Does human endometrial LGR5 gene expression suggest the existence of another hormonally regulated epithelial stem cell niche?

STUDY QUESTION: Is human endometrial leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) gene expression limited to the postulated epithelial stem cell niche, stratum basalis glands, and is it hormonally regulated? SUMMARY ANSWER: LGR5 expressing cells are not limited to the postulated stem cell niche but LGR5 expression is hormonally regulated. WHAT IS KNOWN ALREADY: The human endometrium is a highly regenerative tissue; however, endometrial epithelial stem cell markers are yet to be confirmed. LGR5 is a marker of stem cells in various epithelia. STUDY DESIGN, SIZE, DURATION: The study was conducted at a University Research Institute. Endometrial samples from 50 healthy women undergoing benign gynaecological surgery with no endometrial pathology at the Liverpool Women's hospital were included and analysed in the following six sub-categories; proliferative, secretory phases of menstrual cycle, postmenopausal, those using oral and local progestagens and samples for in vitro explant culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we used the gold standard method, in situ hybridisation (ISH) along with qPCR and a systems biology approach to study the location of LGR5 gene expression in full thickness human endometrium and Fallopian tubes. The progesterone regulation of endometrial LGR5 was examined in vivo and in short-term cultured endometrial tissue explants in vitro. LGR5 expression was correlated with epithelial proliferation (Ki67), and expression of previously reported epithelia progenitor markers (SOX9 and SSEA-1) immunohistochemistry (IHC). MAIN RESULTS AND THE ROLE OF CHANCE: LGR5 gene expression was significantly higher in the endometrial luminal epithelium than in all other epithelial compartments in the healthy human endometrium, including the endometrial stratum basalis (P < 0.05). The strongest SSEA-1 and SOX9 staining was observed in the stratum basalis glands, but the general trend of SOX9 and SSEA-1 expression followed the same cyclical pattern of expression as LGR5. Stratum functionalis epithelial Ki67-LI and LGR5 expression levels correlated significantly (r = 0.74, P = 0.01), however, they did not correlate in luminal and stratum basalis epithelium (r = 0.5 and 0.13, respectively). Endometrial LGR5 demonstrates a dynamic spatiotemporal expression pattern, suggesting hormonal regulation. Oral and local progestogens significantly reduced endometrial LGR5 mRNA levels compared with women not on hormonal treatment (P < 0.01). Our data were in agreement with in silico analysis of published endometrial microarrays. LARGE SCALE DATA: We did not generate our own large scale data but interrogated publically available large scale data sets. LIMITATIONS, REASONS FOR CAUTION: In the absence of reliable antibodies for human LGR5 protein and validated lineage markers for the various epithelial populations that potentially exist within the endometrium, our study does not formally characterise or examine the functional ability of the resident LGR5+ cells as multipotent. WIDER IMPLICATIONS OF THE FINDINGS: These data will facilitate future lineage tracing studies in the human endometrial epithelium; to identify the location of stem cells and further complement the in vitro functional studies, to confirm if the LGR5 expressing epithelial cells indeed represent the epithelial stem cell population. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the Wellbeing of Women project grant (RTF510) and Cancer Research UK (A14895). None of the authors have any conflicts of interest to disclose.

A method to measure predictive ability of an injury risk curve using an observation-adjusted area under the receiver operating characteristic curve.

Area under the receiver operating characteristic curve (AROC) is commonly used to choose a biomechanical metric from which to construct an injury risk curve (IRC). However, AROC may not handle censored datasets adequately. Survival analysis creates robust estimates of IRCs which accommodate censored data. We present an observation-adjusted ROC (oaROC) which uses the survival-based IRC to estimate the AROC. We verified and evaluated this method using simulated datasets of different censoring statuses and sample sizes. For a dataset with 1000 left and right censored observations, the median AROC closely approached the oaROCTrue, or the oaROC calculated using an assumed "true" IRC, differing by a fraction of a percent, 0.1%. Using simulated datasets with various censoring, we found that oaROC converged onto oaROCTrue in all cases. For datasets with right and non-censored observations, AROC did not converge onto oaROCTrue. oaROC for datasets with only non-censored observations converged the fastest, and for a dataset with 10 observations, the median oaROC differed from oaROCTrue by 2.74% while the corresponding median AROC with left and right censored data differed from oaROCTrue by 9.74%. We also calculated the AROC and oaROC for a published side impact dataset, and differences between the two methods ranged between -24.08% and 24.55% depending on metric. Overall, when compared with AROC, we found oaROC performs equivalently for doubly censored data, better for non-censored data, and can accommodate more types of data than AROC. While more validation is needed, the results indicate that oaROC is a viable alternative which can be incorporated into the metric selection process for IRCs.

