A machine learning photon detection algorithm for coherent x-ray ultrafast fluctuation analysis.

X-ray free electron laser experiments have brought unique capabilities and opened new directions in research, such as creating new states of matter or directly measuring atomic motion. One such area is the ability to use finely spaced sets of coherent x-ray pulses to be compared after scattering from a dynamic system at different times. This enables the study of fluctuations in many-body quantum systems at the level of the ultrafast pulse durations, but this method has been limited to a select number of examples and required complex and advanced analytical tools. By applying a new methodology to this problem, we have made qualitative advances in three separate areas that will likely also find application to new fields. As compared to the "droplet-type" models, which typically are used to estimate the photon distributions on pixelated detectors to obtain the coherent x-ray speckle patterns, our algorithm achieves an order of magnitude speedup on CPU hardware and two orders of magnitude improvement on GPU hardware. We also find that it retains accuracy in low-contrast conditions, which is the typical regime for many experiments in structural dynamics. Finally, it can predict photon distributions in high average-intensity applications, a regime which up until now has not been accessible. Our artificial intelligence-assisted algorithm will enable a wider adoption of x-ray coherence spectroscopies, by both automating previously challenging analyses and enabling new experiments that were not otherwise feasible without the developments described in this work.

Mechanism and dynamics of fatty acid photodecarboxylase.

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

Electron Ghost Imaging.

In this Letter we report a demonstration of electron ghost imaging. A digital micromirror device directly modulates the photocathode drive laser to control the transverse distribution of a relativistic electron beam incident on a sample. Correlating the structured illumination pattern to the total sample transmission then retrieves the target image, avoiding the need for a pixelated detector. In our example, we use a compressed sensing framework to improve the reconstruction quality and reduce the number of shots compared to raster scanning a small beam across the target. Compressed electron ghost imaging can reduce both acquisition time and sample damage in experiments for which spatially resolved detectors are unavailable (e.g., spectroscopy) or in which the experimental architecture precludes full frame direct imaging.

Femtosecond mega-electron-volt electron microdiffraction.

To understand and control the basic functions of physical, chemical and biological processes from micron to nano-meter scale, an instrument capable of visualizing transient structural changes of inhomogeneous materials with atomic spatial and temporal resolutions, is required. One such technique is femtosecond electron microdiffraction, in which a short electron pulse with femtosecond-scale duration is focused into a micron-scale spot and used to obtain diffraction images to resolve ultrafast structural dynamics over a localized crystalline domain. In this letter, we report the experimental demonstration of time-resolved mega-electron-volt electron microdiffraction which achieves a 5 µm root-mean-square (rms) beam size on the sample and a 110 fs rms temporal resolution. Using pulses of 10k electrons at 4.2 MeV energy with a normalized emittance 3 nm-rad, we obtained high quality diffraction from a single 10 µm paraffin (C44H90) crystal. The phonon softening mode in optical-pumped polycrystalline Bi was also time-resolved, demonstrating the temporal resolution limits of the instrument. This new characterization capability will open many research opportunities in material and biological sciences.

Data systems for the Linac coherent light source.

The data systems for X-ray free-electron laser (FEL) experiments at the Linac coherent light source (LCLS) are described. These systems are designed to acquire and to reliably transport shot-by-shot data at a peak throughput of 5 GB/s to the offline data storage where experimental data and the relevant metadata are archived and made available for user analysis. The analysis and monitoring implementation (AMI) and Photon Science ANAlysis (psana) software packages are described. Psana is open source and freely available.

Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY.

The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. Here, we report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structure of the excited state ensemble. This prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. Using these results, we then predict incisive single molecule FRET experiments as a means of model validation. This study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.

Imaging Molecular Motion: Femtosecond X-Ray Scattering of an Electrocyclic Chemical Reaction.

