Evolutionary dynamics of autopolyploids in natural populations: the case of Turnera sidoides complex / Dinámica evolutiva de autopoliploides en poblaciones naturales: el caso del complejo Turnera sidoides

ABSTRACT Turnera sidoides (x=7) is one of the few well-studied South American autopolyploid complexes. Since polyploidy has played a prominent role within this complex, ongoing studies in T. sidoides focus on understanding the mechanisms involved in the origin and the establishment of polyploids using integrative approaches. This paper synthesises the results of more than 20 years of research on this topic. Cytogenetics analysis provided evidences for the production of unreduced male and female gametes, supporting the hypothesis of bilateral sexual polyploidization as the mechanism of origin of polyploids in T. sidoides. The finding of viable triploids suggested that unilateral sexual polyploidization could also be an important mechanism for the origin of tetraploids in T. sidoides. The occurrence of plants continuously forming many unreduced gametes would play a key role in the establishment of neopolyploids in natural populations. Also, the higher number of propagules that tetraploids contribute to subsequent generations, the ability to multiply asexually by rhizomes, and the occurrence of occasional cases of self-compatibility and successful illegitimate crosses in polyploids increase the likelihood that a low frequency of neopolyploids can be maintained in natural populations of T. sidoides. In addition, integration of cytogeographic and genetic divergence data together with past niche modelling provided further insights supporting the hypothesis that historical climatic and geomorphological events provided favourable conditions for the establishment of autopolyploids, with the wider distribution of tetraploids of T. sidoides being the result of their range expansion.

RESUMEN Turnera sidoides (x=7) es uno de los pocos complejos autopoliploides sudamericanos bien estudiados. Como la poliploidía ha tenido un papel destacado en el complejo, los estudios en curso en T. sidoides se centraron en la comprensión de los mecanismos implicados en el origen y el establecimiento de los poliploides mediante diferentes enfoques. En este trabajo se sintetizan los resultados de más de 20 años de investigación sobre este tema. El análisis citogenético proporcionó evidencias de la producción de gametos masculinos y femeninos no reducidos, sustentando la hipótesis de la poliploidización sexual bilateral como mecanismo de origen de los poliploides en T. sidoides. Sin embargo, el hallazgo de triploides fértiles sugirió que la poliploidización sexual unilateral también sería un mecanismo importante de origen de tetraploides en T. sidoides. La ocurrencia de plantas que forman continuamente gametos no reducidos desempeñaría un papel clave en el establecimiento de neopoliploides. Además, el mayor número de propágulos que los tetraploides aportan a las siguientes generaciones, la capacidad de multiplicación asexual por rizomas y los casos ocasionales de autocompatibilidad y cruzamientos ilegítimos exitosos aumentarían la probabilidad de que se mantenga una baja frecuencia de neopoliploides en las poblaciones naturales de T. sidoides. Asimismo, la integración de datos citogeográficos y de divergencia genética junto con el modelado de nicho en el pasado aportó información que sustenta la hipótesis de que los eventos climáticos y geomorfológicos históricos proporcionaron las condiciones favorables para el establecimiento y expansión de los tetraploides de T. sidoides.

Evaluation of the Induction of Cell-Mediated Immunity Against Candida albicans in a Model of Cutaneous Infection in Newborn 0-Day-Old Mice.

Candida albicans is a commensal fungus of the skin and mucous membranes in humans, but it is also responsible for mucocutaneous and systemic infections in immunocompromised patients like low birth weight neonates and premature newborns. The epicutaneous application of C. albicans is widely used to study the immune response against this pathogen in adult mice models. However, the immune response of newborns against infections caused by the genus Candida is poorly understood. In order to mimic premature human infection, we developed a model of C. albicans epicutaneous infection in newborn mice. We found that yeasts were able to colonize while the pseudohyphae invaded the epidermis. Recruitment of polymorphonuclear and mononuclear cells at the infection zone was observed. Fungal invasion, fungal burden and cellular infiltration displayed a time- and dose-dependent response. Interestingly, newborn mice were able to control C. albicans primary infection. Finally, we showed that the epicutaneous infection of C. albicans in newborn mice at birth results in the induction of cell-mediated immunity as evinced by delayed-type hypersensitivity assays.

Stromal PTEN inhibits the expansion of mammary epithelial stem cells through Jagged-1.

This corrects the article DOI: 10.1038/onc.2016.383.

