Role for Wee1 in inhibition of G2-to-M transition through the cooperation of distinct human papillomavirus type 1 E4 proteins

The infectious cycle of human papillomavirus type 1 (HPV1) is accompanied by abundant expression of the full-length E1;E4 protein (17-kDa) and smaller E4 polypeptides (16-, 11-, and 10-kDa) that arise by sequential loss of N-terminal E1;E4 sequences. HPV1 E4 inhibits G(2)-to-M transition of the cell cycle. Here, we show that HPV1 E4 proteins mediate inhibition of cell division by more than one mechanism. Cells arrested by coexpression of E1;E4 (E4-17K) and a truncated protein equivalent to the 16-kDa species (E4-16K) contain inactive cyclin B1-cdk1 complexes. Inactivation of cdk1 is through inhibitory Tyr(15) phosphorylation, with cells containing elevated levels of Wee1, the kinase responsible for inhibitory cdk1 phosphorylation. Consistent with these findings, overexpression of Wee1 enhanced the extent to which E4-17K/16K-expressing cells arrest in G(2), indicating that maintenance of Wee1 activity is necessary for inhibition of cell division induced by coexpression of the two E4 proteins. Moreover, we have determined that depletion of Wee1 by small interfering RNA (siRNA) alleviates the G(2) block imposed by E4-17K/16K. In contrast however, maintenance of Wee1 activity is not necessary for G(2)-to-M inhibition mediated by E4-16K alone, as overexpression or depletion of Wee1 does not influence the G(2) arrest function of E4-16K. Cells arrested by E4-16K expression contain low levels of active cyclin B1-cdk1 complexes. We hypothesize that differential expression of HPV1 E4 proteins during the viral life cycle determines the host cell cycle status. Different mechanisms of inhibition of G(2)-to-M transition reinforce the supposition that distinct E4 functions are important for HPV replication.     

CXCL1: A new diagnostic biomarker for human tuberculosis discovered using Diversity Outbred mice

More humans have died of tuberculosis (TB) than any other infectious disease and millions still die each year. Experts advocate for blood-based, serum protein biomarkers to help diagnose TB, which afflicts millions of people in high-burden countries. However, the protein biomarker pipeline is small. Here, we used the Diversity Outbred (DO) mouse population to address this gap, identifying five protein biomarker candidates. One protein biomarker, serum CXCL1, met the World Health Organization’s Targeted Product Profile for a triage test to diagnose active TB from latent M.tb infection (LTBI), non-TB lung disease, and normal sera in HIV-negative, adults from South Africa and Vietnam. To find the biomarker candidates, we quantified seven immune cytokines and four inflammatory proteins corresponding to highly expressed genes unique to progressor DO mice. Next, we applied statistical and machine learning methods to the data, i.e., 11 proteins in lungs from 453 infected and 29 non-infected mice. After searching all combinations of five algorithms and 239 protein subsets, validating, and testing the findings on independent data, two combinations accurately diagnosed progressor DO mice: Logistic Regression using MMP8; and Gradient Tree Boosting using a panel of 4: CXCL1, CXCL2, TNF, IL-10. Of those five protein biomarker candidates, two (MMP8 and CXCL1) were crucial for classifying DO mice; were above the limit of detection in most human serum samples; and had not been widely assessed for diagnostic performance in humans before. In patient sera, CXCL1 exceeded the triage diagnostic test criteria (>90% sensitivity; >70% specificity), while MMP8 did not. Using Area Under the Curve analyses, CXCL1 averaged 94.5% sensitivity and 88.8% specificity for active pulmonary TB (ATB) vs LTBI; 90.9% sensitivity and 71.4% specificity for ATB vs non-TB; and 100.0% sensitivity and 98.4% specificity for ATB vs normal sera. Our findings overall show that the DO mouse population can discover diagnostic-quality, serum protein biomarkers of human TB.     

Biodegradation of N-nitrosodimethylamine in Soil from a Water Reclamation Facility

The potential introduction of N-nitrosodimethylamine (NDMA) into groundwater during water reclamation activities poses a significant risk to groundwater drinking supplies. Greater than 54% biodegradation of N-[methyl-¹⁴C]NDMA to ¹⁴CO₂ or to ¹⁴CO₂ and ¹⁴CH₄ was observed in soil from a water reclamation facility under oxic or anoxic conditions, respectively. Likewise, biodegradation was significant in microcosms containing soil with no history of NDMA contamination. These results indicate that aerobic and anaerobic biodegradation of NDMA may be an effective component of NDMA attenuation in water reclamation facility soils.     

4-(1,3-Thiazol-2-yl)morpholine derivatives as inhibitors of phosphoinositide 3-kinase

4-(1,3-Thiazol-2-yl)morpholine derivatives have been identified as potent and selective inhibitors of phosphoinositide 3-kinase. The SAR data of selected examples are presented and the in vivo profiling of compound 18 is shown to demonstrate the utility of this class of compounds in xenograft models of tumor growth.     

