Mechanical load induced by glass microspheres releases angiogenic factors from neonatal rat ventricular myocytes cultures and causes arrhythmias.

In the present study, we tested the hypothesis that similar to other mechanical loads, notably cyclic stretch (simulating pre-load), glass microspheres simulating afterload will stimulate the secretion of angiogenic factors. Hence, we employed glass microspheres (average diameter 15.7 microm, average mass 5.2 ng) as a new method for imposing mechanical load on neonatal rat ventricular myocytes (NRVM) in culture. The collagen-coated microspheres were spread over the cultures at an estimated density of 3000 microspheres/mm2, they adhered strongly to the myocytes, and acted as small weights carried by the cells during their contraction. NRVM were exposed to either glass microspheres or to cyclic stretch, and several key angiogenic factors were measured by RT-PCR. The major findings were: (1) In contrast to other mechanical loads, such as cyclic stretch, microspheres (at 24 hrs) did not cause hypertrophy. (2) Further, in contrast to cyclic stretch, glass microspheres did not affect Cx43 expression, or the conduction velocity measured by means of the Micro-Electrode-Array system. (3) At 24 hrs, glass microspheres caused arrhythmias, probably resulting from early afterdepolarizations. (4) Glass microspheres caused the release of angiogenic factors as indicated by an increase in mRNA levels of vascular endothelial growth factor (80%), angiopoietin-2 (60%), transforming growth factor-beta (40%) and basic fibroblast growth factor (15%); these effects were comparable to those of cyclic stretch. (5) As compared with control cultures, conditioned media from cultures exposed to microspheres increased endothelial cell migration by 15% (P<0.05) and endothelial cell tube formation by 120% (P<0.05), both common assays for angiogenesis. In conclusion, based on these findings we propose that loading cardiomyocytes with glass microspheres may serve as a new in vitro model for investigating the role of mechanical forces in angiogenesis and arrhythmias.

Robotic assisted spinal surgery--from concept to clinical practice.

After several years of product development, animal trials and human cadaver testing, the SpineAssist--a miniature bone-mounted robotic system--has recently entered clinical use. To the best of the authors' knowledge, this is the only available image-based mechanical guidance system that enables pedicle screw insertion with an overall accuracy in the range of 1 mm in both open and minimally invasive procedures. In this paper, we describe the development and clinical trial process that has brought the SpineAssist to its current state, with an emphasis on the various difficulties encountered along the way and the corresponding solutions. All aspects of product development are discussed, including mechanical design, CT-to-fluoroscopy image registration, and surgical techniques. Finally, we describe a series of preclinical trials with human cadavers, as well as clinical use, which verify the system's accuracy and efficacy.

Febotics - a marriage of fetal surgery and robotics.

For a fetus diagnosed with a severe congenital anomaly, surgery may offer an alternative to abortion, intra-uterine death, or a life with disability. Expertise is limited however, to a few treatment centers worldwide, and there are many technical hurdles to overcome, including requirements for miniaturized instrumentation, real-time high-resolution imaging, and harmless fetal access. This article highlights recent practices in prenatal intervention and various initiatives to integrate robotics into the fetal operating room. While the number of potential patients is low, research for implementation of robotics in the field of fetal surgery is justified by the morbidity rates of current procedures, proven favorable outcomes with intervention, and the educational value with potential for extension to other medical disciplines.

Image-guided system with miniature robot for precise positioning and targeting in keyhole neurosurgery.

This paper describes a novel image-guided system for precise automatic targeting in minimally invasive keyhole neurosurgery. The system consists of the MARS miniature robot fitted with a mechanical guide for needle, probe or catheter insertion. Intraoperatively, the robot is directly affixed to a head clamp or to the patient's skull. It automatically positions itself with respect to predefined targets in a preoperative CT/MRI image following an anatomical registration with an intraoperative 3D surface scan of the patient's facial features and registration jig. We present the system architecture, surgical protocol, custom hardware (targeting and registration jig), and software modules (preoperative planning, intraoperative execution, 3D surface scan processing, and three-way registration). We also describe a prototype implementation of the system and in vitro registration experiments. Our results indicate a system-wide target registration error of 1.7 mm (standard deviation = 0.7 mm), which is close to the required 1.0-1.5 mm clinical accuracy in many keyhole neurosurgical procedures.