Comparison of clinical and radiographic status around immediately loaded versus conventional loaded implants placed in patients with type 2 diabetes: 12- and 24-month follow-up results.

There are no studies that have compared the clinical and radiographic status around immediately loaded (IL) and conventional loaded (CL) implants placed in patients with type 2 diabetes mellitus (T2DM). The aim was to compare the clinical and radiographic status around IL and CL implants placed in T2DM patients. One hundred and eight diabetic patients [55 with IL implants (Group 1) and 53 with CL implants (Group 2)] were included in this cross-sectional study. All implants were placed in healed sites in the maxillary and mandibular premolar and molar regions and supported single restorations. All patients underwent full mouth mechanical debridement biannually. Haemoglobin A1c (HbA1c) levels, clinical [bleeding on probing (BOP) and probing depth (PD) ≥ 4 mm] and radiographic [crestal bone loss (CBL)] peri-implant parameters were measured for both groups at 12- and 24-month follow-up. Group comparisons were performed using the Mann-Whitney U-test (P < 0·05). The mean age and duration of T2DM in groups 1 and 2 were 50·6 ± 2·2 and 51·8 ± 1·7 years, and 9·2 ± 2·4 and 8·5 ± 0·4 years, respectively. At 12- and 24-month follow-up, the mean HbA1c levels in groups 1 and 2 were 5·4% (4·8-5·5%) and 5·1% (4·7-5·4%) and 5·1% (4·7-5·2%) and 4·9% (4·5-5·2%), respectively. At 12- and 24-month follow-up, there was no statistically significant difference in peri-implant BOP, PD and CBL in both groups. It was concluded that clinical and radiographic status is comparable around IL and CL implants placed in patients with T2DM. The contribution of careful case selection, oral hygiene maintenance and glycaemic control is emphasised.

Phase space for the breakdown of the quantum Hall effect in epitaxial graphene.

We report the phase space defined by the quantum Hall effect breakdown in polymer gated epitaxial graphene on SiC (SiC/G) as a function of temperature, current, carrier density, and magnetic fields up to 30 T. At 2 K, breakdown currents (I(c)) almost 2 orders of magnitude greater than in GaAs devices are observed. The phase boundary of the dissipationless state (ρ(xx)=0) shows a [1-(T/T(c))2] dependence and persists up to T(c)>45 K at 29 T. With magnetic field I(c) was found to increase ∝B(3/2) and T(c)∝B2. As the Fermi energy pproaches the Dirac point, the ν=2 quantized Hall plateau appears continuously from fields as low as 1 T up to at least 19 T due to a strong magnetic field dependence of the carrier density.

Estimating risk factors and causes for postpartum febrile morbidity in teenage mothers.

In this cohort study, our objective was to identify potentially modifiable risk factors and causes for febrile morbidity in teenage mothers. We identified all cases of febrile morbidity using the United States Joint Commission on Maternal Welfare definition in a cohort of teenage deliveries over a 4-year period at one institution. Of the 730 included teenage deliveries, 49 (7%) women suffered postpartum febrile morbidity. Higher maternal pre-pregnancy body mass index (BMI: 34.0 ± 8.6 vs 30.3 ± 6.0 kg/m(2), p = 0.0001), caesarean delivery (RR 21.3, 95% CU 8.9-54.9) and postpartum haemorrhage (RR 3.0, 95% CI 1.1-6.7) were associated with postpartum febrile morbidity. Risk factors for febrile morbidity in the teenage parturient include obesity, caesarean delivery and postpartum haemorrhage. Considering the increasing rates of teenage obesity and overall caesarean delivery rates, attention must be focused on these modifiable risk factors to avoid this complication during a tenuous time for the teenage parent.

Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK.