Galectin-1 signaling in leukocytes requires expression of complex-type N-glycans

Dimeric galectin-1 (dGal-1) is a homodimeric lectin with multiple proposed functions. Although dGal-1 binds to diverse glycans, it is unclear whether dGal-1 preferentially binds to specific subsets of glycans on cell surfaces to transmit signals. To explore this question, we selectively inhibited major glycan biosynthetic pathways in human HL60, Molt-4, and Jurkat cells. Inhibition of N-glycan processing blocked surface binding of dGal-1 and prevented dGal-1-induced Ca(2+) mobilization and phosphatidylserine exposure. By contrast, inhibition of O-glycan or glycosphingolipid biosynthesis did not affect dGal-1 binding or dGal-1-induced Ca(2+) mobilization and phosphatidylserine exposure. These results demonstrate that dGal-1 preferentially binds to and signals through glycoproteins containing complex-type N-glycans in at least some leukocyte subsets.     

Identifying unprovoked thromboembolism patients at low risk for recurrence who can discontinue anticoagulant therapy

Background

Whether to continue oral anticoagulant therapy beyond 6 months after an “unprovoked” venous thromboembolism is controversial. We sought to determine clinical predictors to identify patients who are at low risk of recurrent venous thromboembolism who could safely discontinue oral anticoagulants.

Methods

In a multicentre prospective cohort study, 646 participants with a first, unprovoked major venous thromboembolism were enrolled over a 4-year period. Of these, 600 participants completed a mean 18-month follow-up in September 2006. We collected data for 69 potential predictors of recurrent venous thromboembolism while patients were taking oral anticoagulation therapy (5–7 months after initiation). During follow-up after discontinuing oral anticoagulation therapy, all episodes of suspected recurrent venous thromboembolism were independently adjudicated. We performed a multivariable analysis of predictor variables (p < 0.10) with high interobserver reliability to derive a clinical decision rule.

Results

We identified 91 confirmed episodes of recurrent venous thromboembolism during follow-up after discontinuing oral anticoagulation therapy (annual risk 9.3%, 95% CI 7.7%–11.3%). Men had a 13.7% (95% CI 10.8%–17.0%) annual risk. There was no combination of clinical predictors that satisfied our criteria for identifying a low-risk subgroup of men. Fifty-two percent of women had 0 or 1 of the following characteristics: hyperpigmentation, edema or redness of either leg; D-dimer ≥ 250 μg/L while taking warfarin; body mass index ≥ 30 kg/m²; or age ≥ 65 years. These women had an annual risk of 1.6% (95% CI 0.3%–4.6%). Women who had 2 or more of these findings had an annual risk of 14.1% (95% CI 10.9%–17.3%).

Interpretation

Women with 0 or 1 risk factor may safely discontinue oral anticoagulant therapy after 6 months of therapy following a first unprovoked venous thromboembolism. This criterion does not apply to men. (http://Clinicaltrials.gov trial register number {"type":"clinical-trial","attrs":{"text":"NCT00261014","term_id":"NCT00261014"}}NCT00261014)Venous thromboembolism is a common, potentially fatal, yet treatable, condition. The risk of a recurrent venous thromboembolic event after 3–6 months of oral anticoagulant therapy varies. Some groups of patients (e.g., those who had a venous thromboembolism after surgery) have a very low annual risk of recurrence (< 1%),¹ and they can safely discontinue anticoagulant therapy.² However, among patients with an unprovoked thromboembolism who discontine anticoagulation therapy after 3–6 months, the risk of a recurrence in the first year is 5%–27%.^3–6 In the second year, the risk is estimated to be 5%,³ and it is estimated to be 2%–3.8% for each subsequent year.^5,7 The case-fatality rate for recurrent venous thromboembolism is between 5% and 13%.^8,9 Oral anticoagulation therapy is very effective for reducing the risk of recurrence during therapy (> 90% relative risk [RR] reduction);^3,4,10,11 however, this benefit is lost after therapy is discontinued.^3,10,11 The risk of major bleeding with ongoing oral anticoagulation therapy among venous thromboembolism patients is 0.9–3.0% per year,^3,4,6,12 with an estimated case-fatality rate of 13%.¹³Given that the long-term risk of fatal hemorrhage appears to balance the risk of fatal recurrent pulmonary embolism among patients with an unprovoked venous thromboembolism, clinicians are unsure if continuing oral anticoagulation therapy beyond 6 months is necessary.^2,14 Identifying subgroups of patients with an annual risk of less than 3% will help clinicians decide which patients can safely discontinue anticoagulant therapy.We sought to determine the clinical predictors or combinations of predictors that identify patients with an annual risk of venous thromboembolism of less than 3% after taking an oral anticoagulant for 5–7 months after a first unprovoked event.     

Module evolution and substrate specificity of fungal nonribosomal peptide synthetases involved in siderophore biosynthesis

Background  

Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches.     

Effects of available cover and feeding schedule on the behavior and growth of the juvenile African clawed frog (Xenopus laevis)

Though the African clawed frog (Xenopus laevis) is the most commonly used amphibian in biological research, there are no standard protocols for the husbandry of this species. With the goal of developing optimal conditions for raising these frogs, the authors assessed the effects of available cover and feeding schedule on post-metamorphic growth and behavior of juvenile X. laevis. Frogs, which were housed four per tank, had access to varying numbers of shelters (zero, two or four) and were fed either daily or episodically (three times per week) over a period of 6 months. Though X. laevis growth was not influenced by the availability of cover, frogs that were fed daily grew larger than frogs that were fed episodically. Additionally, frogs that were fed daily and had no access to cover were most likely to exhibit foraging behavior when disturbed. These results suggest that feeding frogs daily will promote growth and fecundity. Furthermore, although labs often provide frogs with hiding places, such cover does not enhance X. laevis growth.