Oncologic doses of zoledronic acid induce site specific suppression of bone modelling in rice rats.

OBJECTIVES: To examine the effect of zoledronic acid (ZOL) on cortical bone modelling and healing of extraction sockets in the jaw bones of a rodent model. We hypothesized ZOL suppresses both the bone formation in the modelling mode in the jaw bones and alters the extraction site healing. MATERIAL & METHODS: Rice rats were administered saline solution and two dose regimens of ZOL: 0.1 mg/kg, twice a week, for 4 weeks (n=17, saline=8 & ZOL=9) and a higher dose of 0.4 mg/kg, weekly, for 9 weeks (n=30, saline=15 & ZOL=15). Two pairs of fluorochrome bone labels were administered. Extraction of maxillary teeth was performed in maxilla. Mineral apposition rate, mineralizing surface and bone formation rate (BFR) were quantified on periodontal (PDL), alveolar and basal bone surfaces, and in the trabecular bone of proximal tibia. Bone volume (BV) was evaluated at extraction sockets. Multivariate Gaussian models were used to account for repeated measurements, and analyzes were conducted in SAS V9.3. RESULTS: ZOL suppressed bone modelling (BFR/BS) at the PDL surfaces in the mandible (P<.05), but its effect was not significant at the periosteal surfaces of both jaws. BV for the healing sockets of ZOL treated animals was not significantly different (P=.07) compared to the saline group. ZOL suppressive effect was higher in the tibia compared to the jaws. CONCLUSION: ZOL severely suppresses coupled remodelling in the tibia, and the suppression of bone formation in the modelling mode in the jaws demonstrates the site specific effects of ZOL in rice rats.

Stromal PTEN inhibits the expansion of mammary epithelial stem cells through Jagged-1.

Fibroblasts within the mammary tumor microenvironment are active participants in carcinogenesis mediating both tumor initiation and progression. Our group has previously demonstrated that genetic loss of phosphatase and tensin homolog (PTEN) in mammary fibroblasts induces an oncogenic secretome that remodels the extracellular milieu accelerating ErbB2-driven mammary tumor progression. While these prior studies highlighted a tumor suppressive role for stromal PTEN, how the adjacent normal epithelium transforms in response to PTEN loss was not previously addressed. To identify these early events, we have evaluated both phenotypic and genetic changes within the pre-neoplastic mammary epithelium of mice with and without stromal PTEN expression. We report that fibroblast-specific PTEN deletion greatly restricts mammary ductal elongation and induces aberrant alveolar side-branching. These mice concomitantly exhibit an expansion of the mammary epithelial stem cell (MaSC) enriched basal/myoepithelial population and an increase in in vitro stem cell activity. Further analysis revealed that NOTCH signaling, specifically through NOTCH3, is diminished in these cells. Mechanistically, JAGGED-1, a transmembrane ligand for the NOTCH receptor, is downregulated in the PTEN-null fibroblasts leading to a loss in the paracrine activation of NOTCH signaling from the surrounding stroma. Reintroduction of JAGGED-1 expression within the PTEN-null fibroblasts was sufficient to abrogate the observed increase in colony forming activity implying a direct role for stromal JAGGED-1 in regulation of MaSC properties. Importantly, breast cancer patients whose tumors express both low stromal JAG1 and low stromal PTEN exhibit a shorter time to recurrence than those whose tumors express low levels of either alone suggesting similar stromal signaling in advanced disease. Combined, these results unveil a novel stromal PTEN-to-JAGGED-1 axis in maintaining the MaSC niche, and subsequently inhibiting breast cancer initiation and disease progression.

E2f3 in tumor macrophages promotes lung metastasis.

The Rb-E2F axis is an important pathway involved in cell-cycle control that is deregulated in a number of cancers. E2f transcription factors have distinct roles in the control of cell proliferation, cell survival and differentiation in a variety of tissues. We have previously shown that E2fs are important downstream targets of a CSF-1 signaling cascade involved in myeloid development. In cancer, tumor-associated macrophages (TAMs) are recruited to the tumor stroma in response to cytokines secreted by tumor cells, and are believed to facilitate tumor cell invasion and metastasis. Using the MMTV-Polyoma Middle T antigen (PyMT) mouse model of human ductal carcinoma, we show that the specific ablation of E2f3 in TAMs, but not in tumor epithelial cells, attenuates lung metastasis without affecting primary tumor growth. Histological analysis and gene expression profiling suggest that E2f3 does not impact the proliferation or survival of TAMs, but rather controls a novel gene expression signature associated with cytoskeleton rearrangements, cell migration and adhesion. This E2f3 TAM gene expression signature was sufficient to predict cancer recurrence and overall survival of estrogen receptor (ER)-positive breast cancer patients. Interestingly, we find that E2f3b but not E2f3a levels are elevated in TAMs from PyMT mammary glands relative to controls, suggesting a differential role for these isoforms in metastasis. In summary, these findings identify E2f3 as a key transcription factor in TAMs, which influences the tumor microenvironment and tumor cell metastasis.

