A method for identifying the learning curve for the surgical stabilization of rib fractures.

BACKGROUND: Surgical stabilization of rib fractures (SSRF) is an accepted efficacious treatment modality for patients with severe chest wall injuries. Despite increased adoption of SSRF, surgical learning curves are unknown. We hypothesized intraoperative duration could define individual SSRF learning curves. METHODS: Consecutive SSRF operations between January 2017 and December 2021 at a single institution were reviewed. Operative time, as measured from incision until skin closure, was evaluated by cumulative sum methodology using a range of acceptable "missteps" to determine the learning curves. Misstep was defined by extrapolation of accumulated operative time data. RESULTS: Eighty-three patients underwent SSRF by three surgeons during this retrospective review. Average operative times ranged from 135 minutes for two plates to 247 minutes for seven plates. Using polynomial regression of average operative times, 75 minutes for general procedural requirements plus 35 minutes per plate were derived as the anticipated operative times per procedure. Cumulative sum analyses using 5%, 10%, 15%, and 20% incident rates for not meeting expected operative times, or "missteps" were used. An institutional learning curve between 15 and 55 SSRF operations was identified assuming a 90% performance rate. An individual learning curve of 15 to 20 operations assuming a 90% performance rate was observed. After this period, operative times stabilized or decreased for surgeons A, B, and C. CONCLUSION: The institutional and individual surgeon learning curves for SSRF appears to steadily improve after 15 to 20 operations using operative time as a surrogate for performance. The implementation of SSRF programs by trauma/acute care surgeons is feasible with an attainable learning curve. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level IV.

The Association Between Pulmonary Contusion Severity and Respiratory Failure.

BACKGROUND: Pulmonary contusions (PCs) have historically been viewed as a serious complicating factor in thoracic injury. Recently, there has been conflicting evidence regarding the influence of PCs on outcomes; however, many studies do not stratify contusions by severity and may miss clinical associations. We sought to identify if contusion severity is associated with worse outcomes. METHODS: A previously published chest wall injury database at an urban Level I trauma center was retrospectively reviewed. All severely injured subjects (defined as Injury Severity Score [ISS] ≥ 15) with moderate to severe thoracic injury (defined as a chest wall Abbreviated Injury Scale [AIS] ≥ 3) who required mechanical ventilation for > 24 h were stratified by contusion severity. Moderate to severe contusions were defined as AIS contusion ≥ 3 and Blunt Pulmonary Contusion 18 (BPC18) score ≥ 3. RESULTS: Over 5 y, 3,836 patients presented with chest wall injuries, of which 1,176 (30.6%) had concomitant contusions. When screened for inclusion criteria, 339 subjects with contusions and 211 subjects without contusions (no-PC) were identified. Of these, 234 had moderate to severe contusions defined by AIS contusion ≥ 3 (PC-A) and 230 had moderate to severe contusions by BPC18 ≥ 3 (PC-B). Compared to no-PC, both PC-A and PC-B groups had significantly lower mortality (17.9% and 17.4%, respectively, vs 28.9%); however, PC-A and PC-B groups had longer durations of mechanical ventilation (6 and 7 d, respectively, vs 5 d), longer ICU length of stay (10 and 10 vs 8 d), and longer overall hospital length of stay (15 and 15 vs 13 d). CONCLUSIONS: In severely injured polytrauma patients, PCs are seen with more severe chest injuries. Furthermore, moderate to severe contusions are associated with longer durations of mechanical ventilation, ICU length of stay, and hospital length of stay. Despite practice pattern changes, contusions appear to contribute significantly to the clinical course of the blunt chest wall injured patients.

Does chest wall Organ Injury Scale (OIS) or Abbreviated Injury Scale (AIS) predict outcomes? An analysis of 16,000 consecutive rib fractures.

