical Center in Israel in January-June 2021. The sociodemographic characteristics of the participants, maternal age, gravidity, parity, Pitocin use, epidural analgesia, and neonatal anemia were obtained from the electronic medical records. Gestational age was determined by ultrasound biometric measurements. We classified second-stage labor as normal or prolonged using the WHO guidelines. Samples from umbilical cord blood were drawn using syringes rinsed with anticoagulant by a specialist in pediatrics. The remaining blood was used to determine hs-cTnT levels (ng/L), which was defined as a continuous quantitative variable with the median value and the 25th-75th percentiles. RESULTS: Overall, 184 cord blood samples were performed from healthy newborns (60.6% males) with a median hs-cTnT of 39.03 (25th-75th percentiles = 30.53-54.09) ng/L. A multivariable linear regression model showed no significant association between neonatal anemia and hs-cTnT levels (ng/L) (p = 0.8). Gestational age (B coefficient -4.24, p < 0.001) and gravidity (B coefficient -2.41, p = 0.03) were negatively associated with hs-cTnT levels (ng/L), while Pitocin use (B coefficient 6.91, p = 0.04) and prolonged second stage of labor (B coefficient 18.07, p = 0.02) were positively associated with hs-cTnT levels (ng/L). CONCLUSIONS: High hs-cTnT levels (ng/L) were documented in the cord blood of healthy newborns. Hs-cTnT levels were positively correlated with a prolonged second stage of labor and Pitocin use and negatively correlated with longer gestational age and higher gravidity. Hs-cTnT may signify labor-related fetal distress. A larger surveillance study is mandatory to establish this correlation and assess for possible prognostic significance of elevated hs-cTnT in this context.

Diagnostic Accuracy of a Real-Time Host-Protein Test for Infection.

OBJECTIVE: Determining infection etiology can be difficult because viral and bacterial diseases often manifest similarly. A host protein test that computationally integrates the circulating levels of TNF-related apoptosis-induced ligand, interferon γ-induced protein-10, and C-reactive protein to differentiate between bacterial and viral infection (called MMBV) demonstrated high performance in multiple prospective clinical validation studies. Here, MMBV's diagnostic accuracy is evaluated in febrile children for whom physicians were uncertain about etiology when applied at the physician's discretion. METHODS: Patients aged 3 months to 18 years were retrospectively recruited (NCT03075111; SPIRIT study; 2014-2017). Emergency department physician's etiological suspicion and certainty level were recorded in a questionnaire at blood-draw. MMBV results are based on predefined score thresholds: viral/non-bacterial etiology (0 ≤ score <35), equivocal (35 ≤ score ≤65), and bacterial or coinfection (65 < score ≤100). Reference standard etiology (bacterial/viral/indeterminate) was adjudicated by 3 independent experts based on all available patient data. Experts were blinded to MMBV. MMBV and physician's etiological suspicion were assessed against the reference standard. RESULTS: Of 3003 potentially eligible patients, the physicians were uncertain about infection etiology for 736 of the cases assigned a reference standard (128 bacterial, 608 viral). MMBV performed with sensitivity 89.7% (96/107; 95% confidence interval 82.4-94.3) and specificity 92.6% (498/538; 95% confidence interval 90.0-94.5), significantly outperforming physician's etiological suspicion (sensitivity 49/74 = 66.2%, specificity 265/368 = 72.0%; P < .0001). MMBV equivocal rate was 12.4% (91/736). CONCLUSIONS: MMBV was more accurate in determining etiology compared with physician's suspicion and had high sensitivity and specificity according to the reference standard.

The Diagnostic Value of Human Neutrophilic Peptides 1-3 in Acute Pediatric Febrile Illness.

Background: It is prudent to develop biomarkers that enhance the differentiation between viral and bacterial infection in order to support expeditious and judicious antimicrobial implementation in emergency department admissions. Human neutrophilic peptides 1-3 (HNP1-3) are the major neutrophilic peptides with potent antimicrobial activity. Methods: We tested the performance of the plasma HNP1-3 test in a prospective observational cohort of children admitted to the emergency department for fever. We validated this test with traditionally used biomarkers and final diagnoses. An expert panel reviewed the patient's data and gave a final diagnosis. The final diagnosis was classified as definite, probable, or possible. Results: A total of 111 children (98 with fever and 13 control) were recruited: 55% male, mean age 6.3 years. Plasma HNP1-3 levels were higher with bacterial infections: 10,428 (5789-14,866) vs. 7352 (3762-10,672) pg/mL, p = 0.007. HNP1-3 were negatively correlated with age: r = -0.207, p = 0.029. Of the different categorical variables tested, only c-reactive protein (CRP) (≥42.3 mg/dL), neutrophil count (≥10.2), and age (odds ratio = 1.185, p = 0.013 and 95%CI = 1.037-1.354) had significant diagnostic capability for bacterial disease prediction. Conclusions: Due to its low diagnostic value in febrile patients, the HNP1-3 value is not currently recommended to support pathogen differentiation in children in an emergency setting. Further studies are needed to support its clinical use.

