Adaptable, turn-on maturation (ATOM) fluorescent biosensors for multiplexed detection in cells.

A grand challenge in biosensor design is to develop a single-molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Here, we created a family of adaptable, turn-on maturation (ATOM) biosensors consisting of a monobody (circularly permuted at one of two positions) or a nanobody (circularly permuted at one of three positions) inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells coexpressing cyan, yellow and red ATOM sensors detected biosensor targets that were specifically localized to various subcellular compartments. Fluorescence activation involved ligand-dependent chromophore maturation with turn-on ratios of up to 62-fold in cells and 100-fold in vitro. Endoplasmic reticulum- and mitochondria-localized ATOM sensors detected ligands that were targeted to those organelles. The ATOM design was validated with three monobodies and one nanobody inserted into distinct fluorescent proteins, suggesting that customized ATOM sensors can be generated quickly.

Feasibility of bubble non-invasive positive pressure ventilation, a first-in-human study.

PURPOSE: Infant respiratory distress is  a significant cause of mortality globally. Bubble continuous positive airway pressure (CPAP) is a simple and effective therapy, but sicker infants may require additional support such as non-invasive positive-pressure ventilation (NIPPV). We investigated the feasibility of a simple, low-cost, non-electric bubble NIPPV device. METHODS: In this cross-over feasibility study, seven newborns with moderate respiratory distress (Downes score ≥ 3), weight > 1500 g and gestational age > 32 weeks were randomized to  4 h of treatment with bubble CPAP (5-8 cm H2O) vs. bubble NIPPV (Phigh 8-10 cm H2O/Plow 5-8 cm H2O) followed by 4 h of the alternate treatment. Treatment order (CPAP vs. NIPPV) was randomized. Outcome measures included hourly vital signs, Downes score and O2 saturation. Adverse events including pneumothorax, nasal septal necrosis, necrotizing enterocolitis and death before discharge were also recorded. RESULTS: It took nurses 39 (7.3) s to assemble the bubble NIPPV device. Patients had similar vital signs and Downes scores on both treatments; median (IQR) values on bubble CPAP vs. bubble NIPPV were: heart rate 140 (134.5, 144), 140 (134.5, 144); respiratory rate 70 (56, 80), 65 (58, 82), Downes score 4 (3, 5.75), 4 (3, 5), O2 96 (94, 98), 97 (96, 98). All newborns survived to discharge and there were no adverse events. . CONCLUSIONS: A simple, low-cost, non-electric method of providing NIPPV for newborns with respiratory distress is feasible in limited resource settings. Randomized-controlled trials comparing bubble CPAP and bubble NIPPV are justified.

Bubble bilevel ventilation facilitates gas exchange in anesthetized rabbits.

BACKGROUND: Bubble continuous positive airway pressure is an established therapy for infants in respiratory distress. In resource-limited settings, few treatment options exist for infants requiring further respiratory support. A bubble bilevel device has been developed to provide nonelectric, time-cycled, pressure-limited respiratory support. We compared the efficacy of bubble bilevel ventilation with conventional mechanical ventilation in sedated rabbits. METHODS: Six adult rabbits under inhaled isoflurane general anesthesia were ventilated by alternating intervals of conventional and bubble bilevel ventilation for three 10-15-min periods. During each period, interval arterial blood gas (ABG) measurements were obtained after at least 10 min on the respective mode of ventilation. RESULTS: The bubble bilevel system was able to deliver the following pressures: 20/7, 15/5, 12/5, 8/5 cm H2O. The estimated differences in arterial blood gas values on bubble bilevel vs. ventilator were as follows (normalized values): pH 7.41 vs. 7.40, pCO2 37.7 vs. 40, pO2 97.6 vs. 80. In addition, the bubble bilevel ventilation delivered consistent pressure waveforms without interruption for over 60 min on two rabbits. CONCLUSION: This study demonstrates promising in vivo results on the efficacy of a novel bubble bilevel device, which may prove useful for infants in respiratory distress. IMPACT: Given the lack of personnel, funds or infrastructure to provide neonatal mechanical ventilation in resource-limited settings, additional low-cost, low-tech treatments are necessary to save infant lives. Bubble bilevel ventilation reliably delivers two levels of airway pressure to anesthetized rabbits resulting in normalization of blood gases comparable to those achieved on a traditional ventilator. If proven effective, simple technologies like this device have the potential to significantly impact neonatal mortality due to respiratory distress globally.