BACKGROUND: Chest wall injuries have serious clinical consequences. It is presumed a higher severity of injury correlates with worse outcomes. The 2 most common chest wall injury severity scores, the Organ Injury Scale and the Abbreviated Injury Scale, are based on expert opinion with unknown clinical endpoints. Our aim was to determine if either the Organ Injury Scale or the Abbreviated Injury Scale are associated with clinical outcomes. METHODS: A single institution, 4-year retrospective study of all patients with rib or sternal fractures was conducted. All patients were assessed for both Organ Injury Scale and Abbreviated Injury Scale scores. Outcomes assessed included mortality, complications, tracheostomy, and readmissions. Receiver operating characteristic areas under the curve were calculated to measure discriminatory accuracy of scoring systems for outcomes in chest wall injury. RESULTS: Overall, 3,033 patients presented with a total of 16,055 rib fractures. The median chest wall scores were 2 for Organ Injury Scale and 3 for Abbreviated Injury Scale. Abbreviated Injury Scale scores for the same patients were greater than the Organ Injury Scale in 48.7%, equivalent in 46.7%, and lower in 4.6%. The receiver operating characteristic areas under the curve for in-hospital outcomes were weakly predictive for the Organ Injury Scale over the Abbreviated Injury Scale. The receiver operating characteristic areas under the curve for readmissions were very weakly predictive for the Abbreviated Injury Scale over the Organ Injury Scale. CONCLUSION: There is a very weak association between chest wall Organ Injury Scale score and in-hospital outcomes. The Abbreviated Injury Scale score outperformed the Organ Injury Scale, only being weakly predictive of readmission. Chest wall injury scoring systems may need revision for future outcomes-based research and practice improvements.

Readmission rates and associated factors following rib cage injury.

BACKGROUND: There remains a lack of knowledge about readmission characteristics after sustaining rib fractures. We aimed to determine rates, characteristics, and predictive/protective factors associated with unexpected reevaluation and readmission after rib cage injury. METHODS: A retrospective review was performed based on trauma patients evaluated at an urban Level I trauma center from January 2014 to December 2016. Adult patients sustaining blunt trauma with more than one rib fracture or a sternomanubrial fracture were defined as having moderate to severe rib cage injury. Exclusion criteria included penetrating injury, death during initial hospitalization, and only one rib fracture. Reevaluation was defined as presenting at a hospital within 90 days of discharge urgently or emergently. Demographics, injury characteristics, comorbidities, complications, imaging, and readmission data were collected. Univariate and multivariate analysis was performed with a significance of p less than 0.05. RESULTS: During the study period, 11,667 patients underwent trauma evaluation, of which 1,717 patients were found to have a moderate to severe rib cage injury. Within 90 days, 397 (23.1%) of patients underwent reevaluation, while 177 (10.3%) required readmission. One hundred forty-two (8.3%) patients were reevaluated specifically for chest-related complaints, and 55 (3.2%) required readmission. On univariate analysis, Injury Severity Score greater than 15, hospital length of stay longer than 7 days, intensive care unit (ICU) length of stay longer than 3 days, a worsened chest x-ray at discharge, a psychiatric comorbidity, a smoking comorbidity, deep vein thrombosis, unplanned readmission to the ICU, and unplanned intubation were higher in the overall reevaluation cohort. On multivariate analysis, age of 15 years to 35 years, Risk Assessment Profile score greater than 8, hypertension, psychiatric comorbidity, current smoker, and unplanned return to the ICU on index admission were predictive of reevaluation of overall reevaluation. CONCLUSION: Moderate to severe rib cage injury is associated with high rates of reevaluation and readmission. Younger patients who smoke and required a return to the ICU are at greater risk for readmission. LEVEL OF EVIDENCE: Level IV, Prognostic and Epidemiologic.

Factor VIIa administration in traumatic brain injury: an AAST-MITC propensity score analysis.

BACKGROUND: Recombinant factor VIIa (rFVIIa) has been used off-label as an adjunct in the reversal of warfarin therapy and management of hemorrhage after trauma. Only a handful of these reports are rigorous studies, from which results regarding safety and effectiveness have been mixed. There remains no clear consensus as to the role of rFVIIa in traumatic brain injury (TBI). METHODS: Eleven level 1 trauma centers provided clinical data and head CT scans of patients with a Glasgow Coma Scale (GCS) score of ≤13 and radiographic evidence of TBI. A propensity score (PS) to receive rFVIIa in those surviving ≥2 days was calculated for each patient based on patient demographics, comorbidities, physiology, Injury Severity Score, admission GCS score, and treatment center. Patients receiving rFVIIa within 24 hours of admission were matched to patients who did not receive rFVIIa for outcomes assessment. Subgroup analysis evaluated patients with primary head injury with PS matching. RESULTS: There were 4284 patient observations; 129 received rFVIIa. Groups were comparable after matching. No differences in mortality or morbidity were found. Improvement in GCS score from admission to discharge was less among those receiving rFVIIa (5.5 vs. 2.4; P value 0.001); however, there was no difference in average GCS score at discharge. No significant differences in outcomes were identified in patients with isolated TBI receiving rFVIIa. DISCUSSION: rFVIIa in early management of TBI is not associated with a decreased risk of mortality or morbidity, and may negatively impact recovery and functional status at discharge in the severely injured patient with polytrauma. LEVEL OF EVIDENCE: Level III. STUDY TYPE: Therapeutic/care management.