An immune-protein score combining TRAIL, IP-10 and CRP for predicting severe COVID-19 disease.

COVID-19 patients are oftentimes over- or under-treated due to a deficit in predictive management tools. This study reports derivation of an algorithm that integrates the host levels of TRAIL, IP-10, and CRP into a single numeric score that is an early indicator of severe outcome for COVID-19 patients and can identify patients at-risk to deteriorate. 394 COVID-19 patients were eligible; 29% meeting a severe outcome (intensive care unit admission/non-invasive or invasive ventilation/death). The score's area under the receiver operating characteristic curve (AUC) was 0.86, superior to IL-6 (AUC 0.77; p = 0.033) and CRP (AUC 0.78; p < 0.001). Likelihood of severe outcome increased significantly (p < 0.001) with higher scores. The score differentiated severe patients who further deteriorated from those who improved (p = 0.004) and projected 14-day survival probabilities (p < 0.001). The score accurately predicted COVID-19 patients at-risk for severe outcome, and therefore has potential to facilitate timely care escalation and de-escalation and appropriate resource allocation.

Comparison between School-Age Children with and without Obesity in Nutritional and Inflammation Biomarkers.

Childhood obesity is a major health problem. We examined differences between children with obesity and normal weight in nutritional and inflammation biomarkers. A cross-sectional study was conducted among healthy children aged 10-12 years from Arab villages in Israel. Parents were interviewed regarding sociodemographic and children's health status. Body weight and height measurements were performed and weight categories were defined using the 2007 WHO growth curves. Blood samples were tested for complete blood count, levels of iron, ferritin, lipids, uric acid, and C-reactive protein (CRP). Overall, 146 children (59.0% males, mean age = 11.3 [SD = 0.5]) were enrolled. In total 43.8%, 14.1% and 42.3% of the participants had normal weight, overweight and obesity, respectively. A multivariable logistic regression model showed that children with overweight and obesity had lower iron, and HDL-C levels than children with normal weight. Levels of CRP, uric acid, LDL-C and lymphocytes were higher among children with overweight and obesity. In conclusion, our findings highlight the worse metabolic and nutritional status in overweight and obese children. Such markers play a role in metabolic syndrome, thus suggesting that metabolic syndrome might start in childhood.

BV score differentiates viral from bacterial-viral co-infection in adenovirus PCR positive children.

Background and objectives: Adenovirus causes acute respiratory illness that can mimic bacterial infection, making it challenging to differentiate adenoviral infection from adenoviral-bacterial co-infection. A host-protein score (BV score) for differentiating bacterial from viral infection that combines the expression levels of TNF-related apoptosis-induced ligand, interferon gamma-induced protein-10, and C-reactive protein exhibited a negative predictive value (NPV) of 98% in prior studies. Here we evaluate BV score's diagnostic accuracy in pediatrics with adenovirus PCR detection. Methods: This is a sub-analysis of children aged 3 months to 20 years with adenovirus PCR-positive infection recruited prospectively in two previous cohort studies. Reference standard diagnosis (bacterial, viral or indeterminate) was based on expert adjudication. BV score ranges from 0 to 100 and provides three results based on predefined cutoffs: viral or other non-bacterial etiology (0 ≤ score < 35), equivocal (35 ≤ score ≤ 65), and bacterial or co-infection (65 < score ≤ 100). Experts were blinded to BV results. Results: Out of 1,779 children, 142 had an adenovirus PCR-positive nasopharyngeal swab. Median age was 1.2 years (interquartile range 0.6-1.8), 50.7% were male and 52.8% were hospitalized. 12 cases were reference standard bacterial, 115 reference standard viral and 15 were indeterminate. BV score attained sensitivity of 100.0% (no false negatives), specificity of 89.5% (95% confidence interval: 83.2-95.8), and NPV of 100.0% (92.6-100.0). Equivocal rate was 19.7%. Conclusions: BV score accurately differentiated between adenoviral and bacterial-adenoviral co-infection in this cohort of children with PCR-positive adenovirus detection. This performance supports a potential to improve appropriate antibiotic use.

