Mutation and selection explain why many eukaryotic centromeric DNA sequences are often A + T rich.

We have used chromosome engineering to replace native centromeric DNA with different test sequences at native centromeres in two different strains of the fission yeast Schizosaccharomyces pombe and have discovered that A + T rich DNA, whether synthetic or of bacterial origin, will function as a centromere in this species. Using genome size as a surrogate for the inverse of effective population size (Ne) we also show that the relative A + T content of centromeric DNA scales with Ne across 43 animal, fungal and yeast (Opisthokonta) species. This suggests that in most of these species the A + T content of the centromeric DNA is determined by a balance between selection and mutation. Combining the experimental results and the evolutionary analyses allows us to conclude that A + T rich DNA of almost any sequence will function as a centromere in most Opisthokonta species. The fact that many G/C to A/T substitutions are unlikely to be selected against may contribute to the rapid evolution of centromeric DNA. We also show that a neo-centromere sequence is not simply a weak version of native centromeric DNA and suggest that neo-centromeres require factors either for their propagation or establishment in addition to those required by native centromeres.

Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae.

BACKGROUND: Phage-encoded serine integrases, such as ϕC31 integrase, are widely used for genome engineering but have not been optimized for use in Saccharomyces cerevisiae although this organism is a widely used organism in biotechnology. RESULTS: The activities of derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal were compared using a standardized recombinase mediated cassette exchange reaction. The relative activities of these integrases in S. cerevisiae and in mammalian cells suggested that the major determinant of the activity of an integrase is the enzyme itself and not the cell in which it is working. We used an inducible promoter to show that six integrases were toxic as judged by their effects upon the proliferative ability of transformed yeast. We show that in general the active phage-encoded serine integrases were an order of magnitude more efficient in promoting genome integration reactions than a simple homologous recombination. CONCLUSIONS: The results of our study allow us to identify the integrases of the phage ϕBT1, TP901 ~ nls, R4, Bxb1, MR11, A118, ϕK38, ϕC31 ~ nls, Wß and SPBC ~ nls as active in S. cerevisiae and indicate that vertebrate cells are more restricted than yeast in terms of which integrases are active.

Fecal microbiota transplantation in treating Clostridium difficile infection.

Clostridium difficile infection (CDI) is an increasingly common and severe international health problem. Customary treatment of this infection, usually with antibiotics, is often ineffective and its recurrence is common. In recent years the treatment of recurrent or refractory CDI by the transfer of stool from an uninfected person, so called fecal "microbiota transplantation" has become recognized as effective and generally safe. The effectiveness of this novel treatment is incompletely defined but is likely to be due to its correction of the intestinal dysbiosis that characterizes the disease. Practical methods for the administration of the transplantation have been described. This review summarizes the current reported experiences with fecal microbiota transplantation in the treatment for CDI.

Kinetochore assembly and heterochromatin formation occur autonomously in Schizosaccharomyces pombe.

Kinetochores in multicellular eukaryotes are usually associated with heterochromatin. Whether this heterochromatin simply promotes the cohesion necessary for accurate chromosome segregation at cell division or whether it also has a role in kinetochore assembly is unclear. Schizosaccharomyces pombe is an important experimental system for investigating centromere function, but all of the previous work with this species has exploited a single strain or its derivatives. The laboratory strain and most other S. pombe strains contain three chromosomes, but one recently discovered strain, CBS 2777, contains four. We show that the genome of CBS 2777 is related to that of the laboratory strain by a complex chromosome rearrangement. As a result, two of the kinetochores in CBS 2777 contain the central core sequences present in the laboratory strain centromeres, but lack adjacent heterochromatin. The closest block of heterochromatin to these rearranged kinetochores is ∼100 kb away at new telomeres. Despite lacking large amounts of adjacent heterochromatin, the rearranged kinetochores bind CENP-A(Cnp1) and CENP-C(Cnp3) in similar quantities and with similar specificities as those of the laboratory strain. The simplest interpretation of this result is that constitutive kinetochore assembly and heterochromatin formation occur autonomously.