Safety and efficacy of brain injury guidelines at a Level III trauma center.

BACKGROUND: Patients with mild to moderate traumatic brain injury (TBI) are often primarily managed by emergency medicine and trauma/acute care physicians. The Brain Injury Guidelines (BIG) were developed at an American College of Surgeons-accredited Level 1 trauma center to triage mild to moderate TBI patients and help identify patients who warrant neurosurgical consultation. The BIG have not been validated at a Level III trauma center. We hypothesized that BIG criteria can be safely adapted to an American College of Surgeons-accredited Level III trauma center to guide transfers to a higher echelon of care. METHODS: We reviewed the trauma registry at a Level III trauma center to identify TBI patients who presented with an Abbreviated Injury Severity-Head score greater than zero. Demographic data, injury details, and clinical outcomes were abstracted with primary outcome measures of worsening on second computed tomography of the head, neurosurgical intervention, transfer to a Level I trauma center, and in-hospital mortality. Patients were classified using the BIG criteria. After validating the BIG in our cohort, we reclassified patients using updated BIG criteria. Updated criteria included mechanism of injury, reclassification of anticoagulation or antiplatelet use, and replacement of the neurologic examination component with stratification by admission Glasgow Coma Scale (GCS) score. RESULTS: From July 2013 to June 2016, 332 TBI patients were identified: 115 BIG-1, 25 BIG-2, and 192 BIG-3. Patients requiring neurosurgical intervention (n = 30) or who died (n = 29) were BIG-3 with one exception. Patients with GCS score of less than 12 had worse outcomes than those with a GCS score of 12 or greater, regardless of BIG classification. Anticoagulant or antiplatelet use was not associated with worsened outcomes in patients not meeting other BIG-3 criteria. The updated BIG resulted in more patients in BIG-1 (n = 109) and BIG-2 (n = 100) without negatively affecting outcomes. CONCLUSION: The BIG can be applied in the Level III trauma center setting. Updated BIG criteria can aid triage of mild to moderate TBI patients to a Level I trauma center and may reduce secondary overtriage. LEVEL OF EVIDENCE: Care management, level IV.

Decompressive craniectomy or medical management for refractory intracranial hypertension: an AAST-MIT propensity score analysis.

BACKGROUND: Moderate/severe traumatic brain injury (TBI) management involves minimizing cerebral edema to maintain brain oxygen delivery. While medical therapy (MT) consisting of diuresis, hyperosmolar therapy, ventriculostomy, and barbiturate coma is the standard of care, decompressive craniectomy (DC) for refractory intracranial hypertension (ICH) has gained renewed interest. Since TBI treatment guidelines consider DC a second-tier intervention after MT failure, we sought to determine if early DC (<48 hours) was associated with improved survival in patients with refractory ICH. METHODS: Eleven Level 1 trauma centers provided clinical data and head computed tomographic scans for patients with a Glasgow Coma Scale (GCS) score of 13 or less and radiographic evidence of TBI excluding deaths within 48 hours. Computed tomographic scans were graded according to the Marshall classification. A propensity score to receive DC (regardless of whether DC was performed) was calculated for each patient based on patient characteristics, physiology, injury severity, GCS, severity of intracranial injury, and treatment center. Patients who actually received a DC were matched to patients with similar propensity scores who received MT for analysis. Outcomes were compared between early (<48 hours of injury) primary or secondary DC and matched controls and then between early primary DC only and matched controls. RESULTS: There were 2,602 patients who met the inclusion criteria ,of whom 264 (10.1%) received DC (either primary or secondary to another cranial procedure) and 109 (5%) had a DC that was primary. Variables associated with performing a DC included sex, race, intracranial pressure monitor placement, in-house trauma attending, traumatic subarachnoid hemorrhage, midline shift, and basal cistern compression. There was no survival benefit with early primary DC compared with the controls (relative risk, 1.07; 95% confidence interval, 0.67-1.73; p = 0.77), and resource use was higher. CONCLUSION: Early DC does not seem to significantly improve mortality in patients with refractory ICH compared with MT. Neurosurgeons should pause before entertaining this resource-demanding form of therapy. LEVEL OF EVIDENCE: Therapeutic care/management, level III.

