Bacteria are small but not stupid: cognition, natural genetic engineering and socio-bacteriology.

Forty years' experience as a bacterial geneticist has taught me that bacteria possess many cognitive, computational and evolutionary capabilities unimaginable in the first six decades of the twentieth century. Analysis of cellular processes such as metabolism, regulation of protein synthesis, and DNA repair established that bacteria continually monitor their external and internal environments and compute functional outputs based on information provided by their sensory apparatus. Studies of genetic recombination, lysogeny, antibiotic resistance and my own work on transposable elements revealed multiple widespread bacterial systems for mobilizing and engineering DNA molecules. Examination of colony development and organization led me to appreciate how extensive multicellular collaboration is among the majority of bacterial species. Contemporary research in many laboratories on cell-cell signaling, symbiosis and pathogenesis show that bacteria utilise sophisticated mechanisms for intercellular communication and even have the ability to commandeer the basic cell biology of 'higher' plants and animals to meet their own needs. This remarkable series of observations requires us to revise basic ideas about biological information processing and recognise that even the smallest cells are sentient beings.

Insulin independence of unstable diabetic patient after single living donor islet transplantation.

BACKGROUND: Current success in islet transplantation will lead to a donor shortage. Living donor islet transplantation could be an alternative approach to expand the potential donor pool. In this study we describe the first successful living donor islet transplantation for unstable diabetes, performed at Kyoto University Hospital on January 19, 2005. METHODS: The donor was a healthy 56-year-old woman and mother of the recipient. The recipient was a 27-year-old woman with insulin-dependent diabetes since the age of 15 years. She experienced frequent hypoglycemic unawareness episodes. Her blood glucose concentration was difficult to control and C-peptide level was negative after glucagon stimulation. She needed an average 28 of units of insulin per day. The donor underwent a distal pancreatectomy and islets were isolated from the resected pancreas graft. The total islet yield was 408,114 islet equivalents and isolated islets were immediately transplanted into the recipient's liver. RESULTS: After transplant, the blood glucose level of the recipient was tightly controlled without hypoglycemic episodes. She was discharged on day 37 with a normal oral glucose tolerance test (OGTT). The recipient remained insulin-independent for >3 months, since day 22 posttransplant. The donor's postoperative clinical course was uneventful. She was discharged on postoperative day 18 and returned to her job within 1 month. CONCLUSIONS: We report the first successful living donor islet transplantation for the treatment of unstable diabetes. We believe that living donor islet transplantation may become an option in the treatment of insulin-dependent diabetes.

How repeated retroelements format genome function.

Genomes operate as sophisticated information storage systems. Generic repeated signals in the DNA format expression of coding sequence files and organize additional functions essential for genome replication and accurate transmission to progeny cells. Retroelements comprise a major fraction of many genomes and contain a surprising diversity of functional signals. In this article, we summarize some features of the taxonomic distribution of retroelements, especially mammalian SINEs, tabulate functional roles documented for different classes of retroelements, and discuss their potential roles as genome organizers. In particular, the fact that certain retroelements serve as boundaries for heterochromatin domains and provide a significant fraction of scaffolding/matrix attachment regions (S/MARs) suggests that the reversed transcribed component of the genome plays a major architectonic role in higher order physical structuring. Employing an information science model, the "functionalist" perspective on repetitive DNA leads to new ways of thinking about the systemic organization of cellular genomes and provides several novel possibilities involving retroelements in evolutionarily significant genome reorganization.

A 21(st) Century View of Evolution.

Physicists question whether there are 'universals' in biology. One reason is that the prevailing theory of biological evolution postulates a random walk to each new adaptation. In the last 50 years, molecular genetics has revealed features of DNA sequence organization, protein structure and cellular processes of genetic change that suggest evolution by Natural Genetic Engineering. Genomes are hierarchically organized as systems assembled from DNA modules. Each genome is formatted and integrated by repetitive DNA sequence elements that do not code for proteins, much as a computer drive is formatted. These formatting elements constitute codons in multiple genetic codes for distinct functions such as transcription, replication, DNA compaction and genome distribution to daughter cells. Consequently, there is a computation-ready Genome System Architecture for each species. Whole-genome sequencing indicates that rearrangement of genetic modules plus duplication and reuse of existing genomic systems are fundamental events in evolution. Studies of genetic change show that cells possess mobile genetic elements and other natural genetic engineering activities to carry out the necessary DNA reorganizations. Natural genetic engineering functions are sensitive to biological inputs and their non-random operations help explain how novel genome system architectures can arise in evolution.