Structural rearrangements within single molecules occur on ultrafast time scales. Many aspects of molecular dynamics, such as the energy flow through excited states, have been studied using spectroscopic techniques, yet the goal to watch molecules evolve their geometrical structure in real time remains challenging. By mapping nuclear motions using femtosecond x-ray pulses, we have created real-space representations of the evolving dynamics during a well-known chemical reaction and show a series of time-sorted structural snapshots produced by ultrafast time-resolved hard x-ray scattering. A computational analysis optimally matches the series of scattering patterns produced by the x rays to a multitude of potential reaction paths. In so doing, we have made a critical step toward the goal of viewing chemical reactions on femtosecond time scales, opening a new direction in studies of ultrafast chemical reactions in the gas phase.

Characterization of a variant erythrocyte surface antigen (VESA1) expressed by Babesia bovis during antigenic variation.

Babesia bovis, an intraerythrocytic, protozoal parasite of cattle, undergoes clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91-98). This ability could provide a mechanism by which the parasite escapes host immune defenses to establish chronic infection. Previous work identified two parasite-derived antigens of Mr 128,000 and 113,000 that were present on the surface of the infected erythrocyte and appeared to be associated with clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91 98). Two monoclonal antibodies (mAbs), 3F7.1H11 and 4D9.1G1, which recognize the variant erythrocyte surface antigen (VESA1) have been identified. These mAbs react only with the surface of erythrocytes infected with the B. bovis C9.1 clone in live-cell immunofluorescence assays. In both conventional and surface immunoprecipitations, the mAbs precipitate a variant antigen doublet that matches in mass the infected red blood cell (IRBC) surface antigens precipitated with bovine serum. In contrast, Western blot analysis revealed that only the Mr 128,000 polypeptide is recognized by the mAbs. Neither mAb recognizes antigenically variant progenitor or progeny parasite clones in any of the immunoassays, confirming the involvement of this antigen in rapid clonal antigenic variation. Failure to label this antigen with [9,10(n)-3H]myristic acid, [9,10(n)-3H]palmitic acid or D-[6-3H]glucosamine indicates that these polypeptides are neither N-glycosylated nor fatty acylated. Identity of the variant antigen recognized by the mAbs with that putatively identified with immune serum was confirmed by comparison of partial proteolytic digestion products. Unambiguous identification of the VESA1 antigen as a component of antigenic variation will facilitate characterization of the events leading to antigenic variation on the B. bovis-infected erythrocyte surface and its significance to parasite survival during chronic infection.

Effects of prepubertal gonadectomy on physical and behavioral development in cats.

OBJECTIVE: To determine the effect of prepubertal gonadectomy on physical and behavioral development in cats. DESIGN: Prospective controlled study of kittens randomly assigned to 3 treatment groups: group 1, neutered at 7 weeks of age; group 2, neutered at 7 months of age; and group 3, sexually intact controls. ANIMALS: 31 clinically normal male and female kittens. PROCEDURE: Age at distal radial physeal closure and mature radius length were determined radiographically. Six behavioral characteristics were recorded monthly. At 1 year of age, body weight was recorded and thickness of the falciform ligament was measured from a lateral abdominal radiographic view. Secondary sex characteristics were also examined at 1 year of age. RESULTS: There were no differences between group-1 and group-2 cats for any of the study variables. Sexually intact cats (group 3) weighted significantly less than group-2 cats and had less falciform fat and earlier distal radial physeal closure than cats of both neutered groups. Group-3 cats manifested greater intraspecies aggression, less affection, and greater development of secondary sex characteristics than neutered cats. CLINICAL IMPLICATIONS: Neutering cats at 7 weeks of age had similar effects on physical and behavioral development, compared with neutering at the more traditional age of 7 months. These data lend support to the concept of prepubertal gonadectomy, already performed by many animal shelters/humane organizations, as a method of enhancing the effectiveness of pet population control programs.

Alpha-delta sleep in patients with a chief complaint of chronic fatigue.