CSF1-ETS2-induced microRNA in myeloid cells promote metastatic tumor growth.

Metastasis of solid tumors is associated with poor prognosis and bleak survival rates. Tumor-infiltrating myeloid cells (TIMs) are known to promote metastasis, but the mechanisms underlying their collaboration with tumor cells remain unknown. Here, we report an oncogenic role for microRNA (miR) in driving M2 reprogramming in TIMs, characterized by the acquisition of pro-tumor and pro-angiogenic properties. The expression of miR-21, miR-29a, miR-142-3p and miR-223 increased in myeloid cells during tumor progression in mouse models of breast cancer and melanoma metastasis. Further, we show that these miRs are regulated by the CSF1-ETS2 pathway in macrophages. A loss-of-function approach utilizing selective depletion of the miR-processing enzyme Dicer in mature myeloid cells blocks angiogenesis and metastatic tumor growth. Ectopic expression of miR-21 and miR-29a promotes angiogenesis and tumor cell proliferation through the downregulation of anti-angiogenic genes such as Col4a2, Spry1 and Timp3, whereas knockdown of the miRs impedes these processes. miR-21 and miR-29a are expressed in Csf1r+ myeloid cells associated with human metastatic breast cancer, and levels of these miRs in CD115+ non-classical monocytes correlates with metastatic tumor burden in patients. Taken together, our results suggest that miR-21 and miR-29a are essential for the pro-tumor functions of myeloid cells and the CSF1-ETS2 pathway upstream of the miRs serves as an attractive therapeutic target for the inhibition of M2 remodeling of macrophages during malignancy. In addition, miR-21 and miR-29a in circulating myeloid cells may potentially serve as biomarkers to measure therapeutic efficacy of targeted therapies for CSF1 signaling.

Selective roles of E2Fs for ErbB2- and Myc-mediated mammary tumorigenesis.

Previous studies have demonstrated that cyclin D1, an upstream regulator of the Rb/E2F pathway, is an essential component of the ErbB2/Ras (but not the Wnt/Myc) oncogenic pathway in the mammary epithelium. However, the role of specific E2fs for ErbB2/Ras-mediated mammary tumorigenesis remains unknown. Here, we show that in the majority of mouse and human primary mammary carcinomas with ErbB2/HER2 overexpression, E2f3a is up-regulated, raising the possibility that E2F3a is a critical effector of the ErbB2 oncogenic signaling pathway in the mammary gland. We examined the consequence of ablating individual E2fs in mice on ErbB2-triggered mammary tumorigenesis in comparison to a comparable Myc-driven mammary tumor model. We found that loss of E2f1 or E2f3 led to a significant delay in tumor onset in both oncogenic models, whereas loss of E2f2 accelerated mammary tumorigenesis driven by Myc-overexpression. Furthermore, southern blot analysis of final tumors derived from conditionally deleted E2f3(-/loxP) mammary glands revealed that there is a selection against E2f3(-/-) cells from developing mammary carcinomas, and that such selection pressure is higher in the presence of ErbB2 activation than in the presence of Myc activation. Taken together, our data suggest oncogenic activities of E2F1 and E2F3 in ErbB2- or Myc-triggered mammary tumorigenesis, and a tumor suppressor role of E2F2 in Myc-mediated mammary tumorigenesis.

Comparative effectiveness of antinociceptive gene therapies in animal models of diabetic neuropathic pain.