Development and performance testing of the low-cost, 3D-printed, smartphone-compatible 'Tansen Videolaryngoscope' vs. Pentax-AWS videolaryngoscope vs. direct Macintosh laryngoscope: A manikin study.

BACKGROUND: While videolaryngoscopes help in the management of difficult airways, they remain too expensive for those with limited resources. We have developed a robust, re-usable, low-cost videolaryngoscope at United Mission Hospital Tansen, Nepal, by combining a smartphone-compatible endoscope capable of capturing still and video images with a three dimensional-printed, channelled, hyperangulated blade. The computer-aided design file for the videolaryngoscope blade was emailed and printed in London before evaluation of its performance on a difficult airway manikin. OBJECTIVE: To benchmark the intubation performance of the Tansen Videolaryngoscope (TVL) in a 'difficult airway' manikin (SimMan3G, tongue fully inflated, neck stiff), against a commercially available videolaryngoscope and a conventional Macintosh laryngoscope. DESIGN: A manikin study. SETTING AND PARTICIPANTS: Forty-three experienced videolaryngoscope users in two London teaching hospitals. INTERVENTION AND OUTCOME: Primary outcome: Intubation success rate. SECONDARY OUTCOMES: grade of laryngeal view, median time to intubation and intubator-rated 'ease of use'. RESULTS: Our device was equivalent to Pentax-AWS and superior to Macintosh laryngoscope (TVL vs. Pentax-AWS vs. Macintosh) in overall intubation success rate (88 vs. 98 vs. 67%, P < 0.05); grade of view (median Cormack-Lehane grade 1 vs. 1 vs. 3, P < 0.01); median time to intubation (17.5 vs. 15.5 vs. 27 s, P < 0.01). In subjective 'ease of use' scores, Pentax-AWS was superior to the TVL, which was superior to Macintosh laryngoscope (Likert-type 1 to 5 scale: 4 vs. 4 vs. 1, P < 0.00001). CONCLUSION: In this manikin simulation of a difficult airway, the 'TVL' was superior to the Macintosh laryngoscope, and noninferior to the Pentax-AWS videolaryngoscope in intubation success rate, grade of laryngeal view and time to intubation. Participants found the Pentax device easier to use, and their feedback has given us valuable insights for improving our device. The TVL is well suited to settings in which resources are limited, being inexpensive, simple and re-usable.

Unveiling cytokine charge disparity as a potential mechanism for immune regulation.

Cytokines are small signaling proteins that regulate the immune responses to infection and tissue damage. Surface charges of cytokines determine their in vivo fate in immune regulation, e.g., half-life and distribution. The overall negative charges in the extracellular microenvironment and the acidosis during inflammation and infection may differentially impact cytokines with different surface charges for fine-tuned immune regulation via controlling tissue residential properties. However, the trend and role of cytokine surface charges has yet to be elucidated in the literature. Interestingly, we have observed that most pro-inflammatory cytokines have a negative charge, while most anti-inflammatory cytokines and chemokines have a positive charge. In this review, we extensively examined the surface charges of all cytokines and chemokines, summarized the pharmacokinetics and tissue adhesion of major cytokines, and analyzed the link of surface charge with cytokine biodistribution, activation, and function in immune regulation. Additionally, we identified that the general trend of charge disparity between pro- and anti-inflammatory cytokines represents a unique opportunity to develop precise immune modulation approaches, which can be applied to many inflammation-associated diseases including solid tumors, chronic wounds, infection, and sepsis.

Inferior Frontal Sulcal Hyperintensities on Brain MRI Are Associated with Amyloid Positivity beyond Age-Results from the Multicentre Observational DELCODE Study.

Inferior frontal sulcal hyperintensities (IFSHs) on fluid-attenuated inversion recovery (FLAIR) sequences have been proposed to be indicative of glymphatic dysfunction. Replication studies in large and diverse samples are nonetheless needed to confirm them as an imaging biomarker. We investigated whether IFSHs were tied to Alzheimer's disease (AD) pathology and cognitive performance. We used data from 361 participants along the AD continuum, who were enrolled in the multicentre DELCODE study. The IFSHs were rated visually based on FLAIR magnetic resonance imaging. We performed ordinal regression to examine the relationship between the IFSHs and cerebrospinal fluid-derived amyloid positivity and tau positivity (Aß42/40 ratio ≤ 0.08; pTau181 ≥ 73.65 pg/mL) and linear regression to examine the relationship between cognitive performance (i.e., Mini-Mental State Examination and global cognitive and domain-specific performance) and the IFSHs. We controlled the models for age, sex, years of education, and history of hypertension. The IFSH scores were higher in those participants with amyloid positivity (OR: 1.95, 95% CI: 1.05-3.59) but not tau positivity (OR: 1.12, 95% CI: 0.57-2.18). The IFSH scores were higher in older participants (OR: 1.05, 95% CI: 1.00-1.10) and lower in males compared to females (OR: 0.44, 95% CI: 0.26-0.76). We did not find sufficient evidence linking the IFSH scores with cognitive performance after correcting for demographics and AD biomarker positivity. IFSHs may reflect the aberrant accumulation of amyloid ß beyond age.