Our prospective, standardized cohort study was designed to assess the presence of alpha wave intrusions during non-rapid eye movement sleep (alpha-delta sleep) and its relationship to fibromyalgia, major depression, and chronic fatigue syndrome (CFS) in patients with a chief complaint of chronic fatigue. The study group comprised 30 consecutive patients seen at a university hospital referral clinic for evaluation of chronic fatigue. All patients had nocturnal polysomnography, dolorimetric tender point assessment for fibromyalgia, a comprehensive history, physical, and laboratory evaluation, and a structured psychiatric interview. Alpha-delta sleep was identified in 8 of the 30 patients (26%), major depression in 20 (67%), CFS in 15 (50%), and fibromyalgia in 4 (13%). Ten of the 30 patients (33%) had a primary sleep disorder (sleep apnea, periodic limb movements, or narcolepsy). Alpha-delta sleep was not significantly correlated with fibromyalgia, CFS, major depression, or primary sleep disorders, but was significantly more common among patients who had chronic fatigue without major depression. We conclude that primary sleep disorders are relatively common among patients with chronic fatigue and must be diligently sought and treated. Alpha-delta sleep is not a marker of fibromyalgia or CFS, but may contribute to the illness of nondepressed patients with these conditions.

Antigenic variation of parasite-derived antigens on the surface of Babesia bovis-infected erythrocytes.

The hemoparasite Babesia bovis antigenically alters the bovine erythrocyte membrane surface by expression of isolate-specific, parasite-derived polypeptides. To determine whether antigenic variation also occurred on the infected erythrocyte surface, a calf was infected once with parasitized erythrocytes carrying the C9.1 clonal line of B. bovis. In vitro cultures then were established periodically from the peripheral blood and analyzed with sequentially collected sera from the same animal. The surface reactivity of infected erythrocytes cultured from the infected animal varied over time, on the basis of reactivity in live cell immunofluorescence, surface immunoprecipitation, and panning assays. Subclones C8 and H10, established from day 41 cultures, were analyzed immunochemically. A loss of immunoreactivity was observed in antigens corresponding to the 113- and 128-kDa parasite-derived antigens of clone C9.1, demonstrating epitopic variation in these antigens; the immunochemical recognition of these antigens paralleled the results of live cell immunofluorescence and panning assays. Concomitant size polymorphism suggested polypeptide structural variation of these antigens as well. Calves infected by inoculation of infected blood or by injection of cloned parasites from in vitro cultures rapidly developed antibodies which cross-reacted among the clonal variant lines, suggesting the presence of common as well as unique epitopes. These results demonstrate that antigenic variation occurs on the surface of B. bovis-infected erythrocytes and that the parasite-derived antigens of 113 and 128 kDa compose at least a part of the antigens undergoing variation.

Food intolerance in patients with chronic fatigue.

Intolerance to various foods is reported often by patients seeking evaluation for chronic fatigue, a common symptom in primary care practice. To assess the prevalence and significance of this phenomenon we studied 200 consecutive patients with chronic fatigue who were given a comprehensive medical and psychiatric evaluation. Intolerance to foods from at least three different groups was reported by 27 patients (13.5%). We compared these patients with 27 age- and gender-matched patients from the same cohort of fatigued patients. Physical examination and laboratory testing showed few abnormalities in either group. The two groups were similar with respect to the duration and severity of fatigue, lifetime depressive symptoms, and prevalence of current depressive disorders (67% vs. 63%) and anxiety disorders (11% vs. 15%). Patients with multiple food intolerance had more lifetime functional somatic symptoms (p < .05) and a significantly higher (33% vs. 7%) prevalence of somatization disorder (p < .025). These data suggest that intolerance to multiple foods is probably not a cause or the effect of chronic fatigue, but rather one of the manifestations of the somatization trait expressed in these patients.

Chronic fatigue and chronic fatigue syndrome: clinical epidemiology and aetiological classification.

