Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. This two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively.

Purpose of review Risk assessment tools are essential in COPD care to help clinicians identify patients at higher risk of accelerated lung function decline, respiratory exacerbations, hospitalizations, and death. Recent findings Conventional methods of assessing risk have focused on spirometry, patient-reported symptoms, functional status, and a combination of these tools in composite indices. More recently, qualitatively and quantitatively assessed chest imaging findings, such as emphysema, large and small airways disease, and pulmonary vascular abnormalities have been associated with poor long-term outcomes in COPD patients. Although several blood and sputum biomarkers have been investigated for risk assessment in COPD, most still warrant further validation. Finally, novel remote digital monitoring technologies may be valuable to predict exacerbations but their large-scale performance, ease of implementation, and cost effectiveness remain to be determined. Summary Given the complex heterogeneity of COPD, any single metric is unlikely to fully capture the risk of poor long-term outcomes. Therefore, clinicians should review all available clinical data, including spirometry, symptom severity, functional status, chest imaging, and bloodwork, to guide personalized preventive care of COPD patients. The potential of machine learning tools and remote monitoring technologies to refine COPD risk assessment is promising but remains largely untapped pending further investigation.

Purpose: To evaluate national trends in nonvascular invasive radiology procedures performed by advanced practice providers (APPs), focusing specifically on nurse practitioners and physician assistants.Methods: Nonvascular invasive radiology procedures commonly performed by APPs at our 2 largest hospitals were used to identify procedure groups for national trends analysis. We mapped categories of services annually to then-current Current Procedural Terminology codes from 1994 to 2012 and identified national Medicare Part B beneficiary paid claims frequency using Physician Supplier Procedure Summary Master Files. Trends were studied for APPs, radiologists, and all providers nationally for 7 categories of service: paracentesis, thoracentesis, fine-needle aspiration (FNA), superficial lymph node biopsy, abdominal biopsy, thoracic biopsy, and abdominal drainage.Results: Of 1,352 nonvascular invasive procedures performed by APPs at our facilities over a 1-year period through August 2013, a total of 1,161 (85.9%) fell into the 7 defined categories. Between 1994 and 2012, national Medicare claims by APPs increased dramatically for all of these categories: paracentesis from 0 to 17,967; thoracentesis from 119 to 4,141 (+3,379%); FNA from 0 to 3,921; superficial lymph node biopsy from 0 to 251; abdominal biopsy from 1 to 1,819 (+1,818%); thoracic biopsy from 0 to 552; and abdominal drainage from 37 to 410 (+1,008%). Overall, volumes increased for both radiologists and all providers, with the total fraction of national services performed by APPs increasing from 0% to 10.7% for paracentesis, 0.1% to 5.7% for thoracentesis, 0% to 2.1% for FNA, 0% to 1.4% for superficial lymph node biopsy, 0% to 1.7% for abdominal biopsy, 0% to 1.0% for thoracic biopsy, and 0.1% to 1.2% for abdominal drainage.Conclusions: Although APPs perform a relatively small portion of commonly performed nonvascular invasive radiology procedures nationally, paid Medicare claims for those services have increased dramatically over nearly 2 decades, and at a faster pace than that for all Providers as a whole. Given the multiple hurdles involved in obtaining Medicare reimbursement, that growth indicates increasing acceptance of APPs as procedure service providers at the institutional credentialing, state licensure, and payer policy levels. Copyright (C) 2015 American College of Radiology

Purpose: To evaluate national trends in percutaneous hepatic and renal biopsy procedures with regard to utilization, specialty group roles, and sites of service.Materials and Methods: Service-specific claims data for percutaneous hepatic and renal biopsy procedures were identified using Medicare Physician Supplier Procedure Summary Master Files for the period 1994-2012. Longitudinal national utilization rates were calculated using annual Medicare enrollment data. Procedure volumes by specialty group and site of service were analyzed.Results: Between 1994 and 2012, the number of hepatic and renal biopsies performed on Medicare Part B beneficiaries increased 22% (from 43,478 to 53,055) and 68% (19,508 to 32,762), respectively. Per 100,000 beneficiaries, the utilization of hepatic and renal biopsy increased 19.6% (from 134.6 to 161.0) and 69.3% (from 60.4 to 102.2). Procedures performed by radiologists disproportionately,increased 81% (from 25,484 to 46,181) and 236% (from 6,855 to 23,003), respectively. Although utilization in the inpatient setting declined 28.7% (from 68.2 to 48.6 per 100,000) for hepatic biopsies and 9.4% (from 43.1 to 39.1) for renal biopsies, there Were larger concurrent increases of 73.9% (from 59.2 to 103.0) and 303.9% (from 15.1 to 61.0) in utilization in the outpatient setting.Conclusions: Between 1994 and 2012, national utilization of percutaneous hepatic and renal biopsy procedures in the Medicare population increased as services increasingly shifted from the hospital inpatient to outpatient setting. Radiologists are presently and increasingly the dominant providers of both services.