Colorectal cancer-screening tests and associated health behaviors.

BACKGROUND: Studies have shown that screening reduces colorectal cancer mortality. We analyzed national survey data to determine rates of use of fecal occult blood testing (FOBT) and sigmoidoscopy, and to determine if these rates differ by demographic factors and other health behaviors. METHODS: A total of 52,754 respondents aged >or=50 years were questioned in the 1997 Behavioral Risk Factor Surveillance System (BRFSS) survey (a random-digit-dialing telephone survey of the non-institutionalized U.S. population) about their use of FOBT and sigmoidoscopy. RESULTS: The age-adjusted proportion of respondents who reported having had a colorectal cancer screening test during the recommended time interval (past year for FOBT and past 5 years for sigmoidoscopy) was 19.8% for FOBT, 30.5% for sigmoidoscopy, and 41.1% for either FOBT or sigmoidoscopy. Rates of use of colorectal cancer screening tests were higher for those who had other screening tests (mammography, Papanicolaou smear, and cholesterol check). There were also differences in rates of use of colorectal cancer screening tests according to other health behaviors (smoking, seat belt use, fruit and vegetable intake, and physical activity) and several demographic factors. However, none of the subgroups that we examined reported a rate of FOBT use above 29% within the past year or a rate of sigmoidoscopy use above 41% within the past 5 years. CONCLUSIONS: While rates of use of FOBT and sigmoidoscopy were higher among people who practiced other healthy behaviors, rates of use were still quite low in all subgroups. There is a need for increased awareness of the importance of colorectal cancer screening.

Cigar smoking in men and risk of death from tobacco-related cancers.

BACKGROUND: Cigar consumption in the United States has increased dramatically since 1993, yet there are limited prospective data on the risk of cancer associated with cigar smoking. We examined the association between cigar smoking and death from tobacco-related cancers in a large, prospective cohort of U. S. men. METHODS: We used Cox proportional hazards models to analyze the relationship between cigar smoking at baseline in 1982 and mortality from cancers of the lung, oral cavity/pharynx, larynx, esophagus, bladder, and pancreas over 12 years of follow-up of the American Cancer Society's Cancer Prevention Study II cohort. A total of 137 243 men were included in the final analysis. Women were not included because we had no data on their cigar use. We excluded men who ever smoked cigarettes or pipes and adjusted all rate ratio (RR) estimates for age, alcohol use, and use of snuff or chewing tobacco. RESULTS: Current cigar smoking at baseline, as compared with never smoking, was associated with an increased risk of death from cancers of the lung (RR = 5.1; 95% confidence interval [CI] = 4.0-6.6), oral cavity/pharynx (RR = 4.0 [95% CI = 1.5-10.3]), larynx (RR = 10.3 [95% CI = 2.6-41.0]), and esophagus (RR = 1.8; 95% CI = 0.9-3.7). Although current cigar smokers overall did not appear to be at an increased risk of death from cancer of the pancreas (RR = 1.3; 95% CI = 0.9-1.9) or bladder (RR = 1.0; 95% CI = 0.4-2.3), there was an increased risk for current cigar smokers who reported that they inhaled the smoke (for pancreas, RR = 2.7; 95% CI = 1.5-4.8; for bladder, RR = 3.6; 95% CI = 1.3-9.9). CONCLUSIONS: Results from this large prospective study support a strong association between cigar smoking and mortality from several types of cancer.

Painful intraosseous ganglion of the scaphoid overshadowed by thoracic outlet syndrome. Case report.

We present a case of a woman diagnosed several years previously with thoracic outlet syndrome who had a 2-3 month history of worsening wrist pain. After an intraosseous ganglion was discovered, curettage and bone grafting successfully relieved her symptoms.

