ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering.

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

Reliability and reproducibility analysis of the AOSpine Sacral Fractures Classification System by spinal and pelvic surgeons.

STUDY DESIGN: Retrospective Cohort Study OBJECTIVES: The AOSpine Sacral Classification System was proposed as a comprehensive and universally accepted new classification for Sacral Fractures, and was recently internally validated. However, an external, independent and multidisciplinary reports on validation of this classification is lacking. Aim of the present study is to analyze the interobserver reliability and intraobserver reproducibility of the AOSpine Sacral Classification System for Sacral Fractures between orthopedic spinal and pelvic surgeons with different levels of experience. METHODS: Our institutional database was searched to retrieve patients with acute, traumatic sacral injury admitted from June 2017 to June 2020. For each patients, X-Rays and CT scans were collected. Three Orthopedic Pelvic Surgeons (Group A) and three Spine Surgeons (Group B), with different level of experience (Junior, 〈 5 years; Middle, 5-10 years; Expert 〉 10 years) independently classified all the sacral fractures included in the dataset, with two separate evaluation three weeks apart. Both intra and interobserver reliability were calculated with k-coefficient. RESULTS: Overall, 150 patients were included in the final dataset, for a total of 1800 different assessments, with all the subtypes reported. The intraobserver reproducibility for the whole group was substantial (κ=0.72). Overall, the interobserver reliability was moderate, with a κ=0.57. When only fracture type was taken in account, the κ value became substantial (κ=0.62). No significant differences were found comparing group A and group B (0.55 vs κ 0.55, p>0.05). No significant differences according to surgeon's experience were found; however, the κ value was slightly lower among the junior surgeons. CONCLUSIONS: Our findings confirmed the reliability and reproducibility of this classification in clinical practice. In the current study the surgeon's expertise (pelvic and spinal trauma) and the level of experience does not influence the reliability of the classification system.

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.

The management of emergency spinal surgery during the COVID-19 pandemic in Italy.

AIMS: The current pandemic caused by COVID-19 is the biggest challenge for national health systems for a century. While most medical resources are allocated to treat COVID-19 patients, several non-COVID-19 medical emergencies still need to be treated, including vertebral fractures and spinal cord compression. The aim of this paper is to report the early experience and an organizational protocol for emergency spinal surgery currently being used in a large metropolitan area by an integrated team of orthopaedic surgeons and neurosurgeons. METHODS: An organizational model is presented based on case centralization in hub hospitals and early management of surgical cases to reduce hospital stay. Data from all the patients admitted for emergency spinal surgery from the beginning of the outbreak were prospectively collected and compared to data from patients admitted for the same reason in the same time span in the previous year, and treated by the same integrated team. RESULTS: A total of 19 patients (11 males and eight females, with a mean age of 49.9 years (14 to 83)) were admitted either for vertebral fracture or spinal cord compression in a 19-day period, compared to the ten admitted in the previous year. No COVID-19 patients were treated. The mean time between admission and surgery was 1.7 days, significantly lower than 6.8 days the previous year (p < 0.001). CONCLUSION: The structural organization and the management protocol we describe allowed us to reduce the time to surgery and ultimately hospital stay, thereby maximizing the already stretched medical resources available. We hope that our early experience can be of value to the medical communities that will soon be in the same emergency situation. Cite this article: Bone Joint J 2020;102-B(6):671-676.

Residual mobility after removal of instrumentation in patient, with type a2-a3 vertebral fractures, treated with percutaneous pedicle screw fixation.