Microscopic colitis. A review.

Microscopic colitis (MC) is characterized by a triad of watery diarrhea, usually normal colonoscopic findings and typical microscopic findings. Two distinct histological forms of MC have been defined: lymphocytic colitis and collagenous colitis, but overlapping features may be present. The incidence of MC appears to be rising and in some countries it may account for as many as 10-20% of patients with non-bloody watery diarrhea. The cause of MC remains unknown and is likely to be multifactorial. The pathogenesis is poorly defined, and numerous immunological abnormalities have been reported. MC is commonly associated with autoimmune diseases including celiac disease. Use of various medications, most notably non-steroidal anti-inflammatory agents and proton pump inhibitors, have been etiologically implicated but not firmly established as causative. In imperfect trials several agents have been reported to be effective in the treatment of MC; budesonide is the best studied and evidence supporting its effectiveness is the most persuasive. In cases of otherwise unexplained watery, non-bloody diarrhea, MC should be considered and colonic biopsied specimens should be taken of normal-appearing mucosa.

Serine recombinases as tools for genome engineering.

The serine recombinases differ mechanistically from the tyrosine recombinases and include proteins such as ϕC31 integrase which, unlike Cre and Flp, promote unidirectional reactions. The serine recombinase family is large and includes many other proteins besides ϕC31 integrase with the potential to be widely used in genome engineering. Here we review the details of the mechanism of the reactions promoted by the serine recombinases and discuss how these not only limit the utility of this class of recombinase but also creates opportunities for the engineering of new enzymes. We discuss the unanswered questions posed by genome engineering experiments in a variety of systems in which the serine recombinases have been used and finally describe more recently discovered serine recombinases that have the potential to be used in genome engineering.

A Geographically Diverse Collection of Schizosaccharomyces pombe Isolates Shows Limited Phenotypic Variation but Extensive Karyotypic Diversity.

The fission yeast Schizosaccharomyces pombe has been widely used to study eukaryotic cell biology, but almost all of this work has used derivatives of a single strain. We have studied 81 independent natural isolates and 3 designated laboratory strains of Schizosaccharomyces pombe. Schizosaccharomyces pombe varies significantly in size but shows only limited variation in proliferation in different environments compared with Saccharomyces cerevisiae. Nucleotide diversity, π, at a near neutral site, the central core of the centromere of chromosome II is approximately 0.7%. Approximately 20% of the isolates showed karyotypic rearrangements as detected by pulsed field gel electrophoresis and filter hybridization analysis. One translocation, found in 6 different isolates, including the type strain, has a geographically widespread distribution and a unique haplotype and may be a marker of an incipient speciation event. All of the other translocations are unique. Exploitation of this karyotypic diversity may cast new light on both the biology of telomeres and centromeres and on isolating mechanisms in single-celled eukaryotes.

ERCP with cholangiopancreatoscopy may be associated with higher rates of complications than ERCP alone: a single-center experience.

BACKGROUND: Comparative data regarding complications associated with ERCP alone or when performed with cholangiopancreatoscopy (CP) are lacking. OBJECTIVE: To determine whether ERCP complications are more frequent when concomitant CP is performed. DESIGN: A retrospective query of a prospectively maintained database of ERCP, CP, and complications. MAIN OUTCOME MEASUREMENTS: Consensus criteria complications and additional adverse events (AEs), including unplanned medical evaluation, cardiopulmonary/sedation events, and others. SETTING: Academic, tertiary referral center. RESULTS: A total of 4214 ERCPs were performed (337 sphincter of Oddi manometry cases excluded) during the study period, of which 3475 ERCPs and 402 ERCPs with CP were analyzed. There were 28 of 402 AEs (7.0%) in the ERCP with CP group and 101 of 3475 (2.9%) in the ERCP-only group (odds ratio [OR], 2.50; 95% CI, 1.56-3.89). Subgroup analysis revealed a significantly higher rate of cholangitis in the CP group versus ERCP group (1.0% vs 0.2%; OR, 4.98; 95% CI, 1.06-19.67) and similar rates of pancreatitis (2.2% vs 1.3%; OR, 1.75; 95% CI, 0.74-3.65) and perforation (1.0% vs 0.3%; OR, 3.16; 95% CI, 0.73-10.75). LIMITATIONS: Retrospective review of a complications database that relies on physician self-reporting. CONCLUSIONS: AEs from CP may be more than double those of ERCP alone (when sphincter of Oddi manometry cases are excluded). CP appears to be associated with a significantly higher rate of cholangitis, possibly because of intermittent intraductal irrigation required during the procedure.