Genome system architecture and natural genetic engineering in evolution.

Molecular genetics teaches three lessons relevant to the nature of genetic change during evolution: (1) Genomes are organized as hierarchies of composite systems (multidomain protein-coding sequences; functional loci made up of regulatory, coding, processing, and intervening sequences; and multilocus regulons and replicons) interconnected and organized into specific "system architectures" by repetitive DNA elements. (2) Genetic change often occurs via natural genetic engineering systems (cellular biochemical functions, such as recombination complexes, topoisomerases, and mobile elements, capable of altering DNA sequence information and joining together different genomic components). (3) The activity of natural genetic systems is regulated by cellular control circuits with respect to the timing, activity levels, and specificities of DNA rearrangements (e.g., adaptive mutation, Ty element mobility, and P factor insertions). These three lessons provide plausible molecular explanations for the episodic, multiple, nonrandom DNA rearrangements needed to account for the evolution of novel genomic system architectures and complex multilocus adaptations. This molecular genetic perspective places evolutionary change in the biologically responsive context of cellular biochemistry.

Differential fiu-lacZ fusion regulation linked to Escherichia coli colony development.

Colonies of strains carrying a stable lambdaplacMu15 translational fusion displayed sharply defined intense staining at the centre on Xgal medium. The fusion was in fiu (ferric ion uptake), encoding an iron-regulated outer membrane protein (IROMP) controlled via four overlapping ferric uptake regulator (Fur) boxes in the sigma70 promoter region. Fiu-LacZ was synthesized in low amounts (< 1% of a transcriptional fiu:lacZ+ fusion), localized to membranes, and underwent processing from a large protein to one that co-migrated with native beta-galactosidase. Intact cells synthesizing Fiu-LacZ often displayed greater enzymatic activity than permeabilized cells. The colony centre was insensitive to iron regulation observed in liquid cultures and at the colony edge. Within colonies grown on 36 microM iron citrate medium, fiu'-'lacZ protein fusion strains displayed 60-fold higher beta-galactosidase activity in the centre, and transcriptional fiu:lacZ+ fusion strains displayed a 10-fold centre/edge difference. On medium without added iron citrate, the centre/edge difference collapsed to < 2.2-fold for both translational and transcriptional fusions because activity at the edge was derepressed. Iron-insensitive fiu'-'lacZ expression in the colony centre occurred during a 6-18 h time window at the start of colony morphogenesis, corresponding to the initiation of multilayer microcolony development. A simple model for differential fiu'-'lacZ regulation is proposed whereby iron accessibility changes during colony morphogenesis.

Starvation-induced Mucts62-mediated coding sequence fusion: a role for ClpXP, Lon, RpoS and Crp.

The formation of araB-lacZ coding sequence fusions in Escherichia coli is a particular type of chromosomal rearrangement induced by Mucts62, a thermoinducible mutant of mutator phage Mu. Fusion formation is controlled by the host physiology. It only occurs after aerobic carbon starvation and requires the phage-encoded transposase pA, suggesting that these growth conditions trigger induction of the Mucts62 prophage. Here, we show that thermal induction of the prophage accelerated araB-lacZ fusion formation, confirming that derepression is a rate-limiting step in the fusion process. Nonetheless, starvation conditions remained essential to complete fusions, suggesting additional levels of physiological regulation. Using a transcriptional fusion indicator system in which the Mu early lytic promoter is fused to the reporter E. coli lacZ gene, we confirmed that the Mucts62 prophage was derepressed in stationary phase (S derepression) at low temperature. S derepression did not apply to prophages that expressed the Mu wild-type repressor. It depended upon the host ClpXP and Lon ATP-dependent proteases and the RpoS stationary phase-specific sigma factor, but not upon Crp. None of these four functions was required for thermal induction. Crp was required for fusion formation, but only when the Mucts62 prophage encoded the transposition/replication activating protein pB. Finally, we found that thermally induced cultures did not return to the repressed state when shifted back to low temperature and, hence, remained activated for accelerated fusion formation upon starvation. The maintenance of the derepressed state required the ClpXP and Lon host proteases and the prophage Ner-regulatory protein. These observations illustrate how the cts62 mutation in Mu repressor provides the prophage with a new way to respond to growth phase-specific regulatory signals and endows the host cell with a new potential for adaptation through the controlled use of the phage transposition machinery.