Does mRNA SARS-CoV-2 vaccine in the follicular fluid impact follicle and oocyte performance in IVF treatments?

PROBLEM: The COVID-19 pandemic has many clinical manifestations. Rapid vaccine development raised concerns and speculations about future fertility outcomes and vaccine safety. We evaluated the effect of Pfizer-BioNTech mRNA SARS-CoV-2 vaccine on IVF treatment, oocyte and embryo quality, and pregnancy outcomes. METHOD OF STUDY: This prospective, observational cohort study was conducted in a referral IVF Unit, 3/2021-5/2021. We aimed to recruit all women undergoing IVF/ICSI cycles from 3/1-4/30/2021, 2-8 weeks after the second vaccination, and to analyze 50-60 samples in the 2-month period. Patients were categorized according to serum antibody levels: positive for spike (S), positive for nucleotide (N), or negative for both. On the day of ovum pick-up, follicular fluid and blood samples were analyzed for anti-nucleotide (anti-N) antibodies, and anti-spike (anti-S) antibodies, hormonal profile, C-reactive protein (CRP) and other metabolic parameters. RESULTS: Of 59 women enrolled, 37 reported being vaccinated and 22 were not. We found 97% correlation between anti-S and anti-N in the blood and the follicular fluid. Follicular fluid was analyzed based on antibody categorization. All IVF treatment parameters in the follicular fluids and serum were comparable, except CRP was significantly elevated among patients with anti-N antibodies (2.29 [1.42-6.08] vs. 4.11 [1.62-5.75] vs. 1.44 [.36-8.33]; p < .001). Pregnancy outcomes were comparable (44% vs. 33% vs. 50%; p = .97). CONCLUSION: mRNA SARS-CoV-2 vaccine did not appear to affect treatment outcomes or ovarian reserves in the subsequent IVF cycle.

Rapid identification of uropathogens by combining Alfred 60 system with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry technology.

Rapid identification of uropathogens is needed to determine appropriate antimicrobial therapy. This study evaluated performance of the Alfred 60 system combined with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) technology for rapid identification of uropathogens. The Alfred 60 system was used to screen urine cultures, followed by identifying the microbial pathogen in positive cultures using MALDI-TOF MS. The Alfred 60 detected positive cultures by measuring the turbidity of urine samples, which were transferred automatically to vials containing liquid medium and incubated for 3.5 h at 35 °C in the Alfred 60 system. Vials that showed growth were removed and centrifuged. The pellet was subjected to MALDI-TOF MS identification. In parallel, positive urine samples were inoculated onto agar plates for identification by conventional culture. The time required to detect positive urine cultures with Alfred 60 and identify the uropathogens with MALDI-TOF MS ranged from 15 min to 3.5 h. Among 146 positive urine samples tested, conventional cultures showed three culture groups: group 1 included 101 samples with growth of a single type of microorganism; group 2 included 34 samples with 2 types of microorganisms; and group 3 included 11 samples with ≥ 3 types of microorganisms. Direct identification by MALDI-TOF MS was concordant with 95% of the samples in group 1, 100% of the principal microorganism in group 2, but could not identify microorganisms in group 3. This combination of methods provides rapid, reliable microbial identification for most positive urine cultures.

Serum separator tube method for matrix-assisted laser desorption/ionization time-of-flight analysis.

BACKGROUND: Without appropriate treatment, bloodstream infections have a high mortality rate. Quicker identification of the microbial pathogen allows the clinician to develop an initial strategy of antimicrobial therapy. Sample preparation protocols for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS; Bruker Daltonics for Microflex LT spectrometer) technology were evaluated in an attempt to identify pathogens directly from positive blood culture bottles and thus shorten the time to identify them. This application requires preparatory processing because blood culture bottles contain undesirable proteins. This study aimed to evaluate two methods for microbial preparation for identification by MALDI-ToF MS. METHODS: This study evaluated two methods for microbial preparation from 200 positive blood culture samples, half prepared by the differential centrifugation method and half with the serum separator tube method for identification by MALDI-ToF MS. Both methods were compared to conventional methods such as VITEK II and ChromAgar culture plates. RESULTS: All Gram-negative bacteria tested were identified correctly by MALDI-ToF MS compared to conventional methods, regardless of the preparation method. However, more Gram-positive bacteria were identified when the serum separator tube method was used (83.3%) compared with the differential centrifugation method (65.3  %). Moreover, the serum separator tube protocol requires 12-15 min, while the differential centrifugation protocol requires 30-45 min. CONCLUSIONS: Sample preparation using the serum separator tube method is easy to perform, fast and reliable for accurate microbial identification by MALDI-ToF MS technology.