A review of string vessels or collapsed, empty basement membrane tubes.

String vessels are thin connective tissue strands, remnants of capillaries, with no endothelial cells; they do not carry blood flow. They occur in numerous species, particularly in the central nervous system, but can occur in any tissue where capillaries have died. String vessels are often associated with pathologies such as Alzheimer's disease, ischemia, and irradiation, but are also found in normal human brains from preterm babies to the aged. They provide a record of the original blood vessel location, but gradually disappear after months or years. There have been numerous studies of string vessels (acellular capillaries) in the retina, because retinal vessels can be seen in great detail in whole mounts after trypsin digestion. Capillary regression occurs by apoptosis, synchronously along capillary segments, with macrophages engulfing apoptotic endothelial cells. Macrophages may cause the apoptosis, or the regression may be triggered by loss of the endothelial cell survival factor VEGF. VEGF expression is induced by hypoxia and promotes capillary growth. Cessation of blood flow eliminates the shear stress that helps maintain endothelial cell survival. Capillaries can re-grow by proliferation and migration of endothelial cells into empty basement membrane tubes, which provide a structural scaffold, replete with signaling molecules. This is a problem in tumor control, but useful for recovery from capillary loss. There is an age-related waning of VEGF expression in response to hypoxia. This causes an age-related decline in cerebral angiogenesis and results in neuronal loss. It may also contribute to the proposed age-related loss of brain reserve.

Site-specific recombination by phiC31 integrase and other large serine recombinases.

Most temperate phages encode an integrase for integration and excision of the prophage. Integrases belong either to the lambda Int family of tyrosine recombinases or to a subgroup of the serine recombinases, the large serine recombinases. Integration by purified serine integrases occurs efficiently in vitro in the presence of their cognate (~50 bp) phage and host attachment sites, attP and attB respectively. Serine integrases require an accessory protein, Xis, to promote excision, a reaction in which the products of the integration reaction, attL and attR, recombine to regenerate attP and attB. Unlike other directional recombinases, serine integrases are not controlled by proteins occupying accessory DNA-binding sites. Instead, it is thought that different integrase conformations, induced by binding to the DNA substrates, control protein-protein interactions, which in turn determine whether recombination proceeds. The present review brings together the evidence for this model derived from the studies on phiC31 integrase, Bxb1 integrase and other related proteins.

Reduced ratio of afferent to total vascular density in mesial temporal sclerosis.

Mesial temporal sclerosis (MTS) is the most common cause of drug-resistant temporal lobe epilepsy in adults. Despite nearly 2 centuries since the first reports of MTS, relatively little is known about its etiology and pathogenesis. Increasing attention has been directed toward the potential role of vascular abnormalities in MTS. We evaluated the hippocampal microvasculature in 9 MTS cases and 3 non-MTS controls using celloidin tissue sections and markers for total (collagen type IV) and afferent (enzymatic alkaline phosphatase) vessels. Tissue sections were assessed by light microscopy and quantified by threshold analysis of digital images and stereological analysis using the Space Balls probe. Although consistent alterations in the total microvascular density were not found, there was a significant reduction in the density of afferent vessels using both methodologies; these reductions were in areas CA2 and CA3 by image threshold analysis and in area CA3 using stereological measures of the ratio of afferent to total vessels. Increased numbers of string vessels (i.e. remnants of regressing vasculature) were also observed in Ammon's horn, suggesting vascular degeneration in the MTS hippocampus. These findings may help further our understanding of the pathophysiology of MTS.