Who should we feed? Western Trauma Association multi-institutional study of enteral nutrition in the open abdomen after injury.

BACKGROUND: The open abdomen is a requisite component of a damage control operation and treatment of abdominal compartment syndrome.Enteral nutrition (EN) has proven beneficial for patients with critical injury, but its application in those with an open abdomen has not been defined. The purpose of this study was to analyze the use of EN for patients with an open abdomen after trauma and the effect of EN on fascial closure rates and nosocomial infections. METHODS: We reviewed patients with an open abdomen after injury from January 2002 to January 2009 from 11 trauma centers. RESULTS: During the 7-year study period, 597 patients required an open abdomen after trauma. Most were men (77%) sustaining blunt trauma (72%), with a mean (SD) age of 38 (0.7) years, an Injury Severity Score of 31 (0.6), an abdominal injury score of 3.8(0.1), and an Abdominal Trauma Index score of 26.8 (0.6). Of the patients, 548 (92%) had an open abdomen after a damage control operation, whereas the remainder experienced an abdominal compartment syndrome. Of the 597 patients, 230 (39%)received EN initiated before the closure of the abdomen at mean (SD) day 3.6 (1.2) after injury. EN was started with an open abdomen in one quarter of the 290 patients with bowel injuries. For the 307 patients without a bowel injury, logistic regression indicated that EN is associated with higher fascial closure rates (odds ratio [OR], 5.3; p G 0.01), decreased complication rates(OR, 0.46; p = 0.02), and decreased mortality (OR, 0.30; p = 0.01). For the 290 patients who experienced a bowel injury,regression analysis showed no significant association between EN and fascial closure rate (OR, 0.6; p = 0.2), complication rate (OR, 1.7; p = 0.19), or mortality (OR, 0.79; p = 0.69). CONCLUSION: EN in the open abdomen after injury is feasible. For patients without a bowel injury, EN in the open abdomen is associated with increased fascial closure rates, decreased complication rates, and decreased mortality. EN should be initiated in these patients once resuscitation is completed. Although EN for patients with bowel injuries did not seem to affect the outcome in this study,prospective randomized controlled trials would further clarify the role of EN in this subgroup.

IL-7R alpha versus CCR7 and CD45 as markers of virus-specific CD8+ T cell differentiation: contrasting pictures in blood and tonsillar lymphoid tissue.

In humans, circulating CD8(+) memory T cells to a nonpersistent virus (influenza) lie within CCR7(+)CD45RA(-) central memory, whereas memory to Epstein-Barr virus (EBV) latent, EBV lytic, and cytomegalovirus (CMV) antigens are progressively larger in size and are more biased toward CCR7(-)CD45RA(-) effector memory and CCR7(-)CD45RA(+) terminally differentiated compartments. We found that these populations are also distinguished by progressively lower expression of the interleukin-7 receptor (IL-7R alpha) and by lower IL-7 responsiveness; indeed, percentage IL-7R alpha -positive values showed a tight inverse correlation with population size. However, these relationships among size, differentiation phenotype, and IL-7R alpha status in blood did not hold in tonsillar tissue. In tonsil tissue, although EBV reactivities outnumbered their CMV and influenza counterparts, the distinct CCR7/CD45 isoform signatures of the different virus-specific populations were retained. Moreover, all detectable reactivities showed high levels of IL-7R alpha expression. As a discriminator between different virus-specific populations, IL-7R alpha therefore appears to be more susceptible to tissue location than the classical CCR7/CD45 markers.

Benefits of non invasive ventilation.

Mechanical ventilation of the newborn infant has increased neonatal survival. However, this increased survival has come at the expense of increased morbidity, in the form of bronchopulmonary dysplasia, and at the cost of an expensive technology. Continuous positive airway pressure (CPAP) is accepted as conferring clinical benefit in supporting the recently extubated preterm infant and in the management of apnea of prematurity. Attention is now being drawn to physiologic and clinical evidence to support CPAP use, with or without early surfactant, as a primary treatment of hyaline membrane disease. The purpose of this review is to explore these proposed benefits of non invasive ventilation and place them in the context of current clinical evidence.