Percutaneous techniques for treatment of thoraco-lumbar fractures type A2 and A3 are widely used. These techniques are considered temporary fixations and instrumentation must be removed with fracture healing. The aim of the study is to analyze clinical results, motility of treated segments and any loss of correction after the removal of instrumentation. We evaluated 36 patients who underwent surgery for removal of the instrumentation. Standard and dynamics x-ray before surgery and at 1 and 12 months after surgery were obtained. Radiographic evaluation was performed by comparing loss of correction after removal of the instrumentation, residual mobility of fractured vertebra, upper and lower level with values defined by Dvorak. For clinical assessment were used SF-12, Oswestry Disability Index (ODI) and Visual Analog Scale (VAS), administered before surgery and at 1 and 12 months after the removal. We analyzed a total of 108 levels in 36 patients. After removal of the instrumentation a normal range of motion was restored in the proximal and distal segment of the fracture, while at level of fractured segment we noticed a decrease in motility. Clinically, patients had a significant decrease in VAS and ODI at 1 month after removal. Our study shows that percutaneous fixation for treatment of thoraco-lumbar fractures type A2 and A3, allows to preserve motility of the treated segments after the removal of the instrumentation until 12 months. The removal of instrumentation is associated with good clinical results without of loss of correction in treated segment.

The distribution of mycotoxins in a heterogeneous wheat field in relation to microclimate, fungal and bacterial abundance.

AIM: To observe the variation in accumulation of Fusarium and Alternaria mycotoxins across a topographically heterogeneous field and tested biotic (fungal and bacterial abundance) and abiotic (microclimate) parameters as explanatory variables. METHODS AND RESULTS: We selected a wheat field characterized by a diversified topography, to be responsible for variations in productivity and in canopy-driven microclimate. Fusarium and Alternaria mycotoxins where quantified in wheat ears at three sampling dates between flowering and harvest at 40 points. Tenuazonic acid (TeA), alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), deoxynivalenol (DON), zearalenone (ZEN) and deoxynivalenol-3-Glucoside (DON.3G) were quantified. In canopy temperature, air and soil humidity were recorded for each point with data-loggers. Fusarium spp. as trichothecene producers, Alternaria spp. and fungal abundances were assessed using qPCR. Pseudomonas fluorescens bacteria were quantified with a culture based method. We only found DON, DON.3G, TeA and TEN to be ubiquitous across the whole field, while AME, AOH and ZEN were only occasionally detected. Fusarium was more abundant in spots with high soil humidity, while Alternaria in warmer and drier spots. Mycotoxins correlated differently to the observed explanatory variables: positive correlations between DON accumulation, tri 5 gene and Fusarium abundance were clearly detected. The correlations among the others observed variables, such as microclimatic conditions, varied among the sampling dates. The results of statistical model identification do not exclude that species coexistence could influence mycotoxin production. CONCLUSIONS: Fusarium and Alternaria mycotoxins accumulation varies heavily across the field and the sampling dates, providing the realism of landscape-scale studies. Mycotoxin concentrations appear to be partially explained by biotic and abiotic variables. SIGNIFICANCE AND IMPACT OF THE STUDY: We provide a useful experimental design and useful data for understanding the dynamics of mycotoxin biosynthesis in wheat.

Degenerative facet joint changes in lumbar percutaneous pedicle screw fixation without fusion.

BACKGROUND: Aim of the study was to evaluate degenerative lumbar facet-joints changes after percutaneous pedicle screw fixation (PPSF) in the treatment of lumbar fractures. MATERIALS AND METHODS: Thirty patients underwent short PPSF without fusion. CT-scan was performed in the pre- and post-operative time at four, eight and 12 months. The six zygapophyseal joints adjacent the fracture's level were evaluated. RESULTS: At four months patients showed no differences between pre- and post-operative joint radiographic aspect. At eight and 12 months, CT-scan demonstrated a progressive degeneration only in the middle joints respectively in 21.42% and in 76.92% of the cases. All 10 disrupted facet joints showed progressive degenerative changes at eight and 12 months. CONCLUSION: Lumbar percutaneous fixation without fusion induces little degenerations essentially collocated in the middle joints close to fracture level at eight and 12 months. In the proximal and distal joints adjacent the screws degenerative changes can be seen only when associated to pedicle-screw encroachment.

Observing heme doming in myoglobin with femtosecond X-ray absorption spectroscopy.