Adaptable, Turn-On Monobody (ATOM) Fluorescent Biosensors for Multiplexed Detection in Cells.

A grand challenge in biosensor design is to develop a single molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Conceptually, this can be achieved by fusing a small, antibody-like binding domain to a fluorescent protein in such a way that target binding activates fluorescence. Although this design is simple to envision, its execution is not obvious. Here, we created a family of adaptable, turn-on monobody (ATOM) biosensors consisting of a monobody, circularly permuted at one of two positions, inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells co-expressing cyan, yellow, and red ATOM sensors detected the biosensor targets (WDR5, SH2, and hRAS proteins) that were localized to the nucleus, cytoplasm, and plasma membrane, respectively, with high specificity. ER- and mitochondria-localized ATOM sensors also detected ligands that were targeted to those organelles. Fluorescence activation involved ligand-dependent chromophore maturation with fluorescence turn-on ratios of >20-fold in cells and up to 100-fold in vitro . The sensing mechanism was validated with three arbitrarily chosen monobodies inserted into jellyfish as well as anemone lineages of fluorescent proteins, suggesting that ATOM sensors with different binding specificities and additional colors can be generated relatively quickly.

Engineering a Fluorescent Protein Color Switch Using Entropy-Driven ß-Strand Exchange.

Protein conformational switches are widely used in biosensing. They are often composed of an input domain (which binds a target ligand) fused to an output domain (which generates an optical readout). A central challenge in designing such switches is to develop mechanisms for coupling the input and output signals via conformational changes. Here, we create a biosensor in which binding-induced folding of the input domain drives a conformational shift in the output domain that results in a sixfold green-to-yellow ratiometric fluorescence change in vitro and a 35-fold intensiometric fluorescence increase in cultured cells. The input domain consists of circularly permuted FK506 binding protein (cpFKBP) that folds upon binding its target ligand (FK506 or rapamycin). cpFKBP folding induces the output domain, an engineered green fluorescent protein (GFP) variant, to replace one of its ß-strands (containing T203 and specifying green fluorescence) with a duplicate ß-strand (containing Y203 and specifying yellow fluorescence) in an intramolecular exchange reaction. This mechanism employs the loop-closure entropy principle, embodied by the folding of the partially disordered cpFKBP domain, to couple ligand binding to the GFP color shift. This study highlights the high-energy barriers present in GFP folding which cause ß-strand exchange to be slow and are also likely responsible for the shift from the ß-strand exchange mechanism in vitro to ligand-induced chromophore maturation in cells. The proof-of-concept design has the advantages of full genetic encodability and potential for modularity. The latter attribute is enabled by the natural coupling of binding and folding and circular permutation of the input domain, which theoretically allows different binding domains to be compatible for insertion into the GFP surface loop.

Screening asymptomatic men for prostate cancer: A comparison of international guidelines on prostate-specific antigen testing.

OBJECTIVE: To summarise and compare the key recommendations on prostate-specific antigen (PSA)-based screening for prostate cancer, and so highlight where more evidence is required to facilitate consistent recommendations. METHODS: The Medline database and websites of 18 national screening organisations and professional associations were searched between January 2010 and November 2020 to identify screening guidelines published in English, considering recent clinical trials. RESULTS: Population-based PSA testing of asymptomatic men is not widely recommended. Guidelines emphasize shared patient-clinician decision making. For 'average-risk' men choosing to be screened, the recommended age varies from 50-55 to 70 years, alongside consideration of life expectancy (ranging from 7-15 years). Screening intervals, when specified, are biennial (most common), annual, or determined from baseline PSA. The earliest age for screening high-risk men (frequently defined as of African descent or with a family history of prostate cancer) is 40 years, but recommendations often defer to clinical judgement. CONCLUSIONS: Population screening of asymptomatic men is not widely recommended. Instead, balancing the potential harms and benefits of PSA testing is endorsed. Variation between guidelines stems from differing interpretations of key trials and could lead to clinician-dependent screening views. The development of clinical decision aids and international consensus on guidelines may help reduce national and international variation on how men are counselled.