Factors influencing time from presentation to treatment of colorectal and breast cancer in urban and rural areas.

Stage at diagnosis and survival from cancer vary according to where people live, suggesting some may have delays in diagnosis. The aim of this study was to determine if time from presentation to treatment was longer for colorectal and breast cancer patients living further from cancer centres, and identify other important factors in delay. Data were collected on 1097 patients with breast and 1223 with colorectal cancer in north and northeast Scotland. Women with breast cancer who lived further from cancer centres were treated more quickly than those living closer to cancer centres (P=0.011). Multilevel modelling found that this was largely due to them receiving earlier treatment at hospitals other than cancer centres. Breast lump, change in skin contour, lymphadenopathy, more symptoms and signs, and increasing age predicted faster treatment. Screen detected cancers and private referrals were treated more quickly. For colorectal cancer, time to treatment was similar for people in rural and urban areas. Quicker treatment was associated with palpable rectal or abdominal masses, tenesmus, abdominal pain, frequent GP consultations, age between 50 and 74 years, tumours of the transverse colon, and iron medication at presentation. Delay was associated with past anxiety or depression. There was variation between general practices and treatment appeared quicker at practices with more female general practitioners.

Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping.

Tertiary structure in globular RNA folds can create local environments that lead to pKa perturbation of specific nucleotide functional groups. To assess the prevalence of functionally relevant adenosine-specific pKa perturbation in RNA structure, we have altered the nucleotide analog interference mapping (NAIM) approach to include a series of a phosphorothioate-tagged adenosine analogs with shifted N1 pKa values. We have used these analogs to analyze the hairpin ribozyme, a small self-cleaving/ligating RNA catalyst that is proposed to employ a general acid-base reaction mechanism. A single adenosine (A10) within the ribozyme active site displayed an interference pattern consistent with a functionally significant base ionization. The exocyclic amino group of a second adenosine (A38) contributes substantially to hairpin catalysis, but ionization of the nucleotide does not appear to be important for activity. Within the hairpin ribozyme crystal structure, A10 and A38 line opposite edges of a solvent-excluded cavity adjacent to the 5'-OH nucleophile. The results are inconsistent with the model of ribozyme chemistry in which A38 acts as a general acid-base catalyst, and suggest that the hairpin ribozyme uses an alternative mechanism to achieve catalytic rate enhancement that utilizes functional groups within a solvent-excluded cleft in the ribozyme active site.

Tertiary structure stability of the hairpin ribozyme in its natural and minimal forms: different energetic contributions from a ribose zipper motif.

The hairpin catalytic motif in tobacco ringspot virus satellite RNA consists of two helix-loop-helix elements on two adjacent arms of a four-way helical junction. The bases essential for catalytic activity are located in the loops that are brought into proximity by a conformational change as a prerequisite for catalysis. The two loops interact via a ribose zipper motif involving the 2'-hydroxyls of A10, G11, A24, and C25 [Rupert, P. B., and Ferre d'Amare, A. R. (2001) Nature 401, 780-786]. To quantify the energetic importance of the ribose zipper hydrogen bonds, we have incorporated deoxy modifications at these four positions and determined the resulting destabilization of the docked conformer by means of time-resolved fluorescence resonance energy transfer. In a minimal form of the ribozyme, in which the loops are placed on the arms of a two-way helical junction, all modifications lead to a significant loss in tertiary structure stability and altered Mg2+ binding. Surprisingly, no significant destabilization was seen with the natural four-way junction ribozyme, suggesting that hydrogen bonding interactions involving the 2'-hydroxyls do not contribute to the stability of the docked conformer. These results suggest that the energetic contributions of ribose zipper hydrogen bonds are highly context dependent and differ significantly for the minimal and natural forms of the ribozyme.

Loss of annexin II heavy and light chains in prostate cancer and its precursors.

Annexin II mRNA coding for a calcium binding protein was found to be absent in prostate cancer by subtractive hybridization and Northern analysis. In contrast to high expression in normal and benign hyperplastic glandular and basal epithelium, Annexin II heavy (p36) and light (p11) chains in 31/31 prostate cancer specimens were lost immunohistochemically. In glands involved by prostate intraepithelial neoplasia, 65% lost both chains in glandular epithelial cells, whereas basal cells were all positively stained. Southern analysis of cancer DNA showed no noticeable deletion in p36 gene. LNCaP cells treated with 5-azacytidine re-expressed p36, suggesting methylation could be responsible for the silencing.

Educational opportunities in palliative care: what do general practitioners want?

It is important to support general practitioners (GPs) in maintaining and developing their palliative care skills as most of the final year of a patient's life is spent at home under the care of the primary health care team. The training needs and uptake of GPs have been explored, but little is known about how GP educational preferences vary. The aim of this study was to explore the current educational preferences of GPs in different geographical locations as part of an evaluation of an educational intervention. The methods used included postal questionnaires sent to 1061 GPs. Results from 640 (60%) of GPs revealed that half (51%) wanted education in symptom control for non-cancer patients. More inner-city GPs wanted education in opiate prescribing (43%), controlling nausea and vomiting (45%), and using a syringe driver (38%) than their urban and rural colleagues (26%, 29% and 21%, respectively). Increased educational preference and increased difficulty in accessing information was associated with reduced confidence in symptom control. To maximize educational uptake it will be important for educational strategies to be developed and targeted according to variations in demand, and in particular to respond to the need for palliative care education in symptom control for patients suffering from advanced non-malignant disease.

Cholera toxin and Escherichia coli enterotoxin B-subunits inhibit macrophage-mediated antigen processing and presentation: evidence for antigen persistence in non-acidic recycling endosomal compartments.

Cholera toxin (Ctx) and the closely related Escherichia coli heat-labile enterotoxin (Etx) not only act as mediators of diarrhoeal disease but also exert potent immunomodulatory properties on mammalian immune systems. The toxins normally exert their diarrhoeagenic effects by initiating receptor-mediated uptake into vesicles that enter a retrograde trafficking pathway, circumventing degradative compartments and targeting them to the trans-Golgi network (TGN) and endoplasmic reticulum. Here, we examine whether receptor-mediated binding and cellular entry by the toxin B-subunits also lead to concomitant changes in uptake and trafficking of exogenous antigens that could contribute to the potent immunomodulatory properties of these toxins. Treatment of the macrophage (J774.2) cell line with Etx B-subunit (EtxB) resulted in EtxB transport to the TGN and also led to the formation of large, translucent, non-acidic, EtxB-devoid vacuoles. When exogenous antigens were added, EtxB-treated cells were found to be proficient in both internalization of ovalbumin (OVA) and phagocytosis of bacterial particles. However, the internalized OVA, instead of trafficking along a lysosome-directed endocytic pathway via acidified endosomes, persisted in a non-acidic, light-density compartment that was distinct from the translucent vacuoles. The rerouted OVA did not co-localize with the endosomal markers rab5 or rab11, nor with EtxB, but was retained in a transferrin receptor-positive compartment. The failure of OVA to enter the late endosomal/lysosomal compartments correlated with a striking inhibition of OVA peptide processing and presentation to OVA-responsive CD4+ T-cells. CtxB also modulated OVA trafficking and inhibited antigen presentation. These findings demonstrate that the B-subunits of Ctx and Etx alter the progression of exogenous antigens along the endocytic processing pathway, and prevent or delay efficient epitope presentation and T-cell stimulation. The formation of such 'antigen depots' could contribute to the immunomodulatory properties of these bacterial virulence determinants.

Bcl10 is a positive regulator of antigen receptor-induced activation of NF-kappaB and neural tube closure.

Bcl10, a CARD-containing protein identified from the t(1;14)(p22;q32) breakpoint in MALT lymphomas, has been shown to induce apoptosis and activate NF-kappaB in vitro. We show that one-third of bcl10-/- embryos developed exencephaly, leading to embryonic lethality. Surprisingly, bcl10-/- cells retained susceptibility to various apoptotic stimuli in vivo and in vitro. However, surviving bcl10-/- mice were severely immunodeficient and bcl10-/- lymphocytes are defective in antigen receptor or PMA/Ionomycin-induced activation. Early tyrosine phosphorylation, MAPK and AP-1 activation, and Ca2+ signaling were normal in mutant lymphocytes, but antigen receptor-induced NF-kappaB activation was absent. Thus, Bcl10 functions as a positive regulator of lymphocyte proliferation that specifically connects antigen receptor signaling in B and T cells to NF-kappaB activation.

Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein.

To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination. The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively. Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy. The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation. The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.