Effects of danofloxacin dosing regimen on gastrointestinal pharmacokinetics and fecal microbiome in steers.

Fluoroquinolones are a class of antimicrobial commonly used in human medicine, and deemed critical by the World Health Organization. Nonetheless, two formulations are approved for the treatment of respiratory disease in beef cattle. The objective of this study was to determine the gastrointestinal pharmacokinetics and impact on enteric bacteria of cattle when receiving one of the two dosing regimens (high: 40 mg/kg SC once or low: 20 mg/kg IM q48hr) of danofloxacin, a commonly utilized synthetic fluoroquinolone in veterinary medicine. Danofloxacin was administered to 12 steers (age 7 months) fitted with intestinal ultrafiltration devices at two different dosing regimens to assess the gastrointestinal pharmacokinetics, the shifts in the gastrointestinal microbiome and the development of resistant bacterial isolates. Our results demonstrated high intestinal penetration of danofloxacin for both dosing groups, as well as, significant differences in MIC values for E. coli and Enterococcus between dosing groups at selected time points over a 38 day period. Danofloxacin treatment consistently resulted in the Euryarchaeota phyla decreasing over time, specifically due to a decrease in Methanobrevibacter. Although microbiome differences were minor between dosing groups, the low dose group had a higher number of isolates with MIC values high enough to cause clinically relevant resistance. This information would help guide veterinarians as to appropriate dosing schemes to minimize the spread of antimicrobial resistance.

Massive in Silico Study of Noble Gas Binding to the Structural Proteome.

Noble gases are chemically inert, and it was therefore thought they would have little effect on biology. Paradoxically, it was found that they do exhibit a wide range of biological effects, many of which are target-specific and potentially useful and some of which have been demonstrated in vivo. The underlying mechanisms by which useful pharmacology, such as tissue and neuroprotection, anti-addiction effects, and analgesia, is elicited are relatively unexplored. Experiments to probe the interactions of noble gases with specific proteins are more difficult with gases than those with other chemicals. It is clearly impractical to conduct the large number of gas-protein experiments required to gain a complete picture of noble gas biology. Given the simplicity of atoms as ligands, in silico methods provide an opportunity to gain insight into which noble gas-protein interactions are worthy of further experimental or advanced computational investigation. Our previous validation studies showed that in silico methods can accurately predict experimentally determined noble gas binding sites in X-ray structures of proteins. Here, we summarize the largest reported in silico reverse docking study involving 127 854 protein structures and the five nonradioactive noble gases. We describe how these computational screening methods are implemented, summarize the main types of interactions that occur between noble gases and target proteins, describe how the massive data set that this study generated can be analyzed (freely available at group18.csiro.au), and provide the NDMA receptor as an example of how these data can be used to understand the molecular pharmacology underlying the biology of the noble gases. We encourage chemical biologists to access the data and use them to expand the knowledge base of noble gas pharmacology, and to use this information, together with more efficient delivery systems, to develop "atomic drugs" that can fully exploit their considerable and relatively unexplored potential in medicine.

Feasibility and safety of lumbosacral epiduroscopy in the standing horse.

REASONS FOR PERFORMING STUDY: The large size of the adult horse prevents the use of advanced imaging modalities in most areas of the axial skeleton, including the lumbosacral vertebral column. Traditional imaging techniques are frequently unable to pinpoint the underlying pathology in horses with caudal back pain. In man, lumbosacral epiduroscopy is used to diagnose and treat subjects with chronic back and leg pain. This technique may close the diagnostic gap in horses with similar clinical signs. OBJECTIVES: To evaluate the safety and feasibility of lumbosacral epiduroscopy in the standing adult horse. STUDY DESIGN: Descriptive, experimental study. METHODS: Seven adult horses weighing 504-578 kg were sedated and restrained in stocks in preparation for aseptic surgery. Vascular dilators of increasing size were inserted cranial to the first moveable vertebra caudal to the sacrum to facilitate a minimally invasive approach into the epidural space. A flexible video-endoscope was introduced and advanced as far as its 60-cm working length permitted. Pre-, intra- and post-operative plasma cortisol samples were collected, and neurological and lameness examinations were performed prior to and during the 2 weeks following the procedure. Post-mortem examinations were conducted in 5 of the 7 horses. RESULTS: Standing lumbosacral epiduroscopy was well tolerated by all horses. The anatomic structures in the epidural space (dura mater, spinal nerve roots, fat and blood vessels) were followed as far cranial as the thoracolumbar region. No complications related to the procedure were noted in the 2-week monitoring period following epiduroscopy. Small, organised haematomas were identified in the sacral epidural space during necropsy in one horse. No abnormalities were seen in the other 4 animals. CONCLUSIONS: Lumbosacral epiduroscopy can be performed safely in sedated standing horses. The procedure may become a valuable diagnostic tool in horses with caudal back or hindlimb pain of unknown origin.