We report time-resolved X-ray absorption measurements after photolysis of carbonmonoxy myoglobin performed at the LCLS X-ray free electron laser with nearly 100 fs (FWHM) time resolution. Data at the Fe K-edge reveal that the photoinduced structural changes at the heme occur in two steps, with a faster (∼70 fs) relaxation preceding a slower (∼400 fs) one. We tentatively attribute the first relaxation to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and the second relaxation to a residual Fe motion out of the heme plane that is coupled to the displacement of myoglobin F-helix.

The impact of sagittal balance on low back pain in patients treated with zygoapophysial facet joint injection.

INTRODUCTION: Aim of the study was to evaluate the effectiveness of facet joints injections in lumbar facet syndrome correlating clinical results to the sagittal contour of the spine. METHODS: Facet joints degree degeneration was evaluated using MRI according to Fujiwara classification. Sagittal contour of the spine was evaluated according to Roussouly classification. The clinical results were evaluated with visual analog scale (VAS) at regular intervals. RESULTS: Twenty-eight (70 %) of the 40 patients had clinical symptoms improvement, 12 (30 %) showed no benefit. There was a statistical significant correlation between postoperative VAS value improvement and Roussouly spine type 1 and 3 (p = 0.003). The benefit was more durable in patients with grade 2 or 3 degeneration. CONCLUSIONS: Facet joints injections have a more effective diagnostic than therapeutic value. The procedure could, however, give a temporary pain relief in cases with an overload of the facet joints due to lumbar hyperlordosis.

Posterior percutaneous reduction and fixation of thoraco-lumbar burst fractures.

BACKGROUND: Treatment of A3 thoraco-lumbar and lumbar spinal fractures nowadays remains a controversial issue. Percutaneous techniques are becoming very popular in the last few years to reduce the approach-related morbidity associated with conventional techniques. HYPOTHESIS: Purpose of the study was to analyze the clinical and radiological outcome of patients who underwent percutaneous posterior fixation without fusion for the treatment of thoraco-lumbar and lumbar A3 fractures. MATERIALS AND METHODS: Sixty-three patients, having sustained a single-level thoraco-lumbar fracture, underwent short segment percutaneous instrumentation and were retrospectively analyzed. sagittal index (SI) was calculated in all patients. Clinical and functional outcome were evaluated by Visual Analog Scale (VAS), Oswestry Disability Index (ODI) and Short Form General Health Status (SF-36). RESULTS: Average operative blood loss was 82 mL (50-320). Mean pre-operative SI in the thoraco-lumbar segment was 13.3° decreased to 5.8° in the immediate postoperative with a mean deformity correction of 7.5. Mean pre-operative SI in the lumbar segment was 16.5° decreased to 11.3° in the immediate postoperative with a mean deformity correction of 5.2. Not statistically significant correction loss was registered at 1-year minimum follow-up. Constant clinical conditions improvement in the examined patients was observed. CONCLUSION: Percutaneous pedicle screw fixation for A3 thoraco-lumbar and lumbar spinal fractures is a reliable and safe procedure. LEVEL OF EVIDENCE: Level IV. Retrospective study.

The boson peak of amyloid fibrils: probing the softness of protein aggregates by inelastic neutron scattering.

Proteins and polypeptides are characterized by low-frequency vibrations in the terahertz regime responsible for the so-called "boson peak". The shape and position of this peak are related to the mechanical properties of peptide chains. Amyloid fibrils are ordered macromolecular assemblies, spontaneously formed in nature, characterized by unique biological and nanomechanical properties. In this work, we investigate the effects of the amyloid state and its polymorphism on the boson peak. We used inelastic neutron scattering to probe low-frequency vibrations of the glucagon polypeptide in the native state and in two different amyloid morphologies in both dry and hydrated sample states. The data show that amyloid fibril formation and hydration state affect the softness of the polypeptide not only by changing the distribution of vibrational modes but also, and most significantly, the dissipative mechanisms of collective low-frequency vibrations provided by water-protein and protein-protein interactions. We show how the morphology of the fibril is able to tune these effects. Atomic fluctuations were also measured by elastic neutron scattering. The data confirm that any effect of protein aggregation on fluctuation amplitudes is essentially due to changes in surface exposure to hydration water. The results demonstrate the importance of protein-protein and protein-water interactions in the dynamics and mechanics of amyloid fibrils.

Cervical myelopathy due to ossification of the transverse atlantal ligament: a Caucasian case report operated on and literature analysis.

One case of cervical myelopathy associated to ossification of transverse atlantal ligament (OTAL) and C1 posterior arch hypoplasia in a Caucasian adult female is reported. A 53-year-old female affected by cervical myelopathy was treated with C1 laminectomy and posterior arthrodesis. CT scan demonstrated that the distance between ossification of the ligament and anterior cortex of the posterior arch of atlas was 6,2mm leading to consistent space reduction for spinal cord at this level. Patient underwent spinal cord decompression and fixation with C1 poliaxial screws in lateral masses and two bilateral crossing C2 laminar screws with an improvement of neurological functions at 4-years follow-up. The association between OTAL and C1 hypoplasia was reported in very few cases. The treatment with C1 laminectomy without fusion is reported in medical literature with good clinical outcome. Our patient obtained a neurological improvement at midterm follow-up with spinal cord decompression and fusion.

Transdiscal L5-S1 screws for the treatment of adult spondylolisthesis.

PURPOSE: The aim of the study was to evaluate clinical and radiographic outcome of patients treated with a modified Grob technique analysing the advantages related to increased mechanical stability. METHODS: 30 patients that underwent "in situ" fusion for L5-S1 spondylolisthesis were evaluated. All patients presented a low-dysplastic developmental L5-S1 spondylolisthesis. Patients were divided into two groups: A, in which L5-S1 pedicle instrumentation associated with transsacral screw fixation was performed, and B, in which L5-S1 pedicle instrumentation associated with a posterolateral interbody fusion (PLIF) was performed. RESULTS: Patients treated with transdiscal L5-S1 fixation observed a faster resolution of the symptoms and a more rapid return to daily activities, especially at 3-6 months' follow-up. The technique is reliable in giving an optimal mechanical stability to obtain a solid fusion. CONCLUSIONS: The advantages of this technique are lower incidence of neurologic complications, speed of execution and faster return to normal life.

Impulsive solvent heating probed by picosecond x-ray diffraction.

The time-resolved diffraction signal from a laser-excited solution has three principal components: the solute-only term, the solute-solvent cross term, and the solvent-only term. The last term is very sensitive to the thermodynamic state of the bulk solvent, which may change during a chemical reaction due to energy transfer from light-absorbing solute molecules to the surrounding solvent molecules and the following relaxation to equilibrium with the environment around the scattering volume. The volume expansion coefficient alpha for a liquid is typically approximately 1 x 10(-3) K(-1), which is about 1000 times greater than for a solid. Hence solvent scattering is a very sensitive on-line thermometer. The decomposition of the scattered x-ray signal has so far been aided by molecular dynamics (MD) simulations, a method capable of simulating the solvent response as well as the solute term and solute/solvent cross terms for the data analysis. Here we present an experimental procedure, applicable to most hydrogen containing solvents, that directly measures the solvent response to a transient temperature rise. The overtone modes of OH stretching and CH3 asymmetric stretching in liquid methanol were excited by near-infrared femtosecond laser pulses at 1.5 and 1.7 microm and the ensuing hydrodynamics, induced by the transfer of heat from a subset of excited CH3OH* to the bulk and the subsequent thermal expansion, were probed by 100 ps x-ray pulses from a synchrotron. The time-resolved data allowed us to extract two key differentials: the change in the solvent diffraction from a temperature change at constant density, seen at a very short time delay approximately 100 ps, and a term from a change in density at constant temperature. The latter term becomes relevant at later times approximately 1 mus when the bulk of liquid expands to accommodate its new temperature at ambient pressure. These two terms are the principal building blocks in the hydrodynamic equation of state, and they are needed in a self-consistent reconstruction of the solvent response during a chemical reaction. We compare the experimental solvent terms with those from MD simulations. The use of experimentally determined solvent differentials greatly improved the quality of global fits when applied to the time-resolved data for C2H4I2 dissolved in methanol.