Purpose: The aim of this study was to evaluate changes in diagnostic radiology resident and fellow workloads in recent years.Methods: Berenson-Eggers Type of Service categorization was applied to Medicare Part B Physician/Supplier Procedure Summary Master Files to identify total and resident-specific claims for radiologist imaging services between 1998 and 2010. Data were extracted and subgroup analytics performed by modality. Volumes were annually normalized for active diagnostic radiology trainees.Results: From 1998 to 2010, Medicare claims for imaging services rendered by radiologists increased from 78,901,255 to 105,252,599 (+33.4%). Service volumes increased across all modalities: for radiography from 55,661,683 to 59,654,659 (+7.2%), for mammography from 5,780,624 to 6,570,673 (+13.7%), for ultrasound from 5,851,864 to 9,853,459 (+68.4%), for CT from 9,351,780 to 22,527,488 (+140.9%), and for MR from 2,255,304 to 6,646,320 (+194.7%). Total trainee services nationally increased 3 dines as rapidly. On an average per trainee basis, however, the average number of diagnostic services rendered annually to Medicare Part B beneficiaries increased from 499 to 629 (+26.1%). By modality, this represents an average change from 333 to 306 examinations (-8.1%) for radiography, from 20 to 18 (-7.4%) for mammography, from 37 to 56 (+49.7%) for ultrasound, from 88 to 202 (+129.1%) for CT, and from 20 to 47 (+132.0%) for MRI.Conclusions: Between 1998 and 2010, the number of imaging examinations interpreted by diagnostic radiology residents and fellows on Medicare beneficiaries increased on average by 26% per trainee, with growth largely accounted for by disproportionate increases in more complex services (CT and MRI).

A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na+. The absorption spectrum of GLR is insensitive to Na+ at concentrations of ≤3 M. However, very low concentrations of Na+ cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a “Na+-independent” to a “Na+-dependent” photocycle (or photocycle branch) at ∼60 μM Na+. The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na+ concentration. This suggests that a high-affinity Na+ binding site is created transiently after photoexcitation, and entry of Na+ from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na+ is needed for switching the reaction path at lower pH. The data suggest therefore competition between H+ and Na+ to determine the two alternative pathways. The idea that a Na+ binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na+ binds without photoexcitation. Binding of Na+ to the mutant shifts the chromophore maximum to the red like that of H+, which occurs in the photocycle of the wild type.

The photocycle of the retinal protein from Exiguobacterium sibiricum, which differs from bacteriorhodopsin in both its primary donor and acceptor, is characterized by visible and infrared spectroscopy. At pH above pKa ~6.5, we find a bacteriorhodopsin-like photocycle, which originates from excitation of the all-trans retinal chromophore with K-, L-, M-, and N-like intermediates. At pH below pKa ~6.5, the M state, which reflects Schiff base deprotonation during proton pumping, is not accumulated. However, using the infrared band at ~1760 cm(-1) as a marker for transient protonation of the primary acceptor, we find that Schiff base deprotonation must have occurred at pH not only above but also below the pKa ~6.5. Thus, the M state is formed but not accumulated for kinetic reasons. Further, chromophore reisomerization from the 13-cis to the all-trans conformation occurs very late in the photocycle. The strongly red-shifted states that dominate the second half of the cycle are produced before the reisomerization step, and by this criterion, they are not O-like but rather N-like states. The assignment of photocycle intermediates enables reevaluation of the photocycle; its specific features are discussed in relation to the general mechanism of proton transport in retinal proteins.