Automating a new host-protein assay for differentiating bacterial from viral infection to reduce operator hands-on time.

Distinguishing bacterial from viral infections is often challenging, leading to antibiotic misuse, and detrimental ramifications for the patient, the healthcare system and society. A novel ELISA-based assay that integrates the circulating levels of three host-response proteins (TRAIL, IP-10 and CRP) was developed to assist in differentiation between bacterial and viral etiologies. We developed a new protocol for measuring the host-based assay biomarkers using an automated ELISA workstation. The automated protocol was validated and was able to reduce technician hands-on time by 76%, while maintaining high analytical performance. Following automation, the assay has been incorporated into the routine workflow at a pediatric department, and is performed daily on admitted and emergency department patients. The automation protocol reduces the overall burden on the hospital laboratory performing the assay. This benefit has potential to promote adoption of the host-based assay, facilitating timely triage of febrile patients and prudent use of antibiotics.

Correction to: The mTOR inhibitor rapamycin down-regulates the expression of the ubiquitin ligase subunit Skp2 in breast cancer cells.

After the publication of this work [1], an error was noticed in Fig. 2b, Fig. 3a and Fig. 5b. The Skp1 loading control was accidentally duplicated. We apologize for this error, which did not affect any of the interpretations or conclusions of the article.

Thrombin is a selective inducer of heparanase release from platelets and granulocytes via protease-activated receptor-1.

Heparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.

Regulation of APC/C (Cdh1) ubiquitin ligase in differentiation of human embryonic stem cells.

We have recently shown that Skp2 levels are high in undifferentiated human embryonic stem cells, but decline rapidly following induction of differentiation, thereby leading to accumulation of p27. Changes in Skp2 levels were found to be caused mainly by its rate of degradation. Here we show that the activity of APC/C (Cdh1), the ubiquitin ligase that targets Skp2 for degradation, increases markedly during the differentiation process of human embryonic stem cells. APC/C (Cdh1) is present but inactive in undifferentiated embryonic stem cells and becomes active in the differentiated state. The rise in APC/C (Cdh1) activity with differentiation appears to be due, at least in part, to a dramatic decline in the levels of its inhibitor Emi1. In addition, protein kinase activity also appears to contribute to the suppression of APC/C (Cdh1) activity in undifferentiated stem cells, possibly by inhibitory phosphorylation of Cdh1.

Over-expression of Skp2 is associated with resistance to preoperative doxorubicin-based chemotherapy in primary breast cancer.

INTRODUCTION: Preoperative chemotherapy is often used in patients with locally advanced breast cancer. However, commonly used clinical and pathological parameters are poor predictors of response to this type of therapy. Recent studies have suggested that altered regulation of the cell cycle in cancer may be involved in resistance to chemotherapy. Over-expression of the ubiquitin ligase Skp2 results in loss of the cell cycle inhibitor p27Kip1 and is associated with poor prognosis in early breast cancer. The purpose of the present study was to examine the role of these proteins as predictors of clinical outcome and response to chemotherapy in locally advanced breast cancer. METHODS: The expression levels of Skp2 and p27Kip1 were determined by immunohistochemistry both before and after preoperative chemotherapy in 40 patients with locally advanced breast cancer. All patients were treated with cyclophosphamide/doxorubicin (adriamycin)/5-fluorouracil (CAF) and some patients received additional treatment with docetaxel. Expression data were compared with patients' clinical and pathological features, clinical outcome, and response to chemotherapy. RESULTS: Skp2 expression before preoperative chemotherapy was inversely related to p27Kip1 levels, tumor grade, and expression of estrogen and progesterone receptors. Both Skp2 and p27Kip1 were found to be accurate prognostic markers for disease-free and overall survival. High preoperative expression of Skp2 was associated with resistance to CAF therapy in 94% of patients (P < 0.0001) but not with resistance to docetaxel. CONCLUSION: Skp2 expression may be a useful marker for predicting response to doxorubicin-based preoperative chemotherapy and clinical outcome in patients with locally advanced breast cancer.