To determine the medical and psychiatric diagnoses that have an aetiological role in chronic fatigue we conducted a prospective study of 405 (65% women) patients who presented for evaluation with this chief complaint to an academic medical centre. The average age was 38.1 years and the average duration of fatigue at entry in the study was 6.9 years. All patients were given comprehensive physical and laboratory evaluations and were administered a highly structured psychiatric interview. Psychiatric diagnoses explaining the chronic fatigue were identified in 74% of patients and physical disorders were diagnosed in 7% of patients. The most common psychiatric conditions in this series were major depression, diagnosed in 58% of patients, panic disorder, diagnosed in 14% of patients, and somatization disorder, diagnosed in 10% of patients. Primary sleep disorders, diagnosed in 2% patients, and chronic infections, confirmed in 1.6% patients, explained the majority of cases whose chronic fatigue was attributed to a physical disorder. Thirty per cent of patients met the criteria used to define the chronic fatigue syndrome (CFS). Compared with age- and gender-matched control subjects with chronic fatigue, CFS patients had a similarly high prevalence of current psychiatric disorders (78% versus 82%), but were significantly more likely to have somatization disorder (28% versus 5%) and to attribute their illness to a viral infection (70% versus 33%). We conclude that most patients with a chief complaint of chronic fatigue, including those exhibiting the features of CFS, suffer from standard mood, anxiety and/or somatoform disorders. Careful research is still needed to determine whether CFS is a distinct entity or a variant of these psychiatric illness.

Chronic fatigue syndromes in clinical practice.

Chronic fatigue is a common and difficult challenge in clinical practice. The majority of patients with this chief complaint have treatable mood or anxiety disorders, complicated by a tendency toward somatization. A minority of patients suffer from sleep disorders, endocrinologic abnormalities, or chronic inflammatory conditions. Prolonged recovery after viral infections is only rarely the cause of chronic fatigue. Specific pharmacologic interventions and cognitive-behavioral therapy are effective in an environment that is sensitive to the patient's interpretation of symptoms and avoids unproven medical investigations and therapies.

The pathophysiology of chronic fatigue syndrome: confirmations, contradictions, and conjectures.

OBJECTIVE: To examine published data regarding patient cohorts with the recently defined chronic fatigue syndrome. METHOD: Review of thirty-two peer-assessed research publications that included full disclosure of the methodology employed; classification of the findings as confirmed, contradictory, or non-duplicated. RESULTS: Research studies have confirmed that the majority of patients with the chronic fatigue syndrome: 1) are white middle-aged women, 2) have a high prevalence of current major depression and somatization disorder, 3) have abnormal personality traits, 4) believe that their fatigue has a physical cause, and 5) show mild abnormalities of humoral immunity. Contradictory data have been presented with regard to: 1) the time of onset of depressive disorders, 2) the etiologic role of herpetic and enteroviral infections, 3) the presence of abnormal cellular immunity, and 4) the clinical utility of immunoglobulin therapy. Non-duplicated research has indicated 1) hypothalamic-pituitary-adrenal axis dysfunction, 2) abnormalities on magnetic resonance images of the brain, 3) altered cytokine production, and 4) the possibility of retroviral infection. CONCLUSIONS: As presently defined, the chronic fatigue syndrome has many of the clinical and biological features associated with depressive and somatoform disorders. A specific etiologic role for infections or immune dysfunction has not been confirmed.

Evaluation and management of patients with chronic fatigue.

Chronic fatigue is a common and disabling problem in primary care practice. The differential diagnosis of chronic fatigue is extensive and includes medical disorders, altered physiologic states (eg, pregnancy, exertion), psychiatric disorders, lifestyle derangements, drugs, and controversial entities (eg, chronic candidiasis, food allergies, environmental illness, and chronic fatigue syndrome). The most common diagnoses are psychiatric disorders, including mood, anxiety, and somatoform disorders. A comprehensive approach to diagnosis and management is necessary, including structured psychiatric interviewing, functional assessment, and elicitation of the patient's diagnostic beliefs. Patients often believe they are suffering from an organic medical disorder (eg, viral or immunologic) and resist psychiatric labelling of their symptoms and referral to mental health practitioners. Establishing and maintaining rapport, having a flexible approach, and demonstrating a personal concern for the patient is essential. Drug therapy for specific psychiatric and medical illnesses and cognitive-behavioral approaches for enhancing coping mechanisms are effective.