Peripheral neuropathic pain is one of the most common and debilitating complications of diabetes. Several genes have been shown to be effective in reducing neuropathic pain in animal models of diabetes after transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)1-based vectors, yet there has never been a comparative analysis of their efficacy. We compared four different HSV1-based vectors engineered to produce one of two opioid receptor agonists (enkephalin or endomorphin), or one of two isoforms of glutamic acid decarboxylase (GAD65 or GAD67), alone and in combination, in the streptozotocin-induced diabetic rat and mouse models. Our results indicate that a single subcutaneous hindpaw inoculation of vectors expressing GAD65 or GAD67 reduced diabetes-induced mechanical allodynia to a degree that was greater than daily injections of gabapentin in rats. Diabetic mice that developed thermal hyperalgesia also responded to GAD65 or endomorphin gene delivery. The results suggest that either GAD65 or GAD67 vectors are the most effective in the treatment of diabetic pain. The vector combinations, GAD67+endomorphin, GAD67+enkephalin or endomorphin+enkephalin also produced a significant antinociceptive effect but the combination did not appear to be superior to single gene treatment. These findings provide further justification for the clinical development of antinociceptive gene therapies for the treatment of diabetic peripheral neuropathies.

Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320.

PTEN (Phosphatase and tensin homolog deleted on chromosome 10) expression in stromal fibroblasts suppresses epithelial mammary tumours, but the underlying molecular mechanisms remain unknown. Using proteomic and expression profiling, we show that Pten loss from mammary stromal fibroblasts activates an oncogenic secretome that orchestrates the transcriptional reprogramming of other cell types in the microenvironment. Downregulation of miR-320 and upregulation of one of its direct targets, ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2) are critical events in Pten-deleted stromal fibroblasts responsible for inducing this oncogenic secretome, which in turn promotes tumour angiogenesis and tumour-cell invasion. Expression of the Pten-miR-320-Ets2-regulated secretome distinguished human normal breast stroma from tumour stroma and robustly correlated with recurrence in breast cancer patients. This work reveals miR-320 as a critical component of the Pten tumour-suppressor axis that acts in stromal fibroblasts to reprogramme the tumour microenvironment and curtail tumour progression.

Zoledronic acid decreases bone formation without causing osteocyte death in mice.

Bisphosphonates have been associated with osteonecrosis of the jaw. The purpose of this study was to examine the effect of a potent bisphosphonate, zoledronic acid (ZA) on osteocyte viability and bone formation. Ten experimental C57BL/6 mice were administered ZA (0.1 mg/kg-i.p.) weekly for 9 weeks while four control mice did not receive the drug. A pair of calcein (30 mg/kg) labels was administered 10 and 3 days prior to sacrifice of the 34-week-old mice. Fresh mandibular and femoral sections were obtained to evaluate osteocyte viability using a lactate dehydrogenase (LDH) assay. In addition, sections from the femur, mandible and maxilla were prepared for standard histomorphometry. The operator was blinded for data collection to eliminate bias. Data on necrotic area/total bone area from the LDH sections were collected. In addition, standard histomorphometric variables including bone formation rate were calculated. Mixed models were used to analyse data. The osteocytes were overwhelmingly viable and no necrotic areas were detected in the mandible and femur of both groups. ZA was not directly cytotoxic to the mouse osteocytes. There was suppression in indices of bone formation at all skeletal sites of the ZA group compared to the control group. While ZA administration in mice does not produce necrotic osteocytes, it severely suppresses bone formation. Such reductions can have a profound effect on bone healing.

Tissue level mechanical properties of cortical bone in skeletally immature and mature dogs.

OBJECTIVES: The purpose of this study was to quantify the tissue level mechanical properties of cortical bone of skeletally immature (~five-month-old) Beagle dogs and compare them to data from mature dogs measured in a previous study. METHODS: Eight femoral cross sectional specimens (two bone sections / dog) were obtained from four skeletally immature dogs. A pair of calcein bone labels were administered intravenously to the dogs to mark sites of active mineralization prior to euthanasia. Prepared bone specimens were placed in a nano- indenter specimen holder and the previously identified calcein labelled osteons were located. Labelled (n = 128) and neighbouring unlabelled (n = 127) osteons in skeletally immature femurs were examined by instrumented indentation testing. Indents were made to a depth of 500 nm at a loading rate of 10 nm/s. Indentation modulus (IM) and hardness (H) were obtained. RESULTS: The overall IM of the cortical bone in the skeletally mature groups was significantly greater than in the immature group (p = 0.0011), however overall H was not significantly different. The differences between the groups in IM were significant for the unlabelled osteons (p = 0.001), but not for the labelled osteons (p = 0.56). CONCLUSION: There are differences in the IM of unlabelled osteons in skeletally immature and mature groups of Beagle dogs. In contrast to whole bone mechanical tests, where there are obvious differences between growing and mature bones, there are only small differences in the micro-mechanical properties.

