Fanconi anemia (FA) is a complex cancer susceptibility disorder associated with DNA repair defects and infertility, yet the precise function of the FA proteins in genome maintenance remains unclear. Here we report that C. elegans FANCD2 (fcd-2) is dispensable for normal meiotic recombination but is required in crossover defective mutants to prevent illegitimate repair of meiotic breaks by nonhomologous end joining (NHEJ). In mitotic cells, we show that DNA repair defects of C. elegans fcd-2 mutants and FA-deficient human cells are significantly suppressed by eliminating NHEJ. Moreover, NHEJ factors are inappropriately recruited to sites of replication stress in the absence of FANCD2. Our findings are consistent with the interpretation that FA results from the promiscuous action of NHEJ during DNA repair. We propose that a critical function of the FA pathway is to channel lesions into accurate, as opposed to error-prone, repair pathways.

Nuclear hormone receptors (NHRs) are proteins that regulate gene expression in response to developmental, environmental, and nutritional signals. The activity of some NHRs is selectively and reversibly modulated by small molecular weight compounds. However, for others - termed "orphan" receptors - no such ligands have (yet) been identified, and at least some NHRs may lack natural ligands. NHRs exhibit a stereotyped architecture, with conserved N-terminal DNA-binding domains (DBDs) and more variable C-terminal ligand-binding domains (LBDs). NHRs control the transcription of remarkably diverse and specific gene networks, apparently by integrating multiple regulatory inputs that interact with distinct receptor surfaces; these inputs include small molecule ligands, transcriptional coregulators, and response elements, the genomic sites to which the receptors bind. NHRs comprise an ancient superfamily found in all metazoans, and recent findings have revealed NHR-like regulatory factors in fungi. Here, we consider NHR function and evolution in nematodes, roundworms that inhabit terrestrial, marine, and freshwater habitats; we focus in particular on the well-established experimental organism Caenorhabditis elegans. Interestingly, the C. elegans genome encodes a massively expanded NHR family; we speculate that some of the multiple physiological activities governed by individual mammalian NHRs may be distributed among multiple members of the C. elegans family, potentially focusing and simplifying functional analyses. Accordingly, investigations of relevant NHR cofactors, ligands, and response elements might also prove to be simpler; moreover, the abbreviated intergenic regions of the C. elegans genome will facilitate the assignment of response elements to target genes. Finally, the growing interest in medically relevant nematodes is providing novel insights into the function and evolution of NHRs.

Meiotic crossovers (COs) are tightly regulated to ensure that COs on the same chromosome are distributed far apart (crossover interference, COI) and that at least one CO is formed per homolog pair (CO homeostasis). CO formation is controlled in part during meiotic double-strand break (DSB) creation in Caenorhabditis elegans, but a second level of control must also exist because meiotic DSBs outnumber COs. We show that the antirecombinase RTEL-1 is required to prevent excess meiotic COs, probably by promoting meiotic synthesis-dependent strand annealing. Two distinct classes of meiotic COs are increased in rtel-1 mutants, and COI and homeostasis are compromised. We propose that RTEL-1 implements the second level of CO control by promoting noncrossovers.

Homologous recombination (HR) is essential for repair of meiotic DNA double-strand breaks (DSBs). Although the mechanisms of RAD-51-DNA filament assembly and strand exchange are well characterized, the subsequent steps of HR are less well defined. Here, we describe a synthetic lethal interaction between the C. elegans helicase helq-1 and RAD-51 paralog rfs-1, which results in a block to meiotic DSB repair after strand invasion. Whereas RAD-51-ssDNA filaments assemble at meiotic DSBs with normal kinetics in helq-1, rfs-1 double mutants, persistence of RAD-51 foci and genetic interactions with rtel-1 suggest a failure to disassemble RAD-51 from strand invasion intermediates. Indeed, purified HELQ-1 and RFS-1 independently bind to and promote the disassembly of RAD-51 from double-stranded, but not single-stranded, DNA filaments via distinct mechanisms in vitro. These results indicate that two compensating activities are required to promote postsynaptic RAD-51 filament disassembly, which are collectively essential for completion of meiotic DSB repair.