Hypertension, antihypertensive medication use, and risk of renal cell carcinoma.

To investigate whether diuretic medication use increases risk of renal cell carcinoma (RCC), the authors conducted a case-control study of health maintenance organization members in western Washington State. Cases (n = 238) diagnosed between January 1980 and June 1995 were compared with controls (n = 616) selected from health maintenance organization membership files. The computerized health maintenance organization pharmacy database provided information on medications prescribed after March 1977. Additional exposure information was collected from medical records. For women, use of diuretics was associated with increased risk of RCC (odds ratio (OR) = 1.8, 95% confidence interval (CI) 1.0-3.1), but the association was not independent of a diagnosis of hypertension (adjusted for hypertension, OR = 1.1, 95% CI 0.5-2.1). Similarly, nondiuretic antihypertensive use was associated with increased risk, but only when unadjusted for hypertension. For men, neither diuretic nor nondiuretic antihypertensive use was associated with risk of RCC. A diagnosis of hypertension was clearly associated with RCC risk for women (OR = 2.5, 95% CI 1.2-5.1), but not men (OR = 1.3, 95% CI 0.7-2.5). High systolic and diastolic blood pressures were associated with increased risk in both sexes. These results do not support the hypothesis that use of diuretic medication increases RCC risk; they are more consistent with an association between RCC and high blood pressure.

Body mass index and risk of renal cell carcinoma.

To examine the association between body mass index and renal cell carcinoma risk, we analyzed data from a case-control study of members of a health maintenance organization in western Washington State. We identified cases diagnosed between 1980 and 1995 through a population-based cancer registry. We selected controls from membership files. We collected adult weight and height from medical records. Increased body mass index was associated with increases in risk for both men and women (for the top quartile relative to the bottom quartile of maximum body mass index: in women, OR = 3.3, 95% CI = 1.2-8.7; in men, OR = 2.3, 95% CI = 1.2-4.5).

Long-term outcome of living related liver transplantation for patients with intrapulmonary shunting and strategy for complications.

BACKGROUND: In 320 living related liver transplantation performed between June 1990 and September 1997, there were 21 living related liver transplantation for patients with intrapulmonary shunting, manifested by digital clubbing, cyanosis, and dyspnea. We report the long-term outcome for more 6 months and our strategy to overcome complications in these recipients. PATIENTS: A total of 21 patients (age range 2-33 years, 19 children and 2 adults, 6 males and 15 females) were classified into three grades according to shunt ratio calculated by TcMAA pulmonary scintigraphy; 5 in mild group (shunt ratio: less than 20%), 6 in moderated group (20%-40%), and 10 in severe group (more than 40%). The original underlying liver disease was biliary atresia in all patients. RESULTS: Spearmen's correlation coefficient rank test revealed that shunt ratio correlated significantly with PaO2 in room air (P=0.0001), PaO2 in 100% oxygen (P=0.0004), hematocrit (P=0.0276), and period of dyspnea before transplantation (P=0.023). COMPLICATIONS: Wound infection occurred in 80, 66, and 80%, and bile leakage in 20, 0, 40% in mild, moderate, and severe group, respectively. Patients who had portal vein thrombosis, and intracranial complication were classified as severe group and the incidence was 20 and 20%, respectively. The patient actuarial one year survival was 80, 66.7, and 48%, in mild, moderate, and severe group, respectively, although there was no significant difference. All patients who survived improved hepatopulmonary syndrome and the length of period required for the resolution was significantly correlated to the preoperative shunt ratio (P=0.023). COMMENTS: Patients with severe shunting are susceptible to wound infection and bile leak. The trend of higher incidence of portal thrombosis and intracranial complications in the severe group was closely related high hematocrit. Secure surgical technique to reduce bile leak and delayed primary wound closure to reduce wound infection were found to be effective. Anticoagulant therapy by infusing heparin through the portal vein followed by coumadin could prevent fatal portal vein thrombosis without counter risk of fatal cerebral hemorrhage.