Navigational ultrasound imaging: A novel imaging tool for aiding interventional therapies of equine musculoskeletal injuries.

REASONS FOR PERFORMING STUDY: Navigational ultrasound imaging, also known as fusion imaging, is a novel technology that allows real-time ultrasound imaging to be correlated with a previously acquired computed tomography (CT) or magnetic resonance imaging (MRI) study. It has been used in man to aid interventional therapies and has been shown to be valuable for sampling and assessing lesions diagnosed with MRI or CT that are equivocal on ultrasonography. To date, there are no reports of the use of this modality in veterinary medicine. OBJECTIVES: To assess whether navigational ultrasound imaging can be used to assist commonly performed interventional therapies for the treatment of equine musculoskeletal injuries diagnosed with MRI and determine the appropriateness of regional anatomical landmarks as registration sites. STUDY DESIGN: Retrospective, descriptive clinical study. METHODS: Horses with musculoskeletal injuries of the distal limb diagnosed with MRI scheduled for ultrasound-guided interventional therapies were evaluated (n = 17 horses with a total of 29 lesions). Anatomical landmarks used for image registration for the navigational procedure were documented. Accuracy of lesion location and success of the procedure were assessed subjectively and described using a grading scale. RESULTS: All procedures were accurately registered using regional anatomical landmarks and considered successful based on our criteria. Anatomical landmarks were described for each lesion type. The addition of navigational imaging was considered to greatly aid the procedures in 59% of cases and added information to the remainder of the procedures. The technique was considered to improve the precision of these interventional procedures. CONCLUSIONS: Navigational ultrasound imaging is a complementary imaging modality that can be used for the treatment of equine soft tissue musculoskeletal injuries diagnosed with MRI.

Epiduroscopy of the lumbosacral vertebral canal in the horse: Technique and endoscopic anatomy.

REASONS FOR PERFORMING STUDY: Back pain is a common cause of gait alterations and poor performance in horses, but the available imaging modalities are frequently insufficient to isolate the underlying pathology. In human patients, epidural endoscopy (epiduroscopy) is successfully used to diagnose and treat challenging cases of lower back pain. Endoscopy of the cervical epidural space has previously been reported in anaesthetised horses. OBJECTIVES: To develop a technique for lumbosacral epiduroscopy in standing horses and to describe the endoscopic anatomy of the lumbosacral epidural space. STUDY DESIGN: Pilot study to assess the feasibility of lumbosacral epiduroscopy in 5 horse cadavers. METHODS: The cadavers of 5 horses, weighing 457-694 kg (mean, 570 kg), were suspended in an upright position. Vascular dilators of increasing size were inserted between the first 2 moveable vertebrae caudal to the sacrum to create a minimally invasive approach into the epidural space. A flexible videoendoscope was introduced and advanced as far cranially as the length of the endoscope permitted. The lumbosacral epidural space underwent gross necropsy examination following the procedure. RESULTS: The endoscope was successfully inserted into the epidural space in all horses. Saline injection through the working channel of the endoscope allowed the following anatomical structures to be seen: dura mater, left and right lumbosacral spinal nerves, cauda equina, epidural fat, connective tissue and blood vessels. Using the 60 cm working length of the endoscope, the epidural space could be examined as far cranial as L3-T18, depending on the size of the horse. No gross damage to epidural neurovascular structures was observed on necropsy examination. CONCLUSION: Lumbosacral epiduroscopy is technically feasible in standing horses and may become a valuable diagnostic tool in horses with caudal back or limb pain of unknown origin. Studies in live horses will be necessary to evaluate the safety of the procedure.

Pharmacokinetics of tobramycin following intravenous, intramuscular, and intra-articular administration in healthy horses.