Agonist-specific down regulation of mu-opioid receptors: Different cellular pathways are activated by different opioid agonists.

Opioid agonists are known to induce down regulation of opioid receptors through the classical pathway that involves phosphorylation, clathrin-dependent endocytosis and lysosomal/endosomal degradation of the internalized receptors. As expected, exposure of mu-opioid receptor (MOR)-transfected HEK-293 cells to either DAMGO (a specific mu-opioid agonist) or etorphine (a wide spectrum opioid agonist) resulted in down regulation of the receptors that was blocked by the kinase inhibitor staurosporine, by hypertonic sucrose and by the lysosomal and proteasomal inhibitors chloroquine and lactacystin. High concentration of etorphine, but not of DAMGO, induced an additional process of down regulation that was resistant to staurosporine, to hypertonic sucrose and to chloroquine-lactacystin. Etorphine, but not DAMGO, also induced down regulation of mu-opioid receptors in isolated membranes of HEK cells. This membrane-delimited down regulation was blocked by selective inhibitors of protease enzymes, suggesting the involvement of membranous serine- and amino-peptidases. This membranous down regulation of opioid receptors was dependent on the concentration of etorphine and was blocked by the opioid antagonist naloxone. Etorphine induced similar down regulation in membranes of HEK-293 cells transfected with delta-opioid receptors (DOR) as well in membranes of cells that endogenously express opioid receptors. This agonist-specific membrane-delimited regulatory process appears to be physiologically relevant and should be taken into account when studying long term effects of opioid drugs.

Differential effects of doxorubicin treatment on cell cycle arrest and Skp2 expression in breast cancer cells.

Overexpression of Skp2, the ubiquitin ligase subunit that targets p27 for degradation, is often observed in cancers, and is associated with aggressive tumor proliferation and poor prognosis. As there is no drug at present that specifically targets Skp2, studies were undertaken to examine the effects of commonly used drugs on Skp2 regulation. Doxorubicin is among the most effective antitumor agents used for the management of breast cancer, but its effect on Skp2 expression is unknown. The objective of this study was to examine the effect of doxorubicin on Skp2 expression regulation in breast cancer cell lines. The expression of Skp2 mRNA and the protein levels of Skp2, p27, p21 and cyclin B were examined in doxorubicin-treated MCF-7 and MDA-MB-231 breast cancer cells. The effect of doxorubicin on the cell cycle profile was assessed by fluorescence-activated cell sorting analysis. Doxorubicin decreased Skp2 mRNA and protein levels in MCF-7 cells, but had the opposite effect in MDA-MB-231 cells. p27 levels were slightly decreased, whereas p53 and p21 levels were significantly upregulated in doxorubicin-treated MCF-7 cells. In contrast, p27 levels were unaffected by doxorubicin treatment in MDA-MB-231 cells, but cyclin B levels were markedly increased. Doxorubicin arrested MCF-7 cells at G1/S and G2/M checkpoints, whereas MDA-MB-231 cells were arrested at G2/M only. The differential effects of doxorubicin on Skp2 expression in breast cancer cells depend upon the specific cell cycle checkpoints activated by the drug. These changes induced by doxorubicin, however, do not significantly affect p27 expression in these cell lines, suggesting that the potential of a given drug to alter p27 expression through Skp2 modulation might depend on its specific action on cell cycle arrest.

Regulation of the cell cycle inhibitor p27 and its ubiquitin ligase Skp2 in differentiation of human embryonic stem cells.

Embryonic stem cells combine the features of robust proliferation with precise differentiation capacity. p27 is a cell cycle inhibitor that is involved in the regulation of proliferation and differentiation in many developing tissues. Recent studies in murine embryonic stem cells have suggested that p27 is involved in the progression of normal differentiation programs in these cells. However, the expression and regulation of p27 and its role in the differentiation of human embryonic stem cells (hESc) has not been previously explored. Herein we show that p27 expression was low in undifferentiated hESc, but increased markedly in differentiated cells. The expression of Skp2, the ubiquitin ligase that targets p27 for degradation, was inversely related to p27 expression. Moreover, embryoid bodies (EBs) with low p27 expression and high Skp2/p27 ratio showed poorer differentiation than those with high p27 expression. Modulation of Skp2 expression is mainly regulated by its rate of degradation. In contrast to somatic cells, which have high levels of Skp2 mainly in S and G2/M, in undifferentiated hESc Skp2 levels were also high in G1. These results point to a potentially important role for p27 regulation in hESc.