Morphological study of the spinal canal content for subarachnoid endoscopy.

STUDY DESIGN AND OBJECTIVE: This study was designed to examine the morphology of the spinal dural sac and contents, using magnetic resonance imaging in order to define the inner geometrical dimensions that confine the manoeuvre of an endoscope inserted in the lumbar region and along the thoracic and cervical spine. BACKGROUND: The morphology of the spine has been studied since the development of myelography. However, most studies have measured the diameters of the spinal cord only, not the size of the subarachnoid space. In addition, the few studies available on the subarachnoid space have focused on the cervical spine, leaving a near-complete dearth of data on the subarachnoid space dimensions along the thoracic spine. METHODS: Based on MRI images of the spine from 42 patients, the dimensions of the spinal cord, dural sac, and subarachnoid space were measured at mid-vertebral and inter-vertebral disc levels. RESULTS: It was found that at each selected transverse level, the subarachnoid space tends to be symmetrical on the right and left sides of the cord, and measures 2.5 mm on average. However, the posterior and anterior segments, measured on the mid-sagittal plane, are generally asymmetrical and vary widely in size, ranging from 1 to 5 mm. These measurements match those found in previous studies, where these are available. The coefficient of variance for the dimensions of the subarachnoid space is as high as 42.4%, while that for the dimensions of the spinal cord is 10-15%. CONCLUSIONS: The findings presented here expand our knowledge of the spinal canal's morphology, and show that an endoscope designed to travel within the subarachnoid space must be smaller than 2.5 mm in diameter.

The complex effect of granulocyte colony-stimulating factor on human granulopoiesis analyzed by a new physiologically-based mathematical model.

Neutropenia, frequently a side effect of chemo- and radiotherapy, increases susceptibility to microbial infections and is a life-threatening condition. For realistically predicting drug treatment effects on granulopoiesis, we have constructed a new mathematical model of granulopoiesis in the bone marrow and in the peripheral blood, featuring cell cycle phase transition and detailed granulocyte-colony stimulating factor (G-CSF) pharmacokinetics (PK) and pharmacodynamics (PD), including intracellular second messenger. Using this model, in conjunction with clinical results, we evaluated the system parameters, implemented those in the model and successfully retrieved the results of several independent clinical experiments under a wide range of G-CSF regimens. Our results show that the introduction of G-CSF-controlled intracellular second messenger is indispensable for precise retrieval of the clinical results, and suggest that the half-life of this messenger varies between a single and multiple G-CSF administration schedules. In addition, our model provided reliable steady-state, as well as dynamic, estimations of human granulopoiesis parameters. These included an estimation of apoptosis index in the post-mitotic compartment, which corroborates previous results. At present the model is used for suggesting improved drug regimens.

Robot-assisted image-guided targeting for minimally invasive neurosurgery: planning, registration, and in-vitro experiment.

This paper present a novel image-guided system for precise automatic targeting in keyhole minimally invasive neurosurgery. The system consists of a miniature robot fitted with a mechanical guide for needle/probe insertion. Intraoperatively, the robot is directly affixed to a head clamp or to the patient skull. It automatically positions itself with respect to predefined targets in a preoperative CT/MRI image following an anatomical registration with a intraoperative 3D surface scan of the patient facial features. We describe the preoperative planning and registration modules, and an in-vitro registration experiment of the entire system which yields a target registration error of 1.7 mm (std = 0.7 mm).

Molecular pharmacology and modeling of vasopressin receptors.

AVP receptors represent a logical target for drug development. As a new class of therapeutic agents, orally active AVP analogs could be used to treat several human pathophysiological conditions including neurogenic diabetes insipidus, the syndrome of inappropriate secretion of AVP (SIADH), congestive heart failure, arterial hypertension, liver cirrhosis, nephrotic syndrome, dysmenorrhea, and ocular hypertension. By immunoprecipitation and immunoblotting, we elucidated the phosphorylation pattern of green fluorescent protein-tagged AVP receptors and showed interactions with the specific kinases PKC and GRK5 that are agonist-, time- and receptor subtype-dependent. The tyrosine residue of the NPWIY motif present in the 7th helix of AVP receptors is rapidly and transiently phosphorylated after agonist stimulation. This phosphorylation is instrumental in the genesis of the mitogenic cascade linked to the activation of this receptor, presumably by establishing key intramolecular contacts and by participating in the creation of a scaffold of proteins that produce the activation of downstream kinases. The random screening of chemical entities and optimization of lead compounds recently resulted in the development of orally active non-peptide AVP receptor agonists and antagonists. Furthermore, the identification of the molecular determinants of receptor-ligand interactions should facilitate the development of more potent and very selective orally active compounds via the approach of structure-based drug design. We developed three-dimensional molecular docking models of peptide and non-peptide ligands to the human V1 vascular, V2 renal and V3 pituitary AVP receptors. Docking of the peptide hormone AVP to the receptor ligand binding pockets reflects its dual polar and non-polar structure, but is receptor subtype-specific. The characteristics of non-peptide AVP analogs docking to the receptors are clearly distinct from those of peptide analogs docking. Molecular modeling of the results of site-directed mutagenesis experiments performed in CHO cells stably transfected with the human AVP receptor subtypes revealed that non-peptide antagonists establish key contacts with a few amino acid residues of the receptor subtypes that are different from those involved in agonist binding. Moreover, these interactions are species-specific. These findings provide further understanding of the signal transduction pathways of AVP receptors and new leads for elucidation of drug-receptor interactions and optimization of drug design. NOTE TO THE READER: The recent cloning and molecular characterization of AVP/OT receptor subtypes call for the revision of their nomenclature. For the sake of clarity and reference to their main site of expression, we call the V1a receptor the V1 vascular receptor, the V2 receptor the V2 renal receptor and the V1b or V3 receptor the V3 pituitary receptor in the present review.

Crystal structure of colicin E3: implications for cell entry and ribosome inactivation.

Colicins kill E. coli by a process that involves binding to a surface receptor, entering the cell, and, finally, intoxicating it. The lethal action of colicin E3 is a specific cleavage in the ribosomal decoding A site. The crystal structure of colicin E3, reported here in a binary complex with its immunity protein (IP), reveals a Y-shaped molecule with the receptor binding domain forming a 100 A long stalk and the two globular heads of the translocation domain (T) and the catalytic domain (C) comprising the two arms. Active site residues are D510, H513, E517, and R545. IP is buried between T and C. Rather than blocking the active site, IP prevents access of the active site to the ribosome.

Morphometric study of the human lumbar spine for operation-workspace specifications.

STUDY DESIGN: The anatomy of the lumbar vertebrae of 55 patients was measured by use of data provided by computed tomography. On the basis of these measurements, the location of puncture points and the orientation of the surgical instruments for pedicle, vertebral body, and disc entry points were calculated for open as well as percutaneous surgery. OBJECTIVE: Normal anatomic variations of the lumbar spine were investigated to define the workspace for several spinal procedures and to define the workspace of a robot designed to guide the physician during those procedures. SUMMARY OF BACKGROUND: Several comprehensive studies of vertebrae dimensions have been conducted in the past, but they lack several dimensions that are needed to determine the exact location of the entry point and orientation of the tool, in particular when a computerized guidance system is used. METHODS: Fifty-five spinal columns (L1-L5, total 250 vertebrae) were measured by computed tomography. These data provide geometric relations that determine entry points and tool orientations for different spinal interventions. RESULTS: The workspace for spinal operations was defined on the basis of anatomic data taken from computed tomography scans. The data included 15 measurements for each vertebra that defined its shape. The processed data provided puncture points for several spinal procedures in both open and percutaneous surgery. CONCLUSIONS: This study provides additional information on vertebral structure needed to calculate accurately the entry point and tool orientation in various spinal procedures. These statistical data are also valuable for model and implant designs and for workspace specifications for a robot-assisted surgery system.