Depression and somatization in the chronic fatigue syndrome.

PURPOSE: To report the prevalence, clinical features, and diagnostic associations of the proposed chronic fatigue syndrome (CFS) in a cohort of patients with chronic fatigue and to assess the usefulness of a structured psychiatric interview for detecting previously unrecognized psychiatric morbidity in patients with CFS. PATIENTS AND METHODS: A consecutive sample of 200 adult patients with a chief complaint of chronic fatigue was prospectively evaluated in a referral-based clinic within a university general medicine practice. All patients received a thorough medical history, physical examination, diagnostic laboratory testing, and portions of the Diagnostic Interview Schedule, version III-A. The criteria for CFS were applied, and patients with CFS were compared with matched control subjects from the inception cohort. RESULTS: The 60 patients with CFS had similar likelihoods of current psychiatric disorders (78% versus 82%), active mood disorders (73% versus 77%), and preexisting psychiatric disorders (42% versus 43%) when compared with fatigued control subjects. Patients with CFS were more likely to have somatization disorder (p less than 0.001) and to attribute their illness to a physical cause (p less than 0.005) than fatigued controls. Patients with CFS also displayed functional symptoms, often lifelong, which are not part of the case definition of CFS. Depressive features in patients with CFS were similar to those of control subjects, but a trend toward suicidal behavior was noted. CONCLUSIONS: Patients with CFS have a high prevalence of unrecognized, current psychiatric disorders, which often predate their fatigue syndrome. Assessment of patients with CFS should include a structured psychiatric evaluation.

Substance use disorders in patients with chronic fatigue.

A high prevalence of mood disorders has been found among persons with chronic fatigue. The comorbidity of mood disorders and substance use disorders has long been recognized. In this study, the prevalence of substance use disorders among 100 patients with chronic fatigue was examined. Twenty-eight patients met DSM-III-R criteria for a lifetime diagnosis of substance abuse or dependence; ten of these patients had a current diagnosis. Forty-nine patients had no history of substance abuse or dependence but had other lifetime psychiatric diagnoses. Twenty-three patients had no history of substance abuse or psychiatric illness. No differences in demographic characteristics or in the features of chronic fatigue were found among the three groups. Patients with chronic fatigue who had a lifetime history of a substance use disorder reported more lifetime depressive symptoms and were more likely to have had suicidal ideation or attempts.

Antibodies to Epstein-Barr virus in patients with chronic fatigue.

To clarify the role of Epstein-Barr virus (EBV) infection and the value of EBV antibody testing in evaluating patients with chronic fatigue, we studied 200 consecutive patients with chronic fatigue (mean duration, 9 years). Complete EBV serologic panels were obtained for 154 patients, 35 (23%) of whom met serologic or clinical criteria for chronic or reactivated EBV infection. We compared these patients with chronic EBV infection (CEBV cases) to 35 age- and sex-matched patients who were selected from the same cohort of fatigued patients but who did not meet the criteria (CEBV control subjects). We found few differences between groups in demographic characteristics, clinical features, and symptoms; CEBV cases were more likely to meet criteria for the proposed chronic fatigue syndrome (14% vs 0%), and to report that they suffered from an influenza-like illness at the onset of their fatigue syndrome (34% vs 12%), that they lost their job because of their fatigue (37% vs 11%), and that their fatigue was improved by recreational activity (26% vs 3%). Physical examination and laboratory testing showed few abnormalities in either group. Psychiatric morbidity was common in both groups, including mood disorders (63% of CEBV cases vs 54% of CEBV controls), anxiety (11% vs 9%) and somatization disorder (9% in each group). We conclude that EBV serologic patterns have little clinical usefulness in evaluating patients with chronic fatigue.