The objectives of this study were to examine the pharmacokinetics of tobramycin in the horse following intravenous (IV), intramuscular (IM), and intra-articular (IA) administration. Six mares received 4 mg/kg tobramycin IV, IM, and IV with concurrent IA administration (IV+IA) in a randomized 3-way crossover design. A washout period of at least 7 days was allotted between experiments. After IV administration, the volume of distribution, clearance, and half-life were 0.18 ± 0.04 L/kg, 1.18 ± 0.32 mL·kg/min, and 4.61 ± 1.10 h, respectively. Concurrent IA administration could not be demonstrated to influence IV pharmacokinetics. The mean maximum plasma concentration (Cmax ) after IM administration was 18.24 ± 9.23 µg/mL at 1.0 h (range 1.0-2.0 h), with a mean bioavailability of 81.22 ± 44.05%. Intramuscular administration was well tolerated, despite the high volume of drug administered (50 mL per 500 kg horse). Trough concentrations at 24 h were below 2 µg/mL in all horses after all routes of administration. Specifically, trough concentrations at 24 h were 0.04 ± 0.01 µg/mL for the IV route, 0.04 ± 0.02 µg/mL for the IV/IA route, and 0.02 ± 0.02 for the IM route. An additional six mares received IA administration of 240 mg tobramycin. Synovial fluid concentrations were 3056.47 ± 1310.89 µg/mL at 30 min after administration, and they persisted for up to 48 h with concentrations of 14.80 ± 7.47 µg/mL. Tobramycin IA resulted in a mild chemical synovitis as evidenced by an increase in synovial fluid cell count and total protein, but appeared to be safe for administration. Monte Carlo simulations suggest that tobramycin would be effective against bacteria with a minimum inhibitory concentration (MIC) of 2 µg/mL for IV administration and 1 µg/mL for IM administration based on Cmax :MIC of 10.

Cervical vertebral canal endoscopy in a horse with cervical vertebral stenotic myelopathy.

A 3-year-old Thoroughbred gelding presented with a history of neurological signs, including incoordination in his hindlimbs, of about 7 months' duration. On initial examination, the horse exhibited ataxia and paresis in all limbs with more severe deficits in the hindlimbs. Cervical radiographs displayed severe osteoarthritis of the articular processes between C5 and C6. On subsequent cervical myelography the dorsal contrast column was reduced by 90% at the level of the intervertebral space between C5 and C6. Cervical vertebral canal endoscopy, including epidural (epiduroscopy) and subarachnoid endoscopy (myeloscopy), was performed under general anaesthesia. A substantial narrowing of the subarachnoid space at the level between C6 and C7 was seen during myeloscopy, while no compression was apparent between C5 and C6. Epiduroscopy showed no abnormalities. After completion of the procedure, the horse was subjected to euthanasia and the cervical spinal cord submitted for histopathological examination. Severe myelin and axon degeneration of the white matter was diagnosed at the level of the intervertebral space between C6 and C7, with Wallerian degeneration cranially and caudally, indicating chronic spinal cord compression at this site. Myeloscopy was successfully used to identify the site of spinal cord compression in a horse with cervical vertebral stenotic myelopathy, while myelography results were misleading.

Cervical vertebral canal endoscopy in the horse: intra- and post operative observations.

REASONS FOR PERFORMING STUDY: Despite modern medical diagnostic imaging, it is not possible to identify reliably the exact location of spinal cord compression in horses with cervical vertebral stenotic myelopathy (CVSM). Vertebral canal endoscopy has been successfully used in man and a technique for cervical vertebral canal endoscopy (CVCE) has been described in equine cadavers. OBJECTIVE: To determine the feasibility and safety of CVCE in healthy mature horses. METHODS: Six healthy mature horses were anaesthetised. A flexible videoendoscope was subsequently introduced via the atlanto-occipital space into the epidural space (epiduroscopy, Horses 1-3) or the subarachnoid space (myeloscopy, Horses 4-6) and advanced to the 8th cervical nerve. Neurological examinations were performed after surgery and lumbosacral cerebrospinal fluid (CSF) analysed in horses that had undergone myeloscopy. RESULTS: All procedures were completed successfully and all horses recovered from anaesthesia. Anatomical structures in the epidural space (including the dura mater, nerve roots, fat and blood vessels) and subarachnoid space (including the spinal cord, blood vessels, arachnoid trabeculations, nerve roots and the external branch of the accessory nerve) were identified. During epiduroscopy, a significant increase in mean arterial pressure was recognised, when repeated injections of electrolyte solution into the epidural space were performed. In one horse of the myeloscopy group, subarachnoid haemorrhage and air occurred, resulting in transient post operative ataxia and muscle fasciculations. No complications during or after myeloscopy were observed in the other horses. CSF analysis indicated mild inflammation on Day 7 with values approaching normal 21 days after surgery. CONCLUSIONS: Endoscopic examination of the epidural and subarachnoid space from the atlanto-occipital space to the 8th cervical nerve is possible and can be safely performed in healthy horses. POTENTIAL RELEVANCE: Cervical vertebral canal endoscopy might allow accurate identification of the compression site in horses with CVSM and aid diagnosis of other lesions within the cervical vertebral canal.