Rectal perforation during colonoscopic retroflexion: a large, prospective experience in an academic center.

BACKGROUND: Retroflexion in the rectum is performed routinely during colonoscopy in an attempt to detect lesions that could be missed by forward viewing alone. Case reports of rectal perforation caused by this procedure have been published, but the frequency of this serious complication in large series is unknown. In addition, there are few reports regarding the management of colonoscopic rectal perforations. OBJECTIVE: To determine the colonic perforation rate of rectal retroflexion and the management of this complication at 3 affiliated academic hospitals from July 1, 2001, to June 30, 2008. DESIGN: Multicenter case series. SETTING: A university hospital, a Department of Veterans Affairs hospital, and an urban safety-net hospital. PATIENTS: A diverse population of patients who underwent colonoscopy for various indications. RESULTS: Four cases of rectal perforation occurred during rectal retroflexion in a total of 39,054 consecutive colonoscopies (0.10 per 1000). One of these complications occurred in a colonoscopy performed by a supervised trainee; the other 3 occurred during colonoscopy performed by experienced faculty endoscopists alone. Three of the 4 perforations were managed successfully without surgical intervention. LIMITATIONS: The complication rate relies on physician self-reporting. CONCLUSIONS: Rectal retroflexion during colonoscopy is associated with a low rate of perforation, yet this serious complication can occur even in the hands of experienced endoscopists. Most cases of rectal perforation during retroflexion can be managed nonoperatively.

Microvascular changes in the white mater in dementia.

Our studies of the brain microvascular system have focused on some aspects not commonly studied by other research groups because we use some techniques not often used by others. Our observations tend to add new details to the pathological picture rather than contradict the mainstream findings. We use large, thick celloidin sections which provide a three dimensional view of vascular networks, and alkaline phosphatase (AP) staining which allows one to differentiate between afferent and efferent vessels. We found millions of lipid microemboli in the brains of patients after cardiac surgery, and concluded that they caused vascular dementia in many patients. We previously proposed an animal model of vascular dementia using brain irradiation, which induces capillary loss. Lipid emboli might also be used to create an animal model of vascular dementia. The deep white matter is vulnerable to chronic hypoperfusion because the blood vessels supplying this region arise from the border-zone and have the longest course of all vessels penetrating the cerebrum. In cases with leukoaraiosis (LA), we found periventricular venous collagenosis (PVC), resulting in stenosis. Thirteen of 20 subjects older than 60 years had PVC, and 10 of 13 subjects with severe PVC had LA. Vascular stenosis might induce chronic ischemia and/or edema in the deep white matter, leading to LA. We suggest three mechanisms for a possible genetic predisposition to PVC: i) a predisposition to excessive venous collagenosis; ii) an indirect effect that causes chronic periventricular ischemia with a reactive over-production of collagen; and iii) mechanical damage to small vessels due to increased pulsatile motion. We found tortuous arterioles supplying the deep white matter beginning at about age 50. We also found a trend toward an increase in tortuosity in LA. If tortuosity is a factor in LA, it is probably significant in only a subset of cases. String vessels, remnants of capillaries, occur commonly in the brain, and are increased in ischemia, AD, and irradiation. Capillary injury or shutdown of blood flow can lead to capillary loss and string vessel formation. We found string vessels in brains from preterm babies to the very old. They seem to disappear after some months or years. We found an early loss of capillaries in LA, followed in a few years by the disappearance of string vessels. LA lesions do not progress to cortical cavitating lesions. Our findings raise three questions. 1. Why is the capillary loss arrested before infarction? 2. Why is there a floor below which the vascular density will not fall? 3. Why does the process which initiates string vessels shut down? We explain the vascular changes in LA as follows. LA induces apoptosis with loss of oligodendrocytes. Capillaries and neuropil are lost. Increased oxygen extraction from the blood in the deep white matter in LA implies that there are too many cells for the remaining capillaries. Thus, the capillaries appear to die first. But why do they stop dying? Perhaps a minimum number of capillaries are needed to transport the arterial blood to the venous system. Once the capillaries stop dying, no more string vessels are formed, and the string vessels gradually disappear.