Comparative study of topical anaesthesia with lidocaine 2% vs levobupivacaine 0.75% in cataract surgery.

BACKGROUND: This study compared the efficacy of topical anaesthesia with levobupivacaine 0.75% vs lidocaine 2% during cataract surgery by phacoemulsification. METHODS: A prospective, randomized, double-blind study comparing two agents for topical anaesthesia in cataract surgery. Two hundred and forty-six consecutive patients undergoing corneal phacoemulsification were enrolled into two groups to receive either topical levobupivacaine 0.75% (n=126) or lidocaine 2% (n=120). The main outcome variables of the study were intraoperative and postoperative pain, requirement for additional anaesthesia, patient comfort and cooperation, surgeon satisfaction, and corneal epithelial toxicity induced by topical drugs. RESULTS: evobupivacaine 0.75% provided significantly better analgesia than lidocaine 2% during cataract surgery (P<0.001) at the end of surgery (P<0.002), and up to 30 min after surgery (P<0.001). There were no statistically significant differences between the two groups 5 h after surgery. Epithelial toxicity was similar in both groups, and patient comfort and surgeon assessment of patient cooperation were better in the levobupivacaine group. CONCLUSIONS: Topical anaesthesia with levobupivacaine 0.75% was more effective than lidocaine 2% in preventing pain and improving patient and surgeon comfort during cataract surgery, with similar toxicity.

Comparison of gait performance on different environmental settings for patients with chronic spinal cord injury.

STUDY DESIGN: Observational cross-section study. OBJECTIVES: The objective of our study was to determine if the influence of a community environment would impact on ASIA D spinal cord injured (SCI) gait performance patients. Our main hypothesis is that an outdoor community environment may influence gait speed and endurance on community ambulating patients. METHODS: Ten-Meter Walking (10MWT) and Six-Minute Walking (6MWT) tests were performed on community ambulating SCI research participants (n=18) in two different environmental conditions: (1) Experimental (indoors Gymnasium) and (2) Natural (community setting). Average gait speed and endurance values were obtained for the two different conditions and analyzed for statistical significance on the nonparametric two-tailed Wilcoxon signed rank test. RESULTS: While no difference was observed on the 10MWT we found an improvement on gait performance on the 6 MWT on a community setting. CONCLUSIONS: Our study showed mixed results on environmental influence on gait speed and endurance on ASIA D patient population. While there is no difference on the 10 MWT, there is an improvement on gait performance on the communitary 6MWT.

Transcriptional regulation of parathyroid hormone-related protein promoter P2 by NF-kappaB in adult T-cell leukemia/lymphoma.

Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of patients with adult T-cell leukemia/lymphoma (ATLL) due to human T-cell lymphotropic virus type-1 (HTLV-1) infection. We previously showed that ATLL cells constitutively express high levels of PTHrP via activation of promoters P2 and P3, resulting in HHM. In this study, we characterized a nuclear factor-kappaB (NF-kappaB) binding site in the P2 promoter of human PTHrP. Using electrophoretic mobility shift assays, we detected a specific complex in Tax-expressing human T cells composed of p50/c-Rel, and two distinct complexes in ATLL cells consisting of p50/p50 homodimers and a second unidentified protein(s). Chromatin immunoprecipitation assays confirmed in vivo binding of p50 and c-Rel on the PTHrP P2 promoter. Using transient co-transfection with NF-kappaB expression plasmids and PTHrP P2 luciferase reporter-plasmid, we showed that NF-kappaB p50/p50 alone and p50/c-Rel or p50/Bcl-3 cooperatively upregulated the PTHrP P2 promoter. Furthermore, inhibition of NF-kappaB activity by Bay 11-7082 reduced PTHrP P2 promoter-initiated transcripts in HTLV-1-infected T cells. In summary, the data demonstrated that transcriptional regulation of PTHrP in ATLL cells can be controlled by NF-kappaB activation and also suggest a Tax-independent mechanism of activation of PTHrP in ATLL.

Indentation modulus of the alveolar process in dogs.