Bench Testing of a Bubble Noninvasive Ventilation Device in an Infant Lung Simulator.

BACKGROUND: Infant respiratory distress remains a significant problem worldwide, leading to more than one million neonatal deaths each year. The cost, maintenance, energy, and personnel required to implement ventilators have proven to be a barrier in many resource-limited settings. To address these barriers, a nonelectric bubble noninvasive positive pressure ventilation (NIV) device was developed. This study aims to benchmark the performance of this bubble NIV device against commercially available ventilators. METHODS: The delivered pressure waveforms and tidal volumes of the bubble NIV device were compared with those of 2 conventional ventilators (ie, Dräger Evita Infinity V500 and Hamilton G5) at the following pressure settings: 8/5, 12/5, and 15/5 cm H2O. To simulate the lung mechanics of an infant in respiratory distress, tests were conducted on the IngMar ASL 5000 Test Lung simulator. Resistance was set at 100 cm H2O/L/s, and compliance was tested at 0.5, 1.0, and 2.0 mL/cm H2O to simulate 3 different patients. RESULTS: The delivered pressure waveforms and compliance curves of the bubble NIV device are similar to those of the Hamilton and Dräger ventilators. The mean ± SD differences between delivered versus set pressure gradient (ie, the difference between the high delivered pressure and the low delivered pressure) for each treatment modality across the various values of compliance were -2 ± 8% for the bubble NIV device, 3 ± 4% for the Dräger ventilator, and 7 ± 10% for the Hamilton ventilator. CONCLUSIONS: The similarity of pressure waveforms and delivered tidal volumes in this simulated clinical scenario suggest that the bubble NIV device may provide comparable efficacy compared with traditional ventilator treatment for a range of patients. This may provide clinicians in resource-limited settings with an additional, simple, nonelectric treatment modality for the management of infant respiratory distress.

Reproducing Patient-Specific Hemodynamics in the Blalock-Taussig Circulation Using a Flexible Multi-Domain Simulation Framework: Applications for Optimal Shunt Design.

For babies born with hypoplastic left heart syndrome, several open-heart surgeries are required. During Stage I, a Norwood procedure is performed to construct an appropriate circulation to both the systemic and the pulmonary arteries. The pulmonary arteries receive flow from the systemic circulation, often using a Blalock-Taussig (BT) shunt between the innominate artery and the right pulmonary artery. This procedure causes significantly disturbed flow in the pulmonary arteries. In this study, we use computational hemodynamic simulations to demonstrate its capacity for examining the properties of the flow through and near the BT shunt. Initially, we construct a computational model which produces blood flow and pressure measurements matching the clinical magnetic resonance imaging (MRI) and catheterization data. Achieving this required us to determine the level of BT shunt occlusion; because the occlusion is below the MRI resolution, this information is difficult to recover without the aid of computational simulations. We determined that the shunt had undergone an effective diameter reduction of 22% since the time of surgery. Using the resulting geometric model, we show that we can computationally reproduce the clinical data. We, then, replace the BT shunt with a hypothetical alternative shunt design with a flare at the distal end. Investigation of the impact of the shunt design reveals that the flare can increase pulmonary pressure by as much as 7% and flow by as much as 9% in the main pulmonary branches, which may be beneficial to the pulmonary circulation.

Program of cell survival underlying human and experimental hibernating myocardium.

Hibernating myocardium refers to chronically dysfunctional myocardium in patients with coronary artery disease in which cardiac viability is maintained and whose function improves after coronary revascularization. It is our hypothesis that long-term adaptive genomic mechanisms subtend the survival capacity of this ischemic myocardium. Therefore, the goal of this study was to determine whether chronic repetitive ischemia elicits a gene program of survival protecting hibernating myocardium against cell death. Accordingly, we measured the expression of survival genes in hibernating myocardium, both in patients surgically treated for hibernation and in a chronic swine model of repetitive ischemia reproducing the features of hibernation. Human hibernating myocardium was characterized by an upregulation of genes and corresponding proteins involved in anti-apoptosis (IAP), growth (VEGF, H11 kinase), and cytoprotection (HSP70, HIF-1alpha, GLUT1). In the swine model, the same genes and proteins were upregulated after repetitive ischemia, which was accompanied by a concomitant decrease in myocyte apoptosis. These changes characterize viable tissue, because they were not found in irreversibly injured myocardium. Our report demonstrates a novel mechanism by which the activation of an endogenous gene program of cell survival underlies the sustained viability of the hibernating heart. Potentially, promoting such a program offers a novel opportunity to salvage postmitotic tissues in conditions of ischemia.

