Magnetic force micropiston: an integrated force/microfluidic device for the application of compressive forces in a confined environment.

Cellular biology takes place inside confining spaces. For example, bacteria grow in crevices, red blood cells squeeze through capillaries, and chromosomes replicate inside the nucleus. Frequently, the extent of this confinement varies. Bacteria grow longer and divide, red blood cells move through smaller and smaller passages as they travel to capillary beds, and replication doubles the amount of DNA inside the nucleus. This increase in confinement, either due to a decrease in the available space or an increase in the amount of material contained in a constant volume, has the potential to squeeze and stress objects in ways that may lead to changes in morphology, dynamics, and ultimately biological function. Here, we describe a device developed to probe the interplay between confinement and the mechanical properties of cells and cellular structures, and forces that arise due to changes in a structure's state. In this system, the manipulation of a magnetic bead exerts a compressive force upon a target contained in the confining space of a microfluidic channel. This magnetic force microfluidic piston is constructed in such a way that we can measure (a) target compliance and changes in compliance as induced by changes in buffer, extract, or biochemical composition, (b) target expansion force generated by changes in the same parameters, and (c) the effects of compression stress on a target's structure and function. Beyond these issues, our system has general applicability to a variety of questions requiring the combination of mechanical forces, confinement, and optical imaging.

Brain and testicular tumors in mice with progenitor cells lacking BAX and BAK.

The proapoptotic BCL-2 family proteins BAX and BAK serve as essential gatekeepers of the intrinsic apoptotic pathway and, when activated, transform into pore-forming homo-oligomers that permeabilize the mitochondrial outer membrane. Deletion of Bax and Bak causes marked resistance to death stimuli in a variety of cell types. Bax(-/-)Bak(-/-) mice are predominantly non-viable and survivors exhibit multiple developmental abnormalities characterized by cellular excess, including accumulation of neural progenitor cells in the periventricular, hippocampal, cerebellar and olfactory bulb regions of the brain. To explore the long-term pathophysiological consequences of BAX/BAK deficiency in a stem cell niche, we generated Bak(-/-) mice with conditional deletion of Bax in Nestin-positive cells. Aged Nestin(Cre)Bax(fl/fl)Bak(-/-) mice manifest progressive brain enlargement with a profound accumulation of NeuN- and Sox2-positive neural progenitor cells within the subventricular zone (SVZ). One-third of the mice develop frank masses comprised of neural progenitors, and in 20% of these cases, more aggressive, hypercellular tumors emerged. Unexpectedly, 60% of Nestin(Cre)Bax(fl/fl)Bak(-/-) mice harbored high-grade tumors within the testis, a peripheral site of Nestin expression. This in vivo model of severe apoptotic blockade highlights the constitutive role of BAX/BAK in long-term regulation of Nestin-positive progenitor cell pools, with loss of function predisposing to adult-onset tumorigenesis.

DNA relaxation dynamics as a probe for the intracellular environment.

Investigations into the biophysical properties of single molecules traditionally involve well defined in vitro systems where parameters such as solvent viscosity and applied forces are known a priori. These systems provide means to develop models describing the polymers response to a variety of conditions, including the entropically driven relaxation of a stretched biopolymer upon release of the tension inducing force. While these techniques have proven instrumental for recent advancements in the fields of polymer physics and biophysics, how applicable they are to life inside the cell remains poorly understood. Here we report an investigation of in vivo stretched polymer relaxation dynamics using chromatin relaxation following the breakage of a dicentric chromosome subjected to microtubule-based spindle forces. Additionally, we have developed an in vitro system used to verify the conformations observed during the in vivo relaxation, including the predicted but previously unidentified taut conformation. These observations motivate our use of existing polymer models to determine both the in vivo viscosity as seen by the relaxing chromatin and the tension force applied by the microtubule-based spindle in vivo. As a result, the technique described herein may be used as a biophysical strategy to probe the intranuclear environment.

Thin-foil magnetic force system for high-numerical-aperture microscopy.

Forces play a key role in a wide range of biological phenomena from single-protein conformational dynamics to transcription and cell division, to name a few. The majority of existing microbiological force application methods can be divided into two categories: those that can apply relatively high forces through the use of a physical connection to a probe and those that apply smaller forces with a detached probe. Existing magnetic manipulators utilizing high fields and high field gradients have been able to reduce this gap in maximum applicable force, but the size of such devices has limited their use in applications where high force and high-numerical-aperture (NA) microscopy must be combined. We have developed a magnetic manipulation system that is capable of applying forces in excess of 700 pN on a 1 mum paramagnetic particle and 13 nN on a 4.5 mum paramagnetic particle, forces over the full 4pi sr, and a bandwidth in excess of 3 kHz while remaining compatible with a commercially available high-NA microscope objective. Our system design separates the pole tips from the flux coils so that the magnetic-field geometry at the sample is determined by removable thin-foil pole plates, allowing easy change from experiment to experiment. In addition, we have combined the magnetic manipulator with a feedback-enhanced, high-resolution (2.4 nm), high-bandwidth (10 kHz), long-range (100 mum xyz range) laser tracking system. We demonstrate the usefulness of this system in a study of the role of forces in higher-order chromosome structure and function.

Significance of retroperitoneal air after endoscopic retrograde cholangiopancreatography with sphincterotomy.