Bulk production and functional analyses of mouse CD55's native and deglycosylated active domains.

We report the use of methylotrophic yeast Pichia pastoris as a host to efficiently express complement control protein repeats (CCPs) 1-4 of mouse decay accelerating factor (DAF, CD55) as a soluble protein. With this system, the mouse DAF CCP1-4-active-domain-containing module linked to a 6x His tag at its C terminus was secreted into the culture supernatant at 15 mg/L after 24 h of induction with methanol. A mouse DAF CCP1-4 mutant protein in which its two potential N-glycosylation sites were deleted by changing Asn(187) and Asn(262) to Gln was also produced. Using Ni(2+)-immobilized agarose affinity chromatography, the recombinant mouse DAF modules with their 6x His tags could be one-step isolated to SDS-PAGE purity. Polyclonal antibody against native mouse DAF CCP1-4 was raised by immunizing NZW rabbits with the purified product. Measurements of the bioactivities of the wild-type and mutant mouse DAF proteins in C3b uptake assays showed no differences in regulatory activities in either the classical or the alternative pathways. With the use of the mutant DAF protein, small rod-shaped crystals were produced and preliminary data obtained. The production of large quantities of functional recombinant mouse DAF CCP1-4 modules and their antibody offers the opportunity to study DAF structure and DAF function in vivo.

The basic and clinical pharmacology of nonpeptide vasopressin receptor antagonists.

The neurohypophysial hormone arginine vasopressin (AVP) is a cyclic nonpeptide whose actions are mediated by the stimulation of specific G protein--coupled membrane receptors pharmacologically classified into V1-vascular (V1R), V2-renal (V2R) and V3-pituitary (V3R) AVP receptor subtypes. The random screening of chemical compounds and optimization of lead compounds recently resulted in the development of orally active nonpeptide AVP receptor antagonists. Potential therapeutic uses of AVP receptor antagonists include (a) the blockade of V1-vascular AVP receptors in arterial hypertension, congestive heart failure, and peripheral vascular disease; (b) the blockade of V2-renal AVP receptors in the syndrome of inappropriate vasopressin secretion, congestive heart failure, liver cirrhosis, nephrotic syndrome and any state of excessive retention of free water and subsequent dilutional hyponatremia; (c) the blockade of V3-pituitary AVP receptors in adrenocorticotropin-secreting tumors. The pharmacological and clinical profile of orally active nonpeptide vasopressin receptor antagonists is reviewed here.

A surface-matching technique for robot-assisted registration.

Successful implementation of robot-assisted surgery (RAS) requires coherent integration of spatial image data with sensing and actuating devices, each having its own coordinate system. Hence, accurate estimation of the geometric relationships between relevant reference frames, known as registration, is a crucial procedure in all RAS applications. The purpose of this paper is to present a new registration scheme, along with the results of an experimental evaluation of a robot-assisted registration method for RAS applications in orthopedics. The accuracy of the proposed registration is appropriate for specified orthopedic surgical applications such as Total Knee Replacement. The registration method is based on a surface-matching algorithm that does not require marker implants, thereby reducing surgical invasiveness. Points on the bone surface are sampled by the robot, which in turn directs the surgical tool. This technique eliminates additional coordinate transformations to an external device (such as a digitizer), resulting in increased surgical accuracy. The registration technique was tested on an RSPR six-degrees-of-freedom parallel robot specifically designed for medical applications. A six-axis force sensor attached to the robot's moving platform enables fast and accurate acquisition of positions and surface normal directions at sampled points. Sampling with a robot probe was shown to be accurate, fast, and easy to perform. The whole procedure takes about 2 min, with the robot performing most of the registration procedures, leaving the surgeon's hands free. Robotic registration was shown to provide a flawless link between preoperative planning and robotic assistance during surgery.

A molecular model of agonist and nonpeptide antagonist binding to the human V(1) vascular vasopressin receptor.