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pK(a) of 8.5 +/- 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pK(a) of 8.1 +/- 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the is an element of-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ϵ-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.Background: Lysine rather than a carboxylic residue is in place of the internal proton donor in the E. sibiricum proton pump.Results: H+ uptake precedes reprotonation of the retinal Schiff base. K96A mutation slows it by >100-fold.Conclusion: Lysine 96 facilitates proton delivery to the Schiff base.Significance: This is the first example where lysine mediates proton transfer to the retinal Schiff base. A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ϵ-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein. Background: Lysine rather than a carboxylic residue is in place of the internal proton donor in the E. sibiricum proton pump. Results: H+ uptake precedes reprotonation of the retinal Schiff base. K96A mutation slows it by >100-fold. Conclusion: Lysine 96 facilitates proton delivery to the Schiff base. Significance: This is the first example where lysine mediates proton transfer to the retinal Schiff base. An Unusual Lysine Acts Like a Proton Donor in a Bacterial Proton Pump♦: Breaking the Carboxyl Rule. Lysine 96 Facilitates Reprotonation of the Schiff Base in the Photocycle of a Retinal Protein from Journal of Biological ChemistryVol. 288Issue 29Preview♦ See referenced article, J. Biol. Chem. 2013, 288, 21254–21265 Full-Text PDF Open Access

— 454 — The Auk, Vol. , Number , pages  . ISSN -, electronic ISSN -.  by The American Ornithologists’ Union. All rights reserved. Please direct all requests for permission to photocopy or reproduce article content through the University of California Press’s Rights and Permissions website, http://www.ucpressjournals. com/reprintInfo.asp. DOI: ./auk.. 1E-mail: quecher@yahoo.com Early studies of incubation behavior acknowledged the existence of a less regular form of incubation that can occur from the beginning of egg laying to shortly after clutch completion. It has been variously termed “partial incubation” (Putnam , Seel , Ashkenazie and Safriel ), “brooding” (Barth ), “intermittent incubation” (Samuel ), “irregular incubation” (Beer ), or “nonrhythmic incubation” (Morton et al. ). This form of incubation has been described from visual observations of adults sitting on the nest less regularly, and from egg temperatures that are lower and more variable than those typically attained after clutch completion (Barth , Brackbill , Kendeigh , Barrett , Morton and Pereyra ). We refer to this little-understood behavior as “partial incubation” because it is characterized by a reduced amount of time during egg laying that parents sit on and warm their eggs, compared with the amount and intensity of incubation they provide later in the nesting cycle after the clutch is complete. Partial incubation is a common way for birds to initiate incubation. The onset of incubation, the moment at which a bird begins to incubate a clutch, is a critical event in the avian nesting cycle because it has important effects on fitness, as noted more than half a century ago by David Lack (). Incubation onset can affect hatching success and hatching patterns (Nilsson and Svensson , Wang and Beissinger ), nestling growth and development (Slagsvold et al. , Bitton et al. ), fledging success (Hébert , Hébert and McNeil ), and postfledging survival and recruitment (Cam et al. ). Not only can the onset of incubation affect adult reproductive rates, but it also influences and is influenced by parental body condition and survival (Hanssen et al. ). Yet the onset of incubation is rarely described and even less commonly quantified, and methods of determining incubation onset have not been standardized among studies or species. In this review, we () clarify terminology for the onset of incubation, refine the concept of partial incubation, and summarize the prevalence of partial incubation. We then () examine the PARTIAL INCUBATION IN BIRDS: ITS OCCURRENCE, FUNCTION,

Salinixanthin, a C40-carotenoid acyl glycoside, serves as a light-harvesting antenna in the retinal-based proton pump xanthorhodopsin of Salinibacter ruber. In the crystallographic structure of this protein, the conjugated chain of salinixanthin is located at the protein–lipid boundary and interacts with residues of helices E and F. Its ring, with a 4-keto group, is rotated relative to the plane of the π-system of the carotenoid polyene chain and immobilized in a binding site near the β-ionone retinal ring. We show here that the carotenoid can be removed by oxidation with ammonium persulfate, with little effect on the other chromophore, retinal. The characteristic CD bands attributed to bound salinixanthin are now absent. The kinetics of the photocycle is only slightly perturbed, showing a 1.5-fold decrease in the overall turnover rate. The carotenoid-free protein can be reconstituted with salinixanthin extracted from the cell membrane of S. ruber. Reconstitution is accompanied by restoration of the characteristic vibronic structure of the absorption spectrum of the antenna carotenoid, its chirality, and the excited-state energy transfer to the retinal. Minor modification of salinixanthin, by reducing the carbonyl C=O double bond in the ring to a C-OH, suppresses its binding to the protein and eliminates the antenna function. This indicates that the presence of the 4-keto group is critical for carotenoid binding and efficient energy transfer.