The pediatric neurosurgical mission trips led by physicians at Virginia Commonwealth University (VCU) Health Systems began in 1996 with the formation of Medical Outreach to Children, founded by 1 of the authors (J.D.W.) after a visit to Guatemala. Since then, 19 surgical trips to 4 different countries in Central and South America have been coordinated from 1996 to 2008. This humanitarian work serves a number of purposes. First and foremost, it provides children with access to surgical care that they would otherwise not receive, thereby significantly improving their quality of life. Second, the visiting surgical team participates in the education of local physicians, parents, and caregivers to help improve the healthcare provided to the children. Last, the team works to promote sustainable global health solutions in the countries it travels to by generating a forum for clinical and public health research discourse. Thus far, a total of 414 children have undergone 463 operations, including 154 initial shunt surgeries, 110 myelomeningocele repairs, 39 lipoma resections, 33 tethered cord releases, 18 shunt revisions, 16 encephalocele repairs, 9 lipomyelomeningocele repairs, and 7 diastematomyelia repairs. The complication rate has been 5-8%, and the team has obtained reliable follow-up in approximately 77% of patients. A correlation was found between an increase in the number of trained neurosurgeons in the host countries and a decrease in the average age of patients treated by the visiting surgical team over time. It is also hypothesized that a decrease in the percentage of myelomeningocele repairs performed by the surgical team (as a fraction of total cases between 1996 and 2006) correlates to an increase in the number of local neurosurgeons able to treat common neural tube defects in patients of younger ages. Such analysis can be used by visiting surgical teams to assess the changing healthcare needs in a particular host country.

Homologous recombination (HR) is an important conserved process for DNA repair and ensures maintenance of genome integrity. Inappropriate HR causes gross chromosomal rearrangements and tumorigenesis in mammals. In yeast, the Srs2 helicase eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has been elusive. Here, we identify C. elegans RTEL-1 as a functional analog of Srs2 and describe its vertebrate counterpart, RTEL1, which is required for genome stability and tumor avoidance. We find that rtel-1 mutant worms and RTEL1-depleted human cells share characteristic phenotypes with yeast srs2 mutants: lethality upon deletion of the sgs1/BLM homolog, hyperrecombination, and DNA damage sensitivity. In vitro, purified human RTEL1 antagonizes HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control after deregulation of RTEL1 may be a critical event that drives genome instability and cancer.

BACKGROUND: Many microbes possess restriction-modification systems that protect them from parasitic DNA molecules. Unfortunately, the presence of a restriction-modification system in a given microbe also hampers genetic analysis. Although plasmids can be successfully conjugated into the enteropathogenic Escherichia coli strain E2348/69 and optimized protocols for competent cell preparation have been developed, we found that a large, low copy (approximately 15) bioluminescent reporter plasmid, pJW15, that we modified for use in EPEC, was exceedingly difficult to transform into E2348/69. We reasoned that a restriction-modification system could be responsible for the low transformation efficiency of E2348/69 and sought to identify and inactivate the responsible gene(s), with the goal of creating an easily transformable strain of EPEC that could complement existing protocols for genetic manipulation of this important pathogen. RESULTS: Using bioinformatics, we identified genes in the unfinished enteropathogenic Escherichia coli (EPEC) strain E2348/69 genome whose predicted products bear homology to the HsdM methyltransferases, HsdS specificity subunits, and HsdR restriction endonucleases of type I restriction-modification systems. We constructed a strain carrying a deletion of the conserved enzymatic domain of the EPEC HsdR homologue, NH4, and showed that its transformation efficiency was up to four orders of magnitude higher than that of the parent strain. Further, the modification capacity of NH4 remained intact, since plasmids that were normally recalcitrant to transformation into E2348/69 could be transformed upon passage through NH4. NH4 was unaffected in virulence factor production, since bundle forming pilus (BFP) subunits and type III secreted (T3S) proteins were present at equivalent levels to those seen in E2348/69. Further, NH4 was indistinguishable from E2348/69 in tissue culture infection model assays of localized adherence and T3S. CONCLUSION: We have shown that EPEC strain E2348/69 utilizes a type I restriction-modification system to limit entry of new DNA. This restriction-modification system does not appear to be involved in virulence determinant expression or infection phenotypes. The hsdR mutant strain should prove useful in genetic analysis of the important diarrheal pathogen EPEC.

Repair of the programmed meiotic double-strand breaks (DSBs) that initiate recombination must be coordinated with homolog pairing to generate crossovers capable of directing chromosome segregation. Chromosome pairing and synapsis proceed independently of recombination in worms and flies, suggesting a paradoxical lack of coregulation. Here, we find that the meiotic axis component HTP-3 links DSB formation with homolog pairing and synapsis. HTP-3 forms complexes with the DSB repair components MRE-11/RAD-50 and the meiosis-specific axis component HIM-3. Loss of htp-3 or mre-11 recapitulates meiotic phenotypes consistent with a failure to generate DSBs, suggesting that HTP-3 associates with MRE-11/RAD-50 in a complex required for meiotic DSB formation. Loss of HTP-3 eliminates HIM-3 localization to axes and HIM-3-dependent homolog alignment, synapsis, and crossing over. Our study reveals a mechanism for coupling meiotic DSB formation with homolog pairing through the essential participation of an axis component with complexes mediating both processes.