Association of preterm birth with brain malformations.

This study investigates the rate of preterm birth in babies with congenital brain defects. Autopsy case reports of congenital brain anomalies were obtained from the literature. The control cases were from a large registry, a published report from the Metropolitan Atlanta Congenital Defects Program. From 428 publications, 1168 cases were abstracted that had autopsy studies of congenital brain defects and information on the gestational age (GA) at birth. The control data from Atlanta included 7738 infants with significant birth defects of any kind and 264,392 infants without birth defects. In the autopsy cases with brain defects, the mean GA was 36.6 wk, whereas the Atlanta data showed a mean GA of 39.3 wk for infants with no defects and a significantly shorter gestation of 38.1 wk (p < 0.0001) for infants with defects. In the Atlanta data, the rate of preterm birth was 9.3% for those with no defects compared with 21.5% (p < 0.0001) for those with defects. In the autopsy cases with brain defects, the rate of preterm birth was even greater (33.1%, p < 0.0001). In conclusion, these data show an association of brain defects with preterm births.

The 'kinetochore maintenance loop': the mark of regulation?

Kinetochores can form and be maintained on DNA sequences that are normally non-centromeric. The existence of these so-called neo-centromeres has posed the problem as to the nature of the epigenetic mechanisms that maintain the centromere. Here we highlight results that indicate that the amount of CENP-A at human centromeres is tightly regulated. It is also known that kinetochore assembly requires sister chromatid cohesion at mitosis. We therefore suggest that separation or stretching between the sister chromatids at metaphase reciprocally determines the amount of centromere assembly in the subsequent interphase. This reciprocal relationship forms the basis of a negative feedback loop that could precisely control the amount of CENP-A and faithfully maintain the presence of a kinetochore over many cell divisions. We describe how the feedback loop would work, propose how it could be tested experimentally and suggest possible components of its mechanism.

Modifiable risk factors for colorectal neoplasms and hyperplastic polyps.

PURPOSE: Obesity, smoking and alcohol are modifiable putative risk factors for colorectal neoplasms (CRN) and hyperplastic polyps (HP). The aim of this study was to evaluate the strength of association between these modifiable risk factors and colorectal polyps. METHODS: These risk factors were assessed by using a questionnaire completed by the patient prior to colonoscopy. Eight hundred-seventy consecutive patients satisfying inclusion criteria who had undergone a complete colonoscopy were divided into 4 groups: CRN (n=194), HP (n=132), CRN and HP (n=42) and control (neither CRN nor HP; n=586). Multiple logistic regression was performed. RESULTS: The ORs [95%CI] of both CRN and HP for incremental body mass index expressed in 2 categories (>or=22, >or=25) were 2.12 [1.00, 4.50] and 1.41 [0.53, 3.77], respectively. The ORs [95%CI] of CRN and HP for heavy smoking of over 20 pack-years were 1.66 [1.05, 2.64] and 1.67 [1.01, 2.77], respectively. The ORs of CRN and HP for habitual alcohol drinking (median ethanol intake 32 g/day and interquartile range 18-40 g/day) were 1.31 [0.86, 1.98] and 1.91 [1.06, 3.47], respectively. CRN and HP were correlated with each other (p=0.0043, chi-square test). Aging was a significant risk factor for all three groups of colorectal polyps. CONCLUSION: These findings are especially important since smoking and alcohol consumption are modifiable risk factors. Heavy smokers should be encouraged to quit to reduce their risk of CRN and HP. Habitual drinkers should be warned of the risk of HP. HP can be a marker of coincidence of CRN.