The extracellular, matrix-modifying enzyme lysyl oxidase (LOX) has recently been linked to colorectal cancer (CRC) progression, in particular to the stages of invasion and metastasis. In this report, we use cell lines expressing a catalytically inactive mutant form of LOX to show that catalytic activity is required for LOX-mediated effects on proliferation and invasion in both in vitro and in vivo models of CRC. Furthermore, we use rheology to measure the relative stiffness of modified collagen matrices and subcutaneous tumors, and show that LOX-induced collagen cross-linking results in stiffening of the matrix both in vitro and in vivo. We observe a strong association between matrix stiffness and activation of the FAK (focal adhesion kinase)/SRC-signaling pathway, with a stiffer environment resulting in increased FAK/SRC phosphorylation and a more proliferative and invasive phenotype. We are the first to show a direct relationship between LOX enzymatic activity and tissue stiffness, and to demonstrate a role for stiffness in driving CRC progression. Our findings provide significant evidence to suggest that therapeutic inhibition of LOX activity may provide a novel effective treatment option for patients with metastatic CRC.

Molecular phylogeny supports the paraphyletic nature of the genus Trogoderma (Coleoptera: Dermestidae) collected in the Australasian ecozone.

To date, a molecular phylogenetic approach has not been used to investigate the evolutionary structure of Trogoderma and closely related genera. Using two mitochondrial genes, Cytochrome Oxidase I and Cytochrome B, and the nuclear gene, 18S, the reported polyphyletic positioning of Trogoderma was examined. Paraphyly in Trogoderma was observed, with one Australian Trogoderma species reconciled as sister to all Dermestidae and the Anthrenocerus genus deeply nested within the Australian Trogoderma clade. In addition, time to most recent common ancestor for a number of Dermestidae was calculated. Based on these estimations, the Dermestidae origin exceeded 175 million years, placing the origins of this family in Pangaea.

Evidence for multiple historical colonizations of an endoreic drainage basin by an Australian freshwater fish.

The contemporary and historical colonization capacity of an Australian freshwater fish, north-west glassfish Ambassis sp., was tested using mtDNA sequence data and six newly developed microsatellite loci in an endoreic basin in central Australia. Overall, Ambassis sp. exhibited weak genetic structure within catchments, suggesting some capacity to recolonize extirpated waterholes after disturbance. Genetic structure revealed that the historical pattern of connectivity among catchments in the Lake Eyre Basin was dramatically different from other species studied in this region. Two highly divergent clades were detected in separate catchments in the basin. mtDNA from individuals sampled in catchments north of the Lake Eyre Basin suggest that Ambassis sp. has colonized on two separate occasions from catchments in northern Australia, subsequently generating two highly divergent lineages.

Systematics and biogeography of the Gondwanan Orthocladiinae (Diptera: Chironomidae).

Restrictions to effective dispersal and gene flow caused by the fragmentation of ancient supercontinents are considered to have driven diversification and speciation on disjunct landmasses globally. Investigating the role that these processes have played in the development of diversity within and among taxa is crucial to understanding the origins and evolution of regional biotas. Within the chironomid (non-biting midge) subfamily Orthocladiinae (Diptera: Chironomidae), a group of genera that are distributed across the austral continents (Australia, New Zealand, South America) have been proposed to represent a relict Gondwanan clade. We used a molecular approach to resolve relationships among taxa with the aim to determine the relative roles that vicariance and dispersal may have played in the evolution of this group. Continental biotas did not form monophyletic groups, in accordance with expectations given existing morphological evidence. Patterns of phylogenetic relationships among taxa did not accord with expected patterns based on the geological sequence of break-up of the Gondwanan supercontinent. Likewise, divergence time estimates, particularly for New Zealand taxa, largely post-dated continental fragmentation and implied instead that several transoceanic dispersal events may have occurred post-vicariance. Passive dispersal of gravid female chironomid adults is the most likely mechanism for transoceanic movement, potentially facilitated by West Wind Drift or anti-cyclone fronts. Estimated timings of divergence among Australian and South American Botryocladius, on the other hand, were congruent with the proposed ages of separation of the two continents from Antarctica. Taken together, these data suggest that a complex relationship between both vicariance and dispersal may explain the evolution of this group. The sampling regime we implemented here was the most intensive yet performed for austral members of the Orthocladiinae and unsurprisingly revealed several novel taxa that will require formal description.

Multiple incursions and putative species revealed using a mitochondrial and nuclear phylogenetic approach to the Trogoderma variabile (Coleoptera: Dermestidae) trapping program in Australia.