Pulmonary vein diameter reduction after radiofrequency catheter ablation for paroxysmal atrial fibrillation evaluated by contrast-enhanced three-dimensional magnetic resonance imaging.

BACKGROUND: Radiofrequency catheter ablation (RFCA) is a promising intervention to treat atrial fibrillation. However, pulmonary vein (PV) stenosis after RFCA has been reported. The aim of this study was to investigate the incidence and time course of pulmonary vein stenosis after RFCA within a period of 3 months. Contrast-enhanced magnetic resonance angiography (MRA) was used to visualize pulmonary veins and was compared with radiographic angiography. METHODS AND RESULTS: Forty-six consecutive patients with symptomatic paroxysmal atrial fibrillation had RFCA in the orifice of 138 pulmonary veins. Comparison of diameters measured in 44 untreated vessels either by radiographic angiography or with MRA established the reliability of MRA (r=0.934). MRA measurements revealed an incidence of relevant diameter reductions of > or =25% or stenosis of > or =50% after RFCA of 25 of 138 (18.1%) treated vessels 1 day and/or 3 months after ablation. A progression of diameter reduction after RFCA was observed in 8.3% (maximum 75%), whereas a regression was observed in 6.3% of treated PVs. Ablation at a radial angle of >180 degrees of a pulmonary vein orifice increased the risk of diameter reduction significantly compared with ablation at a radial angle < or =180 degrees (P=0.002). CONCLUSIONS: The occurrence and progression of PV stenosis is a potential significant complication of RFCA in the orifice of pulmonary veins. These findings may have an impact on the technical performance of this intervention. In addition, long-term studies will be necessary to evaluate lumen reduction over time. MRA is a noninvasive, reproducible imaging modality for this purpose.

Magnetic resonance to assess the aortic valve area in aortic stenosis: how does it compare to current diagnostic standards?

OBJECTIVES: The purpose of the present study was to evaluate whether magnetic resonance (MR) planimetry of the aortic valve area (AVA) may prove to be a reliable, non-invasive diagnostic tool in the assessment of aortic valve stenosis, and how the results compare with current diagnostic standards. BACKGROUND: Current standard techniques for assessing the severity of aortic stenosis include transthoracic and transesophageal echocardiography (TEE) as well as transvalvular pressure measurements during cardiac catheterization. METHODS: Forty consecutive patients underwent cardiac catheterization, TEE, and MR. The AVA was estimated by direct planimetry (MR, TEE) or calculated indirectly via the peak systolic transvalvular gradient (catheter). Pressure gradients from cardiac catheterization and Doppler echocardiography were also compared. RESULTS: By MR, the mean AVA(max) was 0.91 +/- 0.25 cm(2); by TEE, AVA(max) was 0.89 +/- 0.28 cm(2); and by catheter, the AVA was calculated as 0.64 +/- 0.26 cm(2). Mean absolute differences in AVA were 0.02 cm(2) for MR versus TEE, 0.27 cm(2) for MR versus catheter, and 0.25 cm(2) for TEE versus catheter. Correlations for AVA(max) were r = 0.96 between MR and TEE, r = 0.47 between TEE and catheter, and r = 0.44 between MR and catheter. The correlation between Doppler and catheter gradients was r = 0.71. CONCLUSIONS: Magnetic resonance planimetry of the AVA correlates well with TEE and less well with the catheter-derived AVA. Invasive and Doppler pressure correlated less well than those obtained from planimetric techniques. Magnetic resonance planimetry of the AVA may provide an accurate, non-invasive, well-tolerated alternative to invasive techniques and transthoracic echocardiography in the assessment of aortic stenosis.

The pathologic basis of Q-wave and non-Q-wave myocardial infarction: a cardiovascular magnetic resonance study.