Early studies of incubation behavior acknowledged the existence of a less regular form of incubation that can occur from the beginning of egg laying to shortly after clutch completion. It has been variously termed “partial incubation” (Putnam , Seel , Ashkenazie and Safriel ), “brooding” (Barth ), “intermittent incubation” (Samuel ), “irregular incubation” (Beer ), or “nonrhythmic incubation” (Morton et al. ). This form of incubation has been described from visual observations of adults sitting on the nest less regularly, and from egg temperatures that are lower and more variable than those typically attained after clutch completion (Barth , Brackbill , Kendeigh , Barrett , Morton and Pereyra ). We refer to this little-understood behavior as “partial incubation” because it is characterized by a reduced amount of time during egg laying that parents sit on and warm their eggs, compared with the amount and intensity of incubation they provide later in the nesting cycle after the clutch is complete. Partial incubation is a common way for birds to initiate incubation. The onset of incubation, the moment at which a bird begins to incubate a clutch, is a critical event in the avian nesting cycle because it has important effects on fitness, as noted more than half a century ago by David Lack (). Incubation onset can affect hatching success and hatching patterns (Nilsson and Svensson , Wang and Beissinger ), nestling growth and development (Slagsvold et al. , Bitton et al. ), fledging success (Hebert , Hebert and McNeil ), and postfledging survival and recruitment (Cam et al. ). Not only can the onset of incubation affect adult reproductive rates, but it also influences and is influenced by parental body condition and survival (Hanssen et al. ). Yet the onset of incubation is rarely described and even less commonly quantified, and methods of determining incubation onset have not been standardized among studies or species. In this review, we () clarify terminology for the onset of incubation, refine the concept of partial incubation, and summarize the prevalence of partial incubation. We then () examine the potential functions of partial incubation, () map individual-level patterns of partial incubation from previous studies onto a recently developed typology of patterns for the onset of incubation (Wang and Beissinger ), and examine intraspecific variability and compare interspecific data with respect to taxonomic and ecological diversity. We also () provide a standardized method for determining the individual onset of full incubation, using complete records of nest attendance or incubation from the start of laying to beyond clutch completion. Our goal is to show how data from disparate studies can be used to test hypotheses about the function of partial incubation. Finally, we () offer questions that may prove to be fertile areas of research and suggest ways to standardize future data collection and analysis to benefit common research goals. TERMINOLOGY FOR THE ONSET OF INCUBATION

The viability of freshly laid avian eggs declines after several days of exposure to ambient temperatures above physiological zero, and declines occur faster in tropical than temperate ecosystems. Microbial infection during preincubation exposure has recently been shown as a second cause of egg viability decline in the tropics, but whether microbial processes influence the viability of wild bird eggs in temperate ecosystems is unknown. We determined the microbial load on eggshells, the incidence of microbial penetration of egg contents, and changes in the viability of wild bird eggs (Sialia mexicana, Tachycineta bicolor, Tachycineta thalassina) experimentally exposed to temperate-zone ambient conditions in situ in a mediterranean climate in northern California. Initial microbial loads on eggshells were generally low, although they were significantly higher on eggs laid in old boxes than in new boxes. Eggshell microbial loads did not increase with exposure to ambient conditions, were not reduced by twice-daily disinfection with alcohol, and were unaffected by parental incubation. The rate of microbial penetration into egg contents was low and unaffected by the duration of exposure. Nevertheless, egg viability declined very gradually and significantly with exposure duration, and the rate of decline differed among species. In contrast to studies performed in the tropics, we found little evidence that temperature or microbial mechanisms of egg viability decline were important at our temperate-zone site; neither temperatures above physiological zero nor alcohol disinfection was significantly related to hatching success. Delaying the onset of incubation until the penultimate or last egg of a clutch at our study site may maintain hatching synchrony without a large trade-off in egg viability. These results provide insight into the environmental mechanisms that may be responsible for large-scale latitudinal patterns in avian clutch size and hatching asynchrony.