The Cpx two-component system regulates an extracytoplasmic stress response that functions to rid the envelope of misfolded and mislocalized proteins that may interfere with normal cellular processes. The Cpx pathway is also involved in pathogenesis. This study investigated the role of the Cpx response in enteropathogenic Escherichia coli (EPEC) type III secretion (T3S). It was determined that a functional Cpx pathway is not required for T3S but that pathway activation inhibits secretion by reducing the cellular pools of T3S substrates. The EPEC T3S system structural components, as well as a number of its substrates, are encoded on the locus of enterocyte effacement (LEE) pathogenicity island. Transcriptional fusions to the five major operons of the LEE were constructed and examined under Cpx pathway-activating conditions. Induction of the Cpx response caused a decrease in the transcription of several LEE operons, with the most pronounced effect on LEE4 and LEE5. Collectively, these two operons encode components of the T3S translocation apparatus, the bacterial adhesin intimin, and the translocated bacterial receptor Tir. These data show for the first time that activation of the Cpx envelope stress response in EPEC inhibits T3S of both translocators and effectors, likely through down regulation of LEE transcription. Coupled with recent findings, our results suggest that Cpx-mediated down regulation of virulence is a conserved theme in a number of bacterial pathogens.

The breast and ovarian cancer susceptibility protein BRCA1 is evolutionarily conserved and functions in DNA double-strand break (DSB) repair through homologous recombination, but its role in meiosis is poorly understood. By using genetic analysis, we investigated the role of the Caenorhabditis elegans BRCA1 orthologue (brc-1) during meiotic prophase. The null mutant in the brc-1 gene is viable, fertile and shows the wild-type complement of six bivalents in most diakinetic nuclei, which is indicative of successful crossover recombination. However, brc-1 mutants show an abnormal increase in apoptosis and RAD-51 foci at pachytene that are abolished by loss of spo-11 function, suggesting a defect in meiosis rather than during premeiotic DNA replication. In genetic backgrounds in which chiasma formation is abrogated, such as him-14/MSH4 and syp-2, loss of brc-1 leads to chromosome fragmentation suggesting that brc-1 is dispensable for crossing over but essential for DSB repair through inter-sister recombination.

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by defective DNA interstrand cross-link (ICL) repair. Here, we show that DOG-1 is the Caenorhabditis elegans homologue of FANCJ, a helicase mutated in FA-J patients. DOG-1 performs a conserved role in ICL repair, as dog-1 mutants are hypersensitive to ICL-inducing agents, but not to UVC irradiation or X rays. Genetic analysis indicated that dog-1 is epistatic with fcd-2 (C. elegans FANCD2) but is nonepistatic with brc-1 (C. elegans BRCA1), thus establishing the existence of two distinct pathways of ICL repair in worms. Furthermore, DOG-1 is dispensable for FCD-2 and RAD-51 focus formation, suggesting that DOG-1 operates downstream of FCD-2 and RAD-51 in ICL repair. DOG-1 was previously implicated in poly(G)/poly(C) (G/C) tract maintenance during DNA replication. G/C tracts remain stable in the absence of ATL-1, CLK-2 (FA pathway activators), FCD-2, BRC-2, and MLH-1 (associated FA components), implying that DOG-1 is the sole FA component required for G/C tract maintenance in a wild-type background. However, FCD-2 is required to promote deletion-free repair at G/C tracts in dog-1 mutants, consistent with a role for FA factors at the replication fork. The functional conservation between DOG-1 and FANCJ suggests a possible role for FANCJ in G/C tract maintenance in human cells.

Homologous recombination (HR) plays a critical role in the restart of blocked replication forks, but how this is achieved remains poorly understood. We show that mutants in the single Rad51 paralog in Caenorhabditis elegans, rfs-1, permit discrimination between HR substrates generated at DNA double-strand breaks (DSBs), or following replication fork collapse from HR substrates assembled at replication fork barriers (RFBs). Unexpectedly, RFS-1 is dispensable for RAD-51 recruitment to meiotic and ionizing radiation (IR)-induced DSBs and following replication fork collapse, yet, is essential for RAD-51 recruitment to RFBs formed by DNA crosslinking agents and other replication blocking lesions. Deletion of rfs-1 also suppresses the accumulation of toxic HR intermediates in him-6; top-3 mutants and accelerates deletion formation at presumed endogenous RFBs formed by poly G/C tracts in the absence of DOG-1. These data suggest that RFS-1 is not a general mediator of HR-dependent DSB repair, but acts specifically to promote HR at RFBs. HR substrates generated at conventional DSBs or following replication fork collapse are therefore intrinsically different from those produced during normal repair of blocked replication forks.

Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.

One of the least well understood DNA repair processes in cells is the repair of DNA interstrand cross-links (ICLs) which present a major obstacle to DNA replication and must be repaired or bypassed to allow fork progression. Fanconi anemia (FA) is an inherited genome instability syndrome characterized by hypersensitivity to ICL damage. Central to the FA repair pathway is FANCD2 that is mono-ubiquitylated in response to replication stress and ICL damage through the action of the FA core complex and its E3-ubiquitin ligase subunit, FANCL. In its mono-ubiquitylated form FANCD2 is recruited to repair foci where it is believed to somehow coordinate ICL repair and restart of impeded replication forks. However, the precise mechanism through which the FA pathway and mono-ubiquitylation of FANCD2 promotes ICL repair remains unclear. Here we report on a functional homologue of FANCD2 in C. elegans (FCD-2). Although fcd-2 mutants are homozygous viable, they are exquisitely sensitive to ICL-inducing agents, but insensitive to ionizing radiation (IR). fcd-2 is dispensable for meiotic recombination and activation of the S-phase checkpoint, indicating that ICL sensitivity is likely due to a repair rather than a signalling defect. Indeed, we show that FCD-2 is mono-ubiquitylated in response to ICL damage and is recruited to nuclear repair foci. Consistent with the sensitivity of fcd-2 mutants, FCD-2 focus formation is induced in response to ICL damage and replication stress, but not following IR, suggesting that FCD-2 responds to lesions that block DNA replication and not DNA double strand breaks per se. The realization that the FA pathway is conserved in a genetically tractable model system will permit the comprehensive analysis of the interplay between the FA, homologous recombination (HR), translesion synthesis (TLS) and nucleotide excision repair (NER) pathways, critical to the understanding of ICL repair.

Three experiments were conducted to investigate the feasibility of using crystalline methionine and lysine as protein supplements for lactating Holstein cows. In the first experiment, Met (dl-methionine) and Lys (l-lysine-HCl) were added to diets used in continuous culture bioreactors to estimate optimal concentrations for use in subsequent in vivo experiments. The second experiment measured ruminal fermentation and nutrient flow to the small intestine when Met and Lys were top-dressed on diets fed to nonlactating cows. The third experiment measured lactation performance when Met and Lys were added to diets fed to late-lactation cows. Providing 0.29 and 2.27% of dry matter as Met and Lys, respectively, provided the largest improvement in fermentation in vitro and these concentrations were used in subsequent experiments. When Met and Lys were top-dressed on diets fed to nonlactating cows, no changes in total tract N digestion were observed. No changes in microbial protein production or ruminal fermentation were observed. Adding Met and Lys did not change production or efficiency of production of milk or milk components by late lactation cows. These data indicate that providing supplemental Met and Lys during late lactation does not significantly improve the protein status of the cow and therefore may not improve milk production.

Results are reported from a large (n = 3,402) four-group randomized trial to increase fruit and vegetable consumption among callers to the National Cancer Institute's (NCI's) Cancer Information Service (CIS) using tailored print materials. Following a baseline telephone interview, which included a brief educational message (BEM), participants were assigned randomly within CIS offices to one of four groups: single untailored (SU) group-one untailored set of materials; single tailored (ST) group-one tailored booklet; multiple tailored (MT) group-four tailored materials; and multiple retailored (MRT) group-four tailored materials with retailoring based on new information obtained at 5 months follow-up. Follow-up telephone interviews were conducted at 5 (n = 2,233) and 12 months (n = 1,927) after baseline. The main outcome measure was self-reported fruit and vegetable consumption using a seven-item food frequency questionnaire. At 12 months follow-up, there was a significant linear trend across groups of 0.21 servings (p = 0.0002). Specific nested hypotheses then were tested and revealed significant mean serving differences between SU (5.07) vs. MT (5.64) (p = 0.002) and SU vs. MRT (5.71; p < 0.001). Although the mean for ST (5.40) was greater than that for SU (5.07), the difference was not statistically significant (p = 0.07), and no difference was found between MT vs. MRT (p = 0.69). A higher proportion of recipients of tailored materials reported reading all of the materials and believing that they were written especially for them. No differences by experimental condition were found for the perceived usefulness or motivational impact of the print materials. In this trial, MT print materials were more effective at increasing fruit and vegetable (FV) consumption than were SU materials. The intervention mechanisms responsible for this effect merit further research. Retailoring did not produce a significant difference when compared with longitudinal baseline tailoring.

The purpose of this study was to assess whether there is an improvement in motor function in persons with cerebral palsy (CP) who have had a reduction of muscle tone by continuous intrathecal baclofen infusion. This was a prospective, open label, non-blinded case series without a control group, conducted at multiple centres. There were 31 subjects, aged 4-29 years. All had a pre-treatment mean lower extremity Ashworth scores of >or= 3 and a significant reduction in tone after a bolus injection of intrathecal baclofen (ITB) and received an implanted pump for continuous delivery of ITB. Motor function was assessed by the Gross Motor Function Measure (GMFM) prior to and 1 year following pump implantation. Significant improvement (p < 0.05) in mean GMFM scores was seen in subjects < 8 years (mean change 4.1) and in those from 8-18 years (mean change 3.7) and in subjects with CP Classes 2 and 5 (mean changes 6.2 and 2.9). There was a statistically significant decrease (p < 0.05) in Ashworth scores in CP classes 2-5. Subjects or their caregivers that completed a survey about perceived changes stated that motor control, positioning and endurance improved.

AIMS/HYPOTHESIS: The early pathological features of human diabetic neuropathy are not clearly defined. Therefore we quantified nerve fibre and microvascular pathology in sural nerve biopsies from diabetic patients with minimal neuropathy. METHODS: Twelve diabetic patients underwent detailed assessment of neuropathy and fascicular sural nerve biopsy at baseline, with repeat assessment of neuropathy 8.7+/-0.6 years later. RESULTS: At baseline, neuropathic symptoms, neurological deficits, quantitative sensory testing, cardiac autonomic function and peripheral nerve electrophysiology showed minimal abnormality, which deteriorated at follow-up. Myelinated fibre density, fibre and axonal area, and g-ratio were normal but teased fibre studies showed paranodal abnormalities (p<0.001), segmental demyelination (p<0.01) and remyelination (p<0.01) without axonal degeneration. Unassociated Schwann cell profile density (p<0.04) and unmyelinated axon density (p<0.001) were increased and axon diameter was decreased (p<0.007). Endoneurial capillaries demonstrated basement membrane thickening (p<0.006), endothelial cell hyperplasia (p<0.004) and a reduction in luminal area (p<0.007). CONCLUSIONS/INTERPRETATION: The early pathological features of human diabetic neuropathy include an abnormality of the myelinated fibre Schwann cell and unmyelinated fibre degeneration with regeneration. These changes are accompanied by a significant endoneurial microangiopathy.

BACKGROUND: Other than glycemic control, there are no treatments for diabetic neuropathy. Thus, identifying potentially modifiable risk factors for neuropathy is crucial. We studied risk factors for the development of distal symmetric neuropathy in 1172 patients with type 1 diabetes mellitus from 31 centers participating in the European Diabetes (EURODIAB) Prospective Complications Study. METHODS: Neuropathy was assessed at baseline (1989 to 1991) and at follow-up (1997 to 1999), with a mean (+/-SD) follow-up of 7.3+/-0.6 years. A standardized protocol included clinical evaluation, quantitative sensory testing, and autonomic-function tests. Serum lipids and lipoproteins, glycosylated hemoglobin, and the urinary albumin excretion rate were measured in a central laboratory. RESULTS: At follow-up, neuropathy had developed in 276 of 1172 patients without neuropathy at baseline (23.5 percent). The cumulative incidence of neuropathy was related to the glycosylated hemoglobin value and the duration of diabetes. After adjustment for these factors, we found that higher levels of total and low-density lipoprotein cholesterol and triglycerides, a higher body-mass index, higher von Willebrand factor levels and urinary albumin excretion rate, hypertension, and smoking were all significantly associated with the cumulative incidence of neuropathy. After adjustment for other risk factors and diabetic complications, we found that duration of diabetes, current glycosylated hemoglobin value, change in glycosylated hemoglobin value during the follow-up period, body-mass index, and smoking remained independently associated with the incidence of neuropathy. Cardiovascular disease at baseline was associated with double the risk of neuropathy, independent of cardiovascular risk factors. CONCLUSIONS: This prospective study indicates that, apart from glycemic control, the incidence of neuropathy is associated with potentially modifiable cardiovascular risk factors, including a raised triglyceride level, body-mass index, smoking, and hypertension.