One mechanism of bone adaptation is alteration in tissue level material properties. We hypothesized that alteration in the indentation modulus of the alveolar process is an adaptive response to the localized mechanical environment. Forty-eight specimens representing anterior and posterior regions of the maxilla and mandible were obtained from 6 mature male beagle dogs. The indentation properties of the alveolar bone proper and more distant osteonal cortical bone were estimated. The bone types were further divided into 3 regions (coronal, middle, and apical), with 27 indents being made in each region of tooth-supporting bone. There was a significant difference (p < 0.001) in the indentation moduli of the jaws (maxilla/mandible), location (anterior/posterior), and bone type (alveolar bone proper vs. cortical bone). However, statistical interactions exist which preclude the simple interpretation of results. The distribution of relative stiffness provides a better understanding of bone adaptations in the alveolar process.

Technical note: determining peeling order using sparse matrix algorithms.

To study the effect of individual genes by segregation or linkage analyses, the likelihood of the model needs to be evaluated. The likelihood can be computed efficiently using the Elston-Stewart algorithm. This algorithm involves summing over the unobserved genotypes in the pedigree, which is called peeling. An important aspect of this algorithm is to determine the order of peeling to maximize efficiency. This paper shows how determining peeling order is related to a problem in solving systems of symmetric sparse linear equations. It also shows how algorithms developed to efficiently solve those systems, can be used to determine the optimal order of peeling in the Elston-Stewart algorithm.

Sampling genotypes in large pedigrees with loops.

Markov chain Monte Carlo (MCMC) methods have been proposed to overcome computational problems in linkage and segregation analyses. This approach involves sampling genotypes at the marker and trait loci. Scalar-Gibbs is easy to implement, and it is widely used in genetics. However, the Markov chain that corresponds to scalar-Gibbs may not be irreducible when the marker locus has more than two alleles, and even when the chain is irreducible, mixing has been observed to be slow. These problems do not arise if the genotypes are sampled jointly from the entire pedigree. This paper proposes a method to jointly sample genotypes. The method combines the Elston-Stewart algorithm and iterative peeling, and is called the ESIP sampler. For a hypothetical pedigree, genotype probabilities are estimated from samples obtained using ESIP and also scalar-Gibbs. Approximate probabilities were also obtained by iterative peeling. Comparisons of these with exact genotypic probabilities obtained by the Elston-Stewart algorithm showed that ESIP and iterative peeling yielded genotypic probabilities that were very close to the exact values. Nevertheless, estimated probabilities from scalar-Gibbs with a chain of length 235 000, including a burn-in of 200 000 steps, were less accurate than probabilities estimated using ESIP with a chain of length 10 000, with a burn-in of 5 000 steps. The effective chain size (ECS) was estimated from the last 25 000 elements of the chain of length 125 000. For one of the ESIP samplers, the ECS ranged from 21 579 to 22 741, while for the scalar-Gibbs sampler, the ECS ranged from 64 to 671. Genotype probabilities were also estimated for a large real pedigree consisting of 3 223 individuals. For this pedigree, it is not feasible to obtain exact genotype probabilities by the Elston-Stewart algorithm. ESIP and iterative peeling yielded very similar results. However, results from scalar-Gibbs were less accurate.

Transfer of the tibialis anterior for calcaneus deformity in myelodysplasia.

We evaluated the results of transfer of the tibialis anterior in the management of calcaneus deformity in young patients who had myelodysplasia; fifteen patients (twenty-two feet) were operated on between 1978 and 1985. The neural deficit was at the fourth and fifth lumbar levels. The average age at the time of the operation was seven years and two months (range, two to nineteen years). The average age at the latest follow-up was thirteen years (range, five to twenty-four years). The average duration of follow-up was five years and ten months (range, two to eleven years). Seventeen feet (twelve patients) had a good result (no ulceration of the heel or osteomyelitis and correction of the calcaneus deformity), and five feet (three patients) had a poor result (persistent ulceration, signs of osteomyelitis, recurrent or persistent calcaneus deformity, or the need for additional operative intervention). Children who were less than five years old had a better outcome, as determined by the Fisher exact test (p less than 0.5).

Neural tube defects: a study in Puerto Rico.

Recent literature reports an apparent decline in the incidence of neural tube defects throughout the world. A revision of stillbirth certificates and surgical reports of closure procedures for open neural tube defects was done in order to establish the incidence and its trend during a nine year period in Puerto Rico. The current prevalence of the syndrome was estimated using the death certificates in addition to the fore-mentioned surgical reports. Our results indicate that Puerto Rico carries probably the highest incidence of the US territories and that the trend is not declining one.