Comparison of conventional and alternative arthroscopic approaches to the palmar/plantar pouch of the equine distal interphalangeal joint.

REASONS FOR PERFORMING THE STUDY: The conventional arthroscopic approach to the palmar/plantar aspect of the distal interphalangeal joint (DIPJ) may result in the inadvertent penetration of the digital flexor tendon sheath (DFTS) and the navicular bursa (NB). This iatrogenic communication would be undesirable subsequent to arthroscopic lavage of a septic DIPJ. HYPOTHESIS: A lateral/medial approach to the palmar/plantar aspect of the DIPJ will result in a significantly lower rate of inadvertent penetration of the DFTS and NB, whilst still providing adequate intra-articular evaluation. METHODS: The conventional palmar/plantar approach or a novel lateral/medial approach to the DIPJ was performed on cadaver fore- and hindlimbs (30 limbs/approach). Subsequently, India ink was injected into the dorsal pouch of the DIPJ, and the DFTS (n = 60) and NB (n = 20) were examined for the presence/absence of ink. In addition, observations of the number of attempts made to access the joint, evidence of iatrogenic intra-articular trauma and occurrence of incomplete visualisation of the palmar/plantar pouch were recorded. RESULTS: With the conventional approach, DFTS penetration was noted in 18/30 (60%) of the limbs, compared to 1/30 (3.3%) with the lateral/medial approach (P≤0.001). NB penetration was seen in 5/10 limbs with the palmar/plantar approach compared to 0/10 with the lateral/medial approach (P = 0.01). No significant differences were found between the approaches in the number of attempts made to access the joint, the incidence of iatrogenic intra-articular trauma, or the occurrence of incomplete visibility of the palmar/plantar pouch. CONCLUSIONS: The novel lateral/medial approach to the DIPJ significantly decreases the risk of inadvertent penetration of the DFTS and NB. POTENTIAL RELEVANCE: The novel lateral/medial approach to the DIPJ is an effective technique to gain access to the palmar/plantar pouches, and is particularly advantageous for arthroscopic lavage of a septic DIPJ.

Endoscopic anatomy of the cervical vertebral canal in the horse: a cadaver study.

REASON FOR PERFORMING STUDY: Localisation of spinal cord compression in horses with cervical vertebral stenotic myelopathy is inexact. Vertebral canal endoscopy has been used in man to localise spinal cord lesions and has the potential to become a useful diagnostic technique in horses. OBJECTIVE: To establish a surgical approach via the atlanto-occipital space to the cervical vertebral canal in equine cadavers and describe the endoscopic anatomy of the cervical epidural and subarachnoid spaces. METHODS: The cadavers of 25 mature horses were used to assess 3 surgical methods to approach the cervical vertebral canal, including 2 minimally invasive and one open technique. Once the approach had been made, a flexible videoendoscope was inserted into the epidural space (epiduroscopy) or the subarachnoid space (myeloscopy) and advanced caudally until the intervertebral space between C7 and T1 was reached. RESULTS: The epidural and subarachnoid spaces could not be accessed reliably using the minimally invasive techniques. Furthermore, damage to the nervous tissues was a frequent complication with these procedures. The open approach allowed successful insertion of the videoendoscope into the epidural and subarachnoid spaces in all horses and no inadvertent damage was observed. Anatomical structures that could be seen in the epidural space included the dura mater, nerve roots, fat and the ventral internal vertebral venous plexus. In the subarachnoid space, the spinal cord, nerve roots, blood vessels, denticulate ligaments and external branch of the accessory nerve were seen. CONCLUSIONS: Using the open approach, epiduroscopy and myeloscopy over the entire length of the cervical vertebral canal are possible in the mature horse. POTENTIAL RELEVANCE: Cervical vertebral canal endoscopy may become a valuable tool to localise the site of spinal cord injury in horses with cervical vertebral stenotic myelopathy and could aid in the diagnosis of other diseases of the cervical spinal cord.

The crystal structure of oxylipin-conjugated barley LTP1 highlights the unique plasticity of the hydrophobic cavity of these plant lipid-binding proteins.

The barley lipid transfer protein (LTP1) adducted by an alpha-ketol, (9-hydroxy-10-oxo-12(Z)-octadecenoic acid) exhibits an unexpected high lipid transfer activity. The crystal structure of this oxylipin-adducted LTP1, (LTP1b) was determined at 1.8A resolution. The covalently bound oxylipin was partly exposed at the surface of the protein and partly buried within the hydrophobic cavity. The structure of the oxylipidated LTP1 emphasizes the unique plasticity of the hydrophobic cavity of these plant lipid-binding proteins when compared to the other members of the family. The plasticity of the hydrophobic cavity and increase of its surface hydrophobicity induced by the oxylipin account for the improvement of the lipid transfer activity of LTP1b. These observations open new perspectives to explore the different biological functions of LTPs, including their allergenic properties.

Destabilizing effect of a fluorouracil extra base in a hybrid RNA duplex compared with bromo and chloro analogues.

In the presence of cobalt, rhodium or iridium hexammine salts, the RNA/DNA hybrid r-GCUUCGGC-d(X)U (with X = F, Cl or Br) crystallizes as a double-stranded helix with four consecutive G-U and C-U mismatches. The deoxy chloro- and bromouracil derivatives are isomorphous, space group C2, unit-cell parameters a = 53.80, b = 19.40, c = 50.31 A, beta = 109.9 degrees, with the same infinite helix arrangement in the packing along the c axis with one extra DNA halogenouracil base included in the stacking. However, the fluorouracil derivative, with unit-cell parameters a = 53.75, b = 19.40, c = 45.84 A, beta = 105.7 degrees, is not isomorphous. The corresponding extra DNA base d(F)U of the second strand is ejected out of the helical stack, leading to a shortening of the c axis. The specific destabilization of the fluorouracil for the duplex building is analyzed in terms of the polarization effect of the halogen atom attached to the 3'-terminal base that modulates its interactions.

High-pressure protein crystallography (HPPX): instrumentation, methodology and results on lysozyme crystals.

A new set-up and associated methodology for the collection of angle-dispersive diffraction data from protein crystals submitted to high hydrostastic pressure have been developed on beamline ID30 at the ESRF. The instrument makes use of intense X-rays of ultra-short wavelength emitted by two collinear undulators, and combines a membrane-driven diamond-anvil cell mounted on a two-axis goniometer and an imaging-plate scanner. Sharp and clean diffraction pictures from tetragonal crystals of hen egg-white lysozyme (tHEWL) and orthorhombic crystals of bovine erythrocyte Cu, Zn superoxide dismutase (SOD) were recorded at room temperature and pressures up to 0.915 and 1.00 GPa, respectively. The compressibility of tHEWL was determined from unit-cell parameters determined at 24 different pressures up to 0.915 GPa. High-pressure diffraction data sets from several crystals of tHEWL were collected and analyzed. Merging of data recorded on different crystals at 0.30 and 0.58 GPa produced two sets of structure amplitudes with good resolution, completeness, redundancy and R(sym) values. A third set at 0.69 GPa was of a similar quality except a lower completeness. The three structures have been refined. The pressure-induced loss of crystalline order in a tHEWL crystal beyond 0.82 GPa was captured through a series of diffraction pictures.

Structural effects of monovalent anions on polymorphic lysozyme crystals.

Understanding direct salt effects on protein crystal polymorphism is addressed by comparing different crystal forms (triclinic, monoclinic, tetragonal and orthorhombic) for hen, turkey, bob white quail and human lysozymes. Four new structures of hen egg-white lysozyme are reported: crystals grown in the presence of NapTS diffracted to 1.85 A, of NaI to 1.6 A, of NaNO(3) to 1.45 A and of KSCN to 1.63 A. These new structures are compared with previously published structures in order to draw a mapping of the surface of different lysozymes interacting with monovalent anions, such as nitrate, chloride, iodide, bromide and thiocyanate. An analysis of the structural sites of these anions in the various lysozyme structures is presented. This study shows common anion sites whatever the crystal form and the chemical nature of anions, while others seem specific to a given geometry and a particular charge environment induced by the crystal packing.