The affinity of the nonpeptide antagonist OPC-21268 is greater for the rat V(1) arginine vasopressin (AVP) receptor (V(1)R) than for the human V(1)R. Site-specific mutagenesis was carried out to identify the residues that determine interspecies selectivity for nonpeptide antagonist binding. The introduction of rat amino acids in position 224, 310, 324, or 337 of the human V(1)R sequence dramatically altered OPC-21268 affinity for the receptor, whereas binding of AVP, the peptide V(1)R antagonist d(CH(2))(5)Tyr(Me)AVP, and the nonpeptide V(1)R antagonist SR49059 was not altered by these mutations. Computer modeling explained the mutagenesis results. Docking of OPC-21268 onto a homology-built model of the V(1)R receptor yielded a model for the bound ligand in which the hydrophobic part is deeply embedded in the transmembrane region, whereas the polar part is located on the surface of the extracellular side. The increased affinity of the G337A mutant is due to two additional van der Waals contacts of the alanine methyl group with carbon atoms on the antagonist. The I310V mutant reduces the hydrophobicity in the vicinity of the polar oxygen atom of the antagonist. The I224V mutant relieves overcrowding in a hydrophobic binding pocket involving the aromatic residues Trp(175), Phe(179), Phe(307), and Trp(304). Finally, the E324D mutant enables the formation of a hydrogen bond of the carboxylate side chain with the amide side chain of Gln(311), which in turn forms a hydrogen bond with the N57 nitrogen atom of OPC-21268. Thus, a few residues, distinct from those involved in agonist binding, control interspecies selectivity toward OPC-21268 nonpeptide antagonist binding.

Crystal structure of colicin E3 immunity protein: an inhibitor of a ribosome-inactivating RNase.

BACKGROUND: Colicins are antibiotic-like proteins of Escherichia coli that kill related strains. Colicin E3 acts as an RNase that specifically cleaves 16S rRNA, thereby inactivating the ribosomes in the infected cell. The producing organism is protected against colicin E3 by a specific inhibitor, the immunity protein Im3, which forms a tight 1:1 complex with colicin E3 and renders it inactive. Crystallographic studies on colicin E3 and Im3 have been undertaken to unravel the structural basis for the ribonucleolytic activity and its inhibition. RESULTS: The crystal structure of Im3 has been determined to a resolution of 1.8 A. The structure consists of a four-standard antiparallel beta sheet flanked by three alpha helices on one side of the sheet. Thr7, Phe9, Phe16 and Phe74 form a hydrophobic cluster on the surface of the protein in the vicinity of Cys47. This cluster is part of a putative binding pocket which also includes nine polar residues. CONCLUSIONS: The putative binding pocket of Im3 is the probable site of interaction with colicin E3. The six acidic residues in the pocket may interact with some of the numerous basic residues of colicin E3. The involvement of hydrophobic moieties in the binding is consistent with the observation that the tight complex can only be dissociated by denaturation. The structure of Im3 resembles those of certain nucleic acid binding proteins, in particular domain II of topoisomerase I and RNA-binding proteins that contain the ribonucleoprotein (RNP) sequence motif. This observation suggests that Im3 has a nucleic acid binding function in addition to binding colicin E3.

Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests determinants of substrate specificity and thermostability.

The crystal structure of a thermophilic alcohol dehydrogenase (TBAD) from Thermoanaerobacter brockii has been determined in a binary complex with sec-butanol as substrate to a resolution of 3.0 A. Van der Waals interactions of the carbon C1 atom of sec-butanol with atoms in His59, Ala85, Trp110, Asp150, and Leu294 account for the substrate preference of this enzyme for secondary over primary alcohols. A crevice from the surface to the active site provides access for substrates and products. This opening is lined with the hydrophobic residues Ile49, Leu107, Trp110, Tyr267, Leu294 as well as Cys283 and Met285 from another molecule within the tetrameric assembly. This might explain the tolerance of this enzyme toward organic solvents. The zinc ion occupies a position in the active site, which is too remote for direct interaction with the alcohol group. A mechanism is suggested whereby the introduction of NADP would trigger a displacement of the zinc ion to its catalytic site. Features important for the unusually high melting temperature of 98 degrees C are suggested by comparison to the crystal structure of a highly homologous mesophilic alcohol dehydrogenase from Clostridium beijerinckii (CBAD). The thermophilic enzyme has a more hydrophilic exterior, a more hydrophobic interior, a smaller surface area, more prolines, alanines, and fewer serines than CBAD. Furthermore, in the thermophilic enzyme the number of all types of intersubunit interactions in these tetrameric enzymes is increased: more salt bridges, hydrogen bonds, and hydrophobic interactions. All these effects combined can account for the higher melting temperature of the thermophilic enzyme.