Low-temperature FTIR spectroscopy of bacteriorhodopsin and xanthorhodopsin was used to elucidate the number of K-like bathochromic states, their sequence, and their contributions to the photoequilibrium mixtures created by illumination at 80-180 K. We conclude that in bacteriorhodopsin the photocycle includes three distinct K-like states in the sequence bR (hv)--> I* --> J --> K(0) --> K(E) --> L --> ..., and similarly in xanthorhodopsin. K(0) is the main fraction in the mixture at 77 K that is formed from J. K(0) becomes thermally unstable above approximately 50 K in both proteins. At 77 K, both J-to-K(0) and K(0)-to-K(E) transitions occur and, contrarily to long-standing belief, cryogenic trapping at 77 K does not produce a pure K state but a mixture of the two states, K(0) and K(E), with contributions from K(E) of approximately 15 and approximately 10% in the two retinal proteins, respectively. Raising the temperature leads to increasing conversion of K(0) to K(E), and the two states coexist (without contamination from non-K-like states) in the 80-140 K range in bacteriorhodopsin, and in the 80-190 K range in xanthorhodopsin. Temperature perturbation experiments in these regions of coexistence revealed that, in spite of the observation of apparently stable mixtures of K(0) and K(E), the two states are not in thermally controlled equilibrium. The K(0)-to-K(E) transition is unidirectional, and the partial transformation to K(E) is due to distributed kinetics, which governs the photocycle dynamics at temperatures below approximately 245 K (Dioumaev and Lanyi, Biochemistry 2008, 47, 11125-11133). From spectral deconvolution, we conclude that the K(E) state, which is increasingly present at higher temperatures, is the same intermediate that is detected by time-resolved FTIR prior to its decay, on a time scale of hundreds of nanoseconds at ambient temperature (Dioumaev and Braiman, J. Phys. Chem. B 1997, 101, 1655-1662), into the K(L) state. We were unable to trap the latter separately from K(E) at low temperature, due to the slow distributed kinetics and the increasingly faster overlapping formation of the L state. Formation of the two consecutive K-like states in both proteins is accompanied by distortion of two different weakly bound water molecules: one in K(0), the other in K(E). The first, well-documented in bacteriorhodopsin at 77 K where K(0) dominates, was assigned to water 401 in bacteriorhodopsin. The other water molecule, whose participation has not been described previously, is disturbed on the next step of the photocycle, in K(E), in both proteins. In bacteriorhodopsin, the most likely candidate is water 407. However, unlike bacteriorhodopsin, the crystal structure of xanthorhodopsin lacks homologous weakly bound water molecules.

We present the first detailed information on egg laying, egg temperature (T-egg), and development of attentiveness and incubation in Western Bluebirds (Sialia mexicana). We used miniature infrared CCD video cameras to record egg laying at three nest boxes and validate attentiveness measurements derived from T-egg in seven other nests. Females entered the nest box on egg-laying days between 0522 and 1037 hrs PST and laid an egg on average 11.4 +/- 4.3 min later (n = 11). Warm weather early in egg laying often elevated T-egg above physiological zero (27 degrees C), despite low parental attendance. Females often began roosting in the nest box 2-3 nights before clutch completion, and most birds began steady night-time incubation with the penultimate egg. Attentiveness, mean T-egg, and number of minutes T-egg exceeded 27 degrees C reached their highest values I day or night after clutch completion. Western Bluebirds exhibit gradual onset to incubation that is attributable to vagaries of weather and varying patterns of parental attentiveness.

Variation among species in the onset of incubation has been attributed to differences in life history traits, and variation within species has been related to individual and environmental factors. We quantified within- and among-species variation in the onset of incubation in five cavity-nesting passerines, using a continuous record of diurnal and nocturnal incubation from clutch initiation through completion. We documented 11 potential patterns for the onset of incubation and showed that onset patterns were significantly related to hatching success. The onset of diurnal partial incubation and nocturnal full incubation generally occurred before diurnal full incubation, which started around clutch completion. Increases in precipitation or wind speed significantly delayed most types of incubation onset, supporting predictions of the energy constraints hypothesis. Ancillary predictions of rain and wind disproportionately delaying incubation for aerial foragers, and for species with male feeding during incubation, were not upheld. Larger clutch size accelerated the timing of full incubation onset in diurnal and nocturnal full incubation, supporting predictions of the egg viability hypothesis. Predictions of both hypotheses for the effects of minimum temperature, proportion of time above 24 degrees C, and seasonality on incubation onset were not supported. We observed egg neglect for up to 4 days or nights; neglect was more common at night, and consecutive nights of neglect occurred during nocturnal full incubation. Egg neglect did not significantly affect hatching success or incubation period. In conclusion, the timing of incubation onset was strongly affected by environmental and individual factors, and patterns of incubation onset affected hatching success. (C) 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Matthew D. Shawkey, Karl L. Kosciuch, Mark Liu, Frank C. Rohwer, Elizabeth R. Loos, Jennifer M. Wang, and Steven R. Beissinger Department of Environmental Science, Policy and Management, 137 Mulford Hall #3114, University of California, Berkeley, CA 94720-3114, USA, Division of Biology, Kansas State University, Manhattan, KS 66506, USA, Department of Biological Sciences, Auburn University, 331 Funchess Hall, Auburn, AL 36849, USA, School of Renewable Natural Resources, Louisiana State University and Louisiana State University Agricultural Center, Baton Rouge, LA 70803, and Delta Waterfowl, PO Box 3124, Bismarck, ND 38502, USA