OBJECTIVES: The purpose of this study was to determine the pathologic basis of Q-wave (QW) and non-Q-wave (NQW) myocardial infarction (MI). BACKGROUND: The QW/NQW distinction remains in wide clinical use but the meaning of the difference remains controversial. We hypothesized that measurement of total MI size and transmural extent by late gadolinium enhancement cardiovascular magnetic resonance (CMR) would identify the pathologic basis of QWs. METHODS: A total of 100 consecutive patients with documented previous MI had electrocardiogram and CMR on the same day. Patients with acute MI within seven days were excluded. Left ventricular function and the size and transmural extent of MI were quantified in the three major arterial territories and correlated with the presence of QW. RESULTS: Subendocardial MI showed QW in 28%. Transmural MI showed NQW in 29%. Of all MIs, 48% were at some point transmural, and 99% of these were at some point non-transmural. As MI size and number of transmural segments increased, the probability of QW increased (anterior: total size chi-square = 53, p < 0.0001, transmural extent chi-square = 36, p < 0.0001; inferior: total size chi-square = 16, p = 0.001, transmural extent chi-square = 10, p = 0.001). These findings did not hold for lateral MI. In a multivariate model, the transmural extent of MI was not an independent predictor of QW when total size of MI was removed. The QW/NQW classification was a good test for size of MI (area under receiver operating characteristic curve: anterior 0.90, inferior 0.77). CONCLUSIONS: The QW/NQW distinction is useful, but it is determined by the total size rather than transmural extent of underlying MI.

Recombinant modular transporters for cell-specific nuclear delivery of locally acting drugs enhance photosensitizer activity.

The search for new pharmaceuticals that are specific for diseased rather than normal cells in the case of cancer and viral disease has raised interest in locally acting drugs that act over short distances within the cell and for which different cell compartments have distinct sensitivities. Thus, photosensitizers (PSs) used in anti-cancer therapy should ideally be transported to the most sensitive subcellular compartments in order for their action to be most pronounced. Here we describe the design, production, and characterization of the effects of bacterially expressed modular recombinant transporters for PSs comprising 1) alpha-melanocyte-stimulating hormone as an internalizable, cell-specific ligand; 2) an optimized nuclear localization sequence of the SV40 large T-antigen; 3) an Escherichia coli hemoglobin-like protein as a carrier; and 4) an endosomolytic amphipathic polypeptide, the translocation domain of diphtheria toxin. These modular transporters delivered PSs into the nuclei, the most vulnerable sites for the action of PSs, of murine melanoma cells, but not non-MSH receptor-overexpressing cells, to result in cytotoxic effects several orders of magnitude greater than those of nonmodified PSs. The modular fusion proteins described here for the first time, capable of cell-specific targeting to particular subcellular compartments to increase drug efficacy, represent new pharmaceuticals with general application.

Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-alpha.

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.

Phosphorylation of insulin-like growth factor binding protein-3 by deoxyribonucleic acid-dependent protein kinase reduces ligand binding and enhances nuclear accumulation.

The IGF binding proteins (IGFBPs) regulate the mitogenic effects of IGFs in the extracellular environment. Several members of this family, including IGFBP-3, also appear to have IGF-independent effects on cell function. For IGFBP-3 and IGFBP-5, both of which are translocated to the cell nuclei, these effects may be related to their putative nuclear actions. Because reversible phosphorylation is an important mechanism for controlling nuclear protein import, we have examined the effect of phosphorylating IGFBP-3 with a number of serine/threonine protein kinases on its nuclear import. Phosphorylation of IGFBP-3 by the double-stranded DNA-dependent protein kinase (DNA-PK) increased both the nuclear import of IGFBP-3 and the binding of IGFBP-3 to components within the nucleus compared with nonphosphorylated IGFBP-3. However, there was no difference in the binding of the nuclear transport factor, importin beta, to nonphosphorylated and phosphorylated IGFBP-3. The ability of the DNA-PK phosphoform of IGFBP-3 to bind IGFs was severely attenuated, and in contrast to nonphosphorylated IGFBP-3, the DNA-PK phosphoform was unable to transport IGF-I to the nucleus. Furthermore, IGFBP-3 was phosphorylated by DNA-PK when complexed to IGF-I causing the phosphoform to release IGF-I. Together, these results suggest that when IGF-I is cotransported into the nucleus by IGFBP-3, phosphorylation of IGFBP-3 by nuclear DNA-PK provides a means for releasing bound IGF-I and creating a phosphoform of IGFBP-3 with increased affinity for nuclear components.