Standing sway: iterative estimation of the kinematics and dynamics of the lower extremities from force-plate measurements.

In this study, a model for the estimation of the dynamics of the lower extremities in standing sway from force plate data only is presented. A three-dimensional, five-segment, four-joint model of the human body was used to describe postural standing sway dynamics. Force-plate data of the reactive forces and centers of pressure were measured bilaterally. By applying the equations of motion to these data, the transversal trajectory of the center of gravity (CG) of the body was resolved in the sagittal and coronal planes. An inverse kinematics algorithm was used to evaluate the kinematics of the body segments. The dynamics of the segments was then resolved by using the Newton-Euler equations, and the model's estimated dynamic quantities of the distal segments were compared with those actually measured. Differences between model and measured dynamics were calculated and minimized, using an iterative algorithm to re-estimate joint positioning and anthropometric properties. The above method was tested with a group of 11 able-bodied subjects, and the results indicated that the relative errors obtained in the final iteration were of the same order of magnitude as those reported for closed loop problems involved in direct kinematic measurements of human gait.

Identification of conserved aromatic residues essential for agonist binding and second messenger production at 5-hydroxytryptamine2A receptors.

Several models of agonist binding to G protein-coupled 5-hydroxytryptamine [5-HT] (serotonin) receptors have highlighted the potential importance of highly conserved aromatic residues for ligand binding and agonist efficacy. In this study, we tested these models by constructing and characterizing a number of point mutations of conserved and nonconserved aromatic residues using the 5-HT2A receptor as a model system. Mutations of three highly conserved tryptophans (W200A, W336A, and W367A) proposed to reside near the binding pocket markedly reduced agonist affinity and efficacy at 5-HT2A receptors. Mutations of two other highly conserved aromatic residues postulated to be near the agonist binding site (F340L and Y370A) also had dramatic effects on agonist binding and efficacy. Point mutations of neighboring conserved phenylalanines (F339L and F365L) had minimal effects on agonist binding, although the F365L mutation diminished agonist efficacy. Finally, mutations of two nonconserved aromatic residues (F125L and F383A) not predicted to be near the binding pocket had no effects on agonist binding, potency, or efficacy. Our results are best explained by models that suggest that helices III, V, VI, and VII can form a unit of interacting helices in which highly conserved aromatic residues are oriented toward the center of the helical aggregate to form an aromatic pocket. In addition, our novel results identify a series of aromatic residues essential for agonist-induced second messenger production. These results demonstrate that highly conserved aromatic residues residing in neighboring helices provide the optimum environment for both agonist binding and activation of 5-HT2A receptors.

Molecular modeling and mechanism of action of human decay-accelerating factor.

A model of the regulatory region of human decay accelerating factor (DAF) was built based on the known coordinates of a fragment of the structurally and functionally homologous serum protein, factor H. According to this model, the four short consensus repeats (SCRs) in DAF are arranged in a helical fashion. A positively charged surface area on SCRs 2 and 3, two of the three repeating units essential for function, is postulated to be the primary recognition site for the C3 convertases C4b2a and C3bBb. This area encompasses a cavity on SCR 2, as well as part of the groove on the SCR 2-SCR 3 interface. Two additional surface depressions are centered around the C-terminal disulfide bridges of SCRs 3 and 4. These are likely to provide additional ligand binding sites. Based on this model in conjunction with sequence homology to the Ba fragment of factor B, a mechanism of DAF's accelerated convertase decay action is postulated.