Xanthorhodopsin is a light-driven proton pump in the extremely halophilic bacterium Salinibacter ruber. Its unique feature is that besides retinal it has a carotenoid, salinixanthin, with a light harvesting function. Tight and specific binding of the carotenoid antenna is controlled by binding of the retinal. Addition of all-trans retinal to xanthorhodopsin bleached with hydroxylamine restores not only the retinal chromophore absorption band, but causes sharpening of the salinixanthin bands reflecting its rigid binding by the protein. In this report we examine the correlation of the changes in the two chromophores during bleaching and reconstitution with native all-trans retinal, artificial retinal analogs and retinol. Bleaching and reconstitution both appear to be multistage processes. The carotenoid absorption changes during bleaching occurred not only upon hydrolysis of the Schiff base but continued while the retinal was leaving its binding site. In the case of reconstitution, the 13-desmethyl analog formed the protonated Schiff base slower than retinal, and provided the opportunity to observe changes in carotenoid binding at various stages. The characteristic sharpening of the carotenoid bands, indicative of its reduced conformational heterogeneity in the binding site, occurs when the retinal occupies the binding site but the covalent bond to Lys-240 via a Schiff base is not yet formed. This is confirmed by the results for retinol reconstitution, where the Schiff base does not form but the carotenoid exhibits its characteristic spectral change from the binding.

The cell membrane of Salinibacter ruber contains xanthorhodopsin, a light-driven transmembrane proton pump with two chromophores: a retinal and the carotenoid, salinixanthin. Action spectra for transport had indicated that light absorbed by either is utilized for function. If the carotenoid is an antenna in this protein, its excited state energy has to be transferred to the retinal and should be detected in the retinal fluorescence. From fluorescence studies, we show that energy transfer occurs from the excited singlet S-2 state of salinixanthin to the S-1 state of the retinal. Comparison of the absorption spectrum with the excitation spectrum for retinal emission yields 45 +/- 5% efficiency for the energy transfer. Such high efficiency would require close proximity and favorable geometry for the two polyene chains, but from the heptahelical crystallographic structure of the homologous retinal protein, bacteriorhodopsin, it is not clear where the carotenoid can be located near the retinal. The fluorescence excitation anisotropy spectrum reveals that the angle between their transition dipole moments is 56 +/- 3 degrees. The protein accommodates the carotenoid as a second chromophore in a distinct binding site to harvest light with both extended wavelength and polarization ranges. The results establish xanthorhodopsin as the simplest biological excited-state donor-acceptor system for collecting light.

Homologous to bacteriorhodopsin and even more to proteorhodopsin, xanthorhodopsin is a light-driven proton pump that, in addition to retinal, contains a noncovalently bound carotenoid with a function of a light-harvesting antenna. We determined the structure of this eubacterial membrane protein-carotenoid complex by X-ray diffraction, to 1.9-A resolution. Although it contains 7 transmembrane helices like bacteriorhodopsin and archaerhodopsin, the structure of xanthorhodopsin is considerably different from the 2 archaeal proteins. The crystallographic model for this rhodopsin introduces structural motifs for proton transfer during the reaction cycle, particularly for proton release, that are dramatically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine-aspartate complex for regulating the pK(a) of the primary proton acceptor not present in archaeal pumps but apparently conserved in eubacterial pumps. In addition to aiding elucidation of a more general proton transfer mechanism for light-driven energy transducers, the structure defines also the geometry of the carotenoid and the retinal. The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as approximately 45%, and the 46 degrees angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer.