The High-Pressure Freezing Laboratory for Macromolecular Crystallography (HPMX), an ancillary tool for the macromolecular crystallography beamlines at the ESRF.

This article describes the High-Pressure Freezing Laboratory for Macromolecular Crystallography (HPMX) at the ESRF, and highlights new and complementary research opportunities that can be explored using this facility. The laboratory is dedicated to investigating interactions between macromolecules and gases in crystallo, and finds applications in many fields of research, including fundamental biology, biochemistry, and environmental and medical science. At present, the HPMX laboratory offers the use of different high-pressure cells adapted for helium, argon, krypton, xenon, nitrogen, oxygen, carbon dioxide and methane.

The TR-icOS setup at the ESRF: time-resolved microsecond UV-Vis absorption spectroscopy on protein crystals.

The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump-probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable.

Evolutionary adaptation from hydrolytic to oxygenolytic catalysis at the α/β-hydrolase fold.

Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading -heteroaromatic substrates belong to the α/β-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. Here, we have integrated crystallographic analyses under normoxic and hyperoxic conditions with molecular dynamics and quantum mechanical calculations to investigate its prototypic 1--3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) member.

Comparative study of the effects of high hydrostatic pressure per se and high argon pressure on urate oxidase ligand stabilization.

The stability of the tetrameric enzyme urate oxidase in complex with excess of 8-azaxanthine was investigated either under high hydrostatic pressure per se or under a high pressure of argon. The active site is located at the interface of two subunits, and the catalytic activity is directly related to the integrity of the tetramer. This study demonstrates that applying pressure to a protein-ligand complex drives the thermodynamic equilibrium towards ligand saturation of the complex, revealing a new binding site.

Accurate and rapid 3D printing of microfluidic devices using wavelength selection on a DLP printer.

The use of microfluidics on synchrotron X-ray beamlines represents an advanced sample preparation and delivery platform for state-of-the-art X-ray characterization of micro-samples. The recent developments of 3D printing technologies have opened possibilities for rapid fabrication of complex microfluidic devices. One of the major challenges in 3D printing of microfluidic devices using a digital light processing (DLP) desktop printer is that the static liquid resin trapped in the channels, once the "ceiling" is printed, still receives small doses of light through the subsequently printed layers.

Exploring the gas access routes in a [NiFeSe] hydrogenase using crystals pressurized with krypton and oxygen.

Hydrogenases are metalloenzymes that catalyse both H evolution and uptake. They are gas-processing enzymes with deeply buried active sites, so the gases diffuse through channels that connect the active site to the protein surface. The [NiFeSe] hydrogenases are a special class of hydrogenases containing a selenocysteine as a nickel ligand; they are more catalytically active and less O-sensitive than standard [NiFe] hydrogenases.

3D-MiXD: 3D-printed X-ray-compatible microfluidic devices for rapid, low-consumption serial synchrotron crystallography data collection in flow.

Serial crystallography has enabled the study of complex biological questions through the determination of biomolecular structures at room temperature using low X-ray doses. Furthermore, it has enabled the study of protein dynamics by the capture of atomically resolved and time-resolved molecular movies. However, the study of many biologically relevant targets is still severely hindered by high sample consumption and lengthy data-collection times.

Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV-visible microspectroscopy , solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15-40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton.

Screening and Stepped Care Targeting Psychological Distress in Patients With Metastatic Colorectal Cancer: The TES Cluster Randomized Trial.

Background: This study evaluated the effectiveness of a screening and stepped care program (the TES program) in reducing psychological distress compared with care as usual (CAU) in patients with metastatic colorectal cancer starting with first-line systemic palliative treatment.

Patients And Methods: In this cluster randomized trial, 16 hospitals were assigned to the TES program or CAU. Patients in the TES arm were screened for psychological distress with the Hospital Anxiety and Depression Scale and the Distress Thermometer/Problem List (at baseline and 10 and 18 weeks).

Tracking the route of molecular oxygen in O-tolerant membrane-bound [NiFe] hydrogenase.

[NiFe] hydrogenases catalyze the reversible splitting of H into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of , is able to overcome aerobic inactivation by catalytic reduction of O to water.

Incidence, prevalence, and management of MRSA bacteremia across patient populations-a review of recent developments in MRSA management and treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is still a major global healthcare problem. Of concern is S. aureus bacteremia, which exhibits high rates of morbidity and mortality and can cause metastatic or complicated infections such as infective endocarditis or sepsis.

A compact and versatile dynamic flow cryostat for photon science.

We have developed a helium gas flow cryostat for use on synchrotron tender to hard X-ray beamlines. Very efficient sample cooling is achieved because the sample is placed directly in the cooling helium flow on a removable sample holder. The cryostat is compact and easy to operate; samples can be changed in less than 5 min at any temperature.

Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies.

CA 19-9 as a marker in addition to CEA to monitor colorectal cancer.

Background: Carcinoembryonic antigen is the commonly used tumor marker in patients with colorectal cancer, and CA 19-9 might be an additional marker. The aim of this retrospective study was to investigate whether CA 19-9 levels can be used to monitor the disease process in patients with colorectal cancer who had no elevated CEA levels. The secondary aim was to determine if preoperative increased levels of CEA and CA 19-9 were associated with mortality.

Cryogenic x-ray diffraction microscopy utilizing high-pressure cryopreservation.

We present cryo x-ray diffraction microscopy of high-pressure-cryofixed bacteria and report high-convergence imaging with multiple image reconstructions. Hydrated D. radiodurans cells were cryofixed at 200 MPa pressure into ∼10-μm-thick water layers and their unstained, hydrated cellular environments were imaged by phasing diffraction patterns, reaching sub-30-nm resolutions with hard x-rays.

SPINE-compatible `carboloops': a new microshaped vitreous carbon sample mount for X-ray and neutron crystallography.

A novel vitreous carbon mount for macromolecular crystallography, suitable for neutron and X-ray crystallographic studies, has been developed. The technology described here is compatible both with X-ray and neutron cryo-crystallography. The mounts have low density and low background scattering for both neutrons and X-rays.

Multi-frame acquisition scheme for efficient energy-dispersive X-ray magnetic circular dichroism in pulsed high magnetic fields at the Fe K-edge.

Using a fast silicon strip detector, a multi-frame acquisition scheme was implemented to perform energy-dispersive X-ray magnetic circular dichroism at the iron K-edge in pulsed high magnetic fields. The acquisition scheme makes use of the entire field pulse. The quality of the signal obtained from samples of ferrimagnetic erbium iron garnet allows for quantitative evaluation of the signal amplitude.

Approach to the immunocompromised host with infection in the intensive care unit.

Despite significant advances in the prevention, diagnosis, and treatment of infection in the immunocompromised host, it remains a major cause of morbidity, increased length of stay, total costs, and of course mortality. Intensive care mortality rates are significantly higher among immunocompromised hosts in part due to the higher incidence of infection severity. The superimposition of the compromised host defenses and critical illness makes the detection and management of infections in such patients more difficult, but crucial toward salvaging patient outcome.

History of solid organ transplantation and organ donation.

Solid organ transplantation is one of the most remarkable and dramatic therapeutic advances in medicine during the past 60 years. This field has progressed initially from what can accurately be termed a "clinical experiment" to routine and reliable practice, which has proven to be clinically effective, life-saving and cost-effective. This remarkable evolution stems from a serial confluence of: cultural acceptance; legal and political evolution to facilitate organ donation, procurement and allocation; technical and cognitive advances in organ preservation, surgery, immunology, immunosuppression; and management of infectious diseases.

Vancomycin resistance: are there better glycopeptides coming?

The appearance and dissemination of vancomycin resistance among clinically important Gram-positive bacteria was an important watershed in antimicrobial resistance trends that drastically narrows therapeutic options, particularly among the enterococci. Clinical resistance despite apparent susceptibility has also become an increasingly recognized issue with vancomycin treatment of methicillin-resistant Staphylococcus aureus pneumonia and endocarditis, which may be, in part, due to vancomycin-heteroresistant strains. The newly developed glycopeptides telavancin, dalbavancin and oritavancin have superior in vitro activity, enhanced bactericidality and unique pharmacokinetic properties compared with vancomycin and teicoplanin.

Health care-associated infection (HAI): a critical appraisal of the emerging threat-proceedings of the HAI Summit.

During the Health Care-Associated Pneumonia Summit conducted in June 2007, it was found that there is a need for educational efforts in several areas of health care-associated infections (HAI) that extend beyond pneumonia. This supplement to Clinical Infectious Diseases represents the proceedings of the HAI Summit, a diverse panel of clinical investigators whose goal was to assess the quality of evidence regarding issues surrounding HAI and to discuss potential implications for its diagnosis and treatment in the future.

Miniature pulsed magnet system for synchrotron x-ray measurements.

We have developed a versatile experimental apparatus for synchrotron x-ray measurements in pulsed high magnetic fields. The apparatus consists of a double cryostat incorporating a liquid nitrogen bath to cool the miniature pulsed coil and an independent helium flow cryostat allowing sample temperatures from 4 up to 250 K. The high duty cycle miniature pulsed coils can generate up to 38 T.