OBJECTIVE: We designed a prospective study to determine the frequency of retroperitoneal air after endoscopic retrograde cholangiopancreatography (ERCP) with sphincterotomy. We sought to elucidate the relationship of retroperitoneal air with endoscopic maneuvers, clinical findings, the length of sphincterotomy, and the time spent during the procedure. We also endeavored to determine the importance of retroperitoneal air and its most appropriate clinical management. METHODS: Twenty-one consecutive patients who had undergone ERCP with sphincterotomy had abdominal computed tomography (CT) examinations within 24 h after completion of the procedure. The CT findings were unknown to the clinicians, and none of the patients received postprocedural antibiotics. RESULTS: Six (29%) of 21 patients exhibited CT findings of retroperitoneal air. All six patients had uneventful postprocedural courses, and none had abnormal clinical signs or symptoms. The occurrence of retroperitoneal air was not influenced by the presence of hyperamylasemia, the duration of the procedure, or the length of the sphincterotomy. CONCLUSIONS: Retroperitoneal air is not an uncommon finding after ERCP with sphincterotomy. Moreover, the finding of retroperitoneal air in the absence of physical findings, is not a cause for alarm and does not require surgical intervention.

Giant sigmoid diverticulum: a rare manifestation of diverticular disease.

We report two unusual cases of giant sigmoid diverticulum, which is a rare manifestation of diverticular disease. Giant diverticula are 3 to 4 cm or greater in size and are produced by gradual enlargement of an acquired pseudodiverticulum that has had superimposed infection, abscess formation, and healing. For more than 3 years, we have observed and managed the first case conservatively, without surgery. The second case represents the largest recorded giant sigmoid diverticulum (33 cm) in the literature. We review the pathogenesis, clinical features, differential diagnosis, and management of this condition.

Pneumothorax detection in patients with apical pleural caps.

The usual small apical pneumothorax has been previously described as being typified on chest radiographs by a pencil-thin white line that represents the retracted visceral pleura. Occasionally the apical visceral pleura can become thickened, known as the apical cap, which leads to a variant presentation of apical pneumothorax. To our knowledge, this variant presentation has never been described in the literature, and should improve the detection of apical pneumothorax.

TNF-alpha and the pathophysiology of endotoxin-induced acute respiratory failure in sheep.

We studied changes in cardiovascular and pulmonary function during prolonged endotoxemia in conscious sheep. Sheep with chronic lung lymph fistulas received a 12-h infusion of Escherichia coli endotoxin (10 ng x kg-1 x min-1) and allowed to recover for 12 h. Supportive therapies were withheld. Prolonged endotoxemia without volume support caused systemic hypotension associated with reduced cardiac output and increased systemic vascular resistance, pulmonary hypertension, and acute lung injury with progressive respiratory failure. Plasma tumor necrosis factor-alpha (TNF-alpha) concentrations increased transiently. Sustained pulmonary hypertension and increased pulmonary and systemic vascular resistances contributed to a poor outcome in 9 of 34 sheep (26%). Plasma TNF-alpha concentrations were significantly greater in survivors with sustained pulmonary hypertension and in nonsurviving sheep than in surviving sheep without pulmonary hypertension. Endotoxin (1 ng/ml) increased neutrophil expression of TNF-alpha in vitro. Addition of interleukin-6 prevented this response. Synthesis and release of TNF-alpha may be an important proximal event influencing the development of sustained pulmonary hypertension and progressive respiratory failure with endotoxemia. Interleukin-6 may contribute to the phasic nature of the TNF-alpha response.

What kind of general practitioner for the twenty-first century?

In this article a recently qualified physician, now in family practice in the USA, gives expression to the widely held view that a new breed of general practitioner with expanded roles and skills is required if the health-for-all targets are to be attained.

Endotoxin infusion in anesthetized sheep is associated with intrapulmonary sequestration of leukocytes that immunohistochemically express tumor necrosis factor-alpha.

Plasma levels of tumor necrosis factor-alpha (TNF-alpha) peak between 2 and 4 h during a 12-h continuous infusion of endotoxin in awake sheep. We hypothesized that a source of this TNF-alpha is the pool of leukocytes that accumulate in the pulmonary circulation. To test this hypothesis, we physiologically monitored six anesthetized sheep during baseline and 4-h endotoxin infusion periods (10 ng/kg x min). We obtained open-lung biopsies at baseline and at 20 min and 2 and 4 h during the endotoxin infusion period for immunohistochemical localization of TNF-alpha. The plasma concentration of TNF-alpha increased from an average baseline concentration of 0.06 +/- 0.03 ng/ml (mean +/- SD) to a peak of 1.40 +/- 0.28 ng/ml at 2 h of the endotoxin infusion. We observed increased cytoplasmic TNF-alpha immunoreactivity in situ among neutrophils and intravascular mononuclear phagocytes during the endotoxin infusion compared with baseline. Also, the number of immunopositive leukocytes increased in the pulmonary circulation during the continuous infusion of endotoxin. We conclude that TNF-alpha-producing leukocytes accumulate in the pulmonary circulation during endotoxemia. These cells probably contribute to both the rise in the circulating levels of TNF-alpha and the development of acute lung injury.

Abnormal colonic wall thickening on computed tomography.

There are many disease processes, both inflammatory and neoplastic, which can alter the colonic bowel wall. A retrospective study was done on surgically proven cases of colonic disease in which colonic bowel wall abnormalities were identified by computed tomography. Colonic bowel wall thickening was the most common finding, but was nonspecific. Finger-like projections extending from the bowel wall into the surrounding mesentery did help distinguish inflammatory change from neoplastic change. Examples of diverticulitis, appendicitis, Crohn's disease, carcinoma, lymphoma, and metastatic disease are presented.