Fatty acids bind to the fungal elicitor cryptogein and compete with sterols.

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid-cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.

Crystallization and preliminary X-ray studies of oligandrin, a sterol-carrier elicitor from Pythium oligandrum.

Oligandrin is a 10 kDa acidic protein produced by the fungus micromycete Pythium oligandrum and is a member of the alpha-elicitin group, with sterol- and lipid-carrier properties. Oligandrin has been crystallized at 290 K using PEG 4000 as a precipitant. A cholesterol complex was obtained under the same conditions. The space group of the crystals at low temperature (100 K) is C222, with unit-cell parameters a = 94.0, b = 171.1, c = 55.3 A. Four molecules are present in the asymmetric unit. Data from the free and cholesterol-complexed forms were recorded at synchrotron sources to resolutions of 2.4 (uncomplexed) and 1.9 A (complexed), respectively.

Crystal structure of a D-aminopeptidase from Ochrobactrum anthropi, a new member of the 'penicillin-recognizing enzyme' family.

BACKGROUND: beta-Lactam compounds are the most widely used antibiotics. They inactivate bacterial DD-transpeptidases, also called penicillin-binding proteins (PBPs), involved in cell-wall biosynthesis. The most common bacterial resistance mechanism against beta-lactam compounds is the synthesis of beta-lactamases that hydrolyse beta-lactam rings. These enzymes are believed to have evolved from cell-wall DD-peptidases. Understanding the biochemical and mechanistic features of the beta-lactam targets is crucial because of the increasing number of resistant bacteria. DAP is a D-aminopeptidase produced by Ochrobactrum anthropi. It is inhibited by various beta-lactam compounds and shares approximately 25% sequence identity with the R61 DD-carboxypeptidase and the class C beta-lactamases. RESULTS: The crystal structure of DAP has been determined to 1.9 A resolution using the multiple isomorphous replacement (MIR) method. The enzyme folds into three domains, A, B and C. Domain A, which contains conserved catalytic residues, has the classical fold of serine beta-lactamases, whereas domains B and C are both antiparallel eight-stranded beta barrels. A loop of domain C protrudes into the substrate-binding site of the enzyme. CONCLUSIONS: Comparison of the biochemical properties and the structure of DAP with PBPs and serine beta-lactamases shows that although the catalytic site of the enzyme is very similar to that of beta-lactamases, its substrate and inhibitor specificity rests on residues of domain C. DAP is a new member of the family of penicillin-recognizing proteins (PRPs) and, at the present time, its enzymatic specificity is clearly unique.

The BPTI decamer observed in acidic pH crystal forms pre-exists as a stable species in solution.

Bovine pancreatic trypsin inhibitor (BPTI) crystallizes under acidic pH conditions in the presence of thiocyanate, chloride and sulfate ions, yielding three different polymorphs in P2(1), P6(4)22 and P6(3)22 space groups, respectively. In all three crystal forms, the same decamer is found in the packing (ten BPTI molecules organized through two perpendicular 2-fold and 5-fold axes as a well-defined and compact object) in contrast to the monomeric crystal forms observed at basic pH conditions. The crystallization of BPTI under acidic conditions (pH 4.5) was investigated by small angle X-ray scattering with both under- and supersaturated BPTI solutions. Data showed the oligomerization of BPTI molecules under all investigated conditions. Accordingly, various mixtures of discrete oligomers (n=1 to 10) were considered. Calculated scattering curves were obtained using models based on the crystallographic structures, and the experimental patterns were analyzed as a linear combination of the model curves using a non-linear curve fitting procedure. The results, confirmed by gel filtration experiments, unambiguously demonstrate the co-existence of two different BPTI particles in solution: a monomer and a decamer, with no evidence of any other intermediates. Moreover, using both approaches, the fraction of decamers was found to increase with increasing salt concentration, even beyond the solubility curve. We therefore propose that at acidic pH, BPTI crystallizes following a two step process: decamers are first built in under- and supersaturated solutions, upon which crystal growth proceeds by decamer stacking. Indeed, those BPTI crystals should best be described as "BPTI decamer" crystals.