Transposable elements as the key to a 21st century view of evolution.

Cells are capable of sophisticated information processing. Cellular signal transduction networks serve to compute data from multiple inputs and make decisions about cellular behavior. Genomes are organized like integrated computer programs as systems of routines and subroutines, not as a collection of independent genetic 'units'. DNA sequences which do not code for protein structure determine the system architecture of the genome. Repetitive DNA elements serve as tags to mark and integrate different protein coding sequences into coordinately functioning groups, to build up systems for genome replication and distribution to daughter cells, and to organize chromatin. Genomes can be reorganized through the action of cellular systems for cutting, splicing and rearranging DNA molecules. Natural genetic engineering systems (including transposable elements) are capable of acting genome-wide and not just one site at a time. Transposable elements are subject to regulation by cellular signal transduction/computing networks. This regulation acts on both the timing and extent of DNA rearrangements and (in a few documented cases so far) on the location of changes in the genomes. By connecting transcriptional regulatory circuits to the action of natural genetic engineering systems, there is a plausible molecular basis for coordinated changes in the genome subject to biologically meaningful feedback.

Menopausal hormone use and endometrial cancer, by tumor grade and invasion.

We analyzed data from a population-based case-control study to investigate whether combined hormone replacement therapy influences the incidence of high-grade and -stage endometrial cancer. Subjects were women with epithelial endometrial cancer (N = 730) diagnosed during 1985-1991 and controls identified through random digit dialing (N = 1,002). Relative to hormone nonusers, women who took unopposed estrogens (mostly conjugated estrogens) for 3 or more years had a fivefold increase in the risk of tumors with myometrial invasion; the corresponding relative risk associated with combined therapy (estrogen and cyclic or continuous progestogen) for 3 or more years was only 1.3 (95% confidence interval = 0.8-2.2). We found a similar pattern of association for high-grade tumors.

Thinking about bacterial populations as multicellular organisms.

It has been a decade since multicellularity was proposed as a general bacterial trait. Intercellular communication and multicellular coordination are now known to be widespread among prokaryotes and to affect multiple phenotypes. Many different classes of signaling molecules have been identified in both Gram-positive and Gram-negative species. Bacteria have sophisticated signal transduction networks for integrating intercellular signals with other information to make decisions about gene expression and cellular differentiation. Coordinated multicellular behavior can be observed in a variety of situations, including development of E. coli and B. subtilis colonies, swarming by Proteus and Serratia, and spatially organized interspecific metabolic cooperation in anaerobic bioreactor granules. Bacteria benefit from multicellular cooperation by using cellular division of labor, accessing resources that cannot effectively be utilized by single cells, collectively defending against antagonists, and optimizing population survival by differentiating into distinct cell types.

Different structures of selected and unselected araB-lacZ fusions.

Formation of araB-lacZ coding-sequence fusions is a key adaptive mutation system. Eighty-four independent araB-lacZ fusions were sequenced. All fusions carried rearranged MuR linker sequences between the araB and lacZ domains indicating that they arose from the standard intermediate of the well-characterized Mu DNA rearrangement process, the strand transfer complex (STC). Five non-standard araB-lacZ fusions isolated after indirect sib selection had novel structures containing back-to-back inverted MuR linkers. The observation that different isolation procedures gave rise to standard and non-standard fusions indicates that cellular physiology can influence late steps in the multi-step biochemical sequence leading to araB-lacZ fusions. Each araB-lacZ fusion contained two novel of DNA junctions. The MuR-lacZ junctions showed 'hot-spotting' according to established rules for Mu target selection. The araB-MuR and MuR-MuR junctions all involved exchanges at regions of short sequence homology. More extensive homology between MuR and araB sequences indicates potential STC isomerization a resolvable four-way structure analogous to a Holliday junction. These results highlight the molecular complexity of araB-lacZ fusion formation, which may be thought of as a multi-step cell biology process rather than a unitary biochemical reaction.

Genome organization, natural genetic engineering and adaptive mutation.

Bacterial evolution is considered in the light of molecular discoveries about genome organization, biochemical mechanisms of genetic change, and cellular control networks. Prokaryotic genetic determinants are organized as modular composites of coding sequences and protein-factor binding sites joined together during evolution. Studies of genetic change have revealed the existence of biochemical functions capable of restructuring the bacterial genome at various levels and joining together different sequence elements. These natural genetic engineering systems can be subject to regulation by signal transduction networks conveying information about the extracellular and intracellular environments. Mu-mediated araB-lacZ coding sequence fusions provide one example of adaptive mutation (increased formation of useful mutations under selection) and illustrate how physiological regulation can modulate the activity of a natural genetic engineering system under specific conditions.

Periodic phenomena in Proteus mirabilis swarm colony development.

Proteus mirabilis colonies exhibit striking geometric regularity. Basic microbiological methods and imaging techniques were used to measure periodic macroscopic events in swarm colony morphogenesis. We distinguished three initial phases (lag phase, first swarming phase, and first consolidation phase) followed by repeating cycles of subsequent swarming plus consolidation phases. Each Proteus swarm colony terrace corresponds to one swarming-plus-consolidation cycle. The duration of the lag phase was dependent upon inoculation density in a way that indicated the operation of both cooperative and inhibitory multicellular effects. On our standard medium, the second and subsequent swarm phases displayed structure in the form of internal waves visible with reflected and dark-field illumination. These internal waves resulted from organization of the migrating bacteria into successively thicker cohorts of swarmer cells. Bacterial growth and motility were independently modified by altering the composition of the growth medium. By varying the glucose concentration in the substrate, it was possible to alter biomass production without greatly affecting the kinetics of colony surface area expansion. By varying the agar concentration in the substrate, initial bacterial biomass production was unaffected but colony expansion dynamics were significantly altered. Higher agar concentrations led to slower, shorter swarm phases and longer consolidation phases. Thus, colony growth was restricted by higher agar concentrations but the overall timing of the swarming-plus-consolidation cycles remained constant. None of a variety of factors which had significant effects on colony expansion altered terracing frequencies at 32 degrees C, but the length of the swarming-plus-consolidation cycle was affected by temperature and medium enrichment. Some clinical isolates displayed significant differences in terracing frequencies at 32 degrees C. Our results defined a number of readily quantifiable parameters in swarm colony development. The data showed no connection between nutrient (glucose) depletion and the onset of different phases in swarm colony morphogenesis. Several observations point to the operation of density-dependent thresholds in controlling the transitions between distinct phases.

Efficacy of lamivudine in chronic hepatitis B patients with active viral replication and decompensated cirrhosis undergoing liver transplantation.

Liver transplantation for endstage hepatitis B virus (HBV) infection has been associated with survival inferior to that of liver transplantation in other chronic liver diseases due to HBV reinfection of the graft. Lamivudine is a new nucleoside analog with potent antiviral effects against hepatitis B. Our aim was to test its efficacy when used pre- and posttransplantation in HBV-DNA positive patients with endstage liver disease. Patients received oral lamivudine 100 mg daily both pretransplant and posttransplant. Viral serology, serum and tissue HBV-DNA and liver histology were assessed sequentially. Five consecutive patients with endstage hepatitis B were entered into the trial. Serum HBV-DNA was cleared pretransplant in all patients. Three of four transplanted patients cleared HBeAg and HBsAg postoperatively, whereas all four became negative for serum HBV-DNA (dot-blot and PCR). Liver biopsies were negative for HBV-DNA by PCR in 3 of 4 cases. Lymphocytes were negative for HBV-DNA by PCR in all cases. With follow-up of 3, 14, 16, and 26 months, two patients have normal liver enzymes and normal liver histology and two have developed recurrent hepatitis B. No significant side effects were seen. This pilot study shows that lamivudine can effectively inhibit hepatitis B virus in cirrhotic patients pretransplant and posttransplant. A lamivudine resistant mutant developed in two patients. Transplant recipients with actively replicating HBV related cirrhosis may achieve a good outcome after liver transplantation using lamivudine, but viral resistance is likely to be a significant problem.