The Warehouse beetle, Trogoderma variabile (Coleoptera: Dermestidae), is an internationally significant invasive pest of packed goods and stored grain. When it was first documented in Australia at Griffith, New South Wales, in 1977, an eradication campaign was initiated. After several years and considerable effort, the eradication campaign was abandoned. To monitor the presence and spread of T. variabile, surveys were carried out by government agencies in 1992 and 2002. When survey data was compared, it was concluded that the distribution of morphologically identified T. variabile had doubled in most Australian states. Here, we used samples from the 2002 survey to conduct a phylogenetic study using partial sequences of mitochondrial genes Cytochrome oxidase I and Cytochrome B, and the nuclear gene 18S, to examine the distribution and dispersal of T. variabile and detect the presence of misidentified species. Based on our molecular results, we show that only 47% of the samples analysed were T. variabile, and the remaining were a mixture of six putative species. In addition, T. variabile was found in only 78% of the trapping sites. We discuss the importance of correct diagnosis in relation to the eradication campaign.

Population subdivision in Siamese mud carp Henicorhynchus siamensis in the Mekong River basin: implications for management.

A molecular approach was employed to investigate stock structure in Siamese mud carp Henicorhynchus siamensis populations collected from 14 sites across mainland south-east Asia, with the major focus being the lower Mekong River basin. Spatial analysis of a mitochondrial DNA fragment (ATPase 6 and 8) identified four stocks in the Mekong River basin that were all significantly differentiated from a population in the nearby Khlong River, Thailand. In the Mekong River basin, populations in northern Lao People's Democratic Republic and northern Thailand represent two independent stocks, and samples from Thai tributaries group with those from adjacent Mekong sites above the Khone Falls to form a third stock. All sites below the Khone Falls constituted a single vast stock that includes Cambodia and the Mekong Delta in Vietnam. While H. siamensis is considered currently to undertake extensive annual migrations across the Mekong River basin, the data presented here suggest that natural gene flow may occur over much more restricted geographical scales within the basin, and hence populations may need to be managed at finer spatial scales than at the whole-of-drainage-basin level.

Biogeographic history of an Australian freshwater shrimp, Paratya australiensis (Atyidae): the role life history transition in phylogeographic diversification.

The widespread distribution of the freshwater shrimp Paratya australiensis in eastern Australia suggests that populations of this species have been connected in the past. Amphidromy is ancestral in these shrimps, although many extant populations are known to be restricted to freshwater habitats. In this study, we used a fragment of the cytochrome c oxidase I mitochondrial DNA (mtDNA) gene to examine diversity within P. australiensis and to assess the relative importance of amphidromy in its evolutionary history. We hypothesized that if transitions from an amphidromous to a freshwater life history were important, then we would find a number of divergent lineages restricted to single or groups of nearby drainages. Alternatively, if amphidromy was maintained within the species historically, we expected to find lineages distributed over many drainages. We assumed that the only way for divergence to occur within amphidromous lineages was if dispersal was limited to between nearby estuaries, which, during arid periods in the earth's history, became isolated from one another. We found nine highly divergent mtDNA lineages, estimated to have diverged from one another in the late Miocene/early Pliocene, when the climate was more arid than at present. Despite this, the geographic distribution of lineages and haplotypes within lineages did not support the notion of a stepping-stone model of dispersal between estuaries. We conclude that the extensive divergence has most likely arisen through a number of independent amphidromy-freshwater life history transitions, rather than via historical isolation of amphidromy populations. We also found evidence for extensive movement between coastal and inland drainages, supporting the notion that secondary contact between lineages may have occurred as a result of drainage rearrangements. Finally, our data indicate that P. australiensis is likely a complex of cryptic species, some of which are widely distributed, and others geographically restricted.

Central penetration and stability of N-terminal tripeptide of insulin-like growth factor-I, glycine-proline-glutamate in adult rat.

Insulin-like growth factor-I is a neurotrophic factor and can prevent neurons from ischemic brain injury. However, the large molecular weight and metabolic effects can be problematic in its central delivery. Glycine-proline-glutamate (GPE) is the N-terminal tripeptide of insulin-like growth factor-I, which is naturally cleaved in the plasma and brain tissues. GPE reduces neuronal loss from hypoxic-ischemic brain injury following central administration. Central penetration and the stability of GPE in the plasma and central nervous system were examined in rats using radioimmunoassay and HPLC. GPE was rapidly metabolised in the plasma (8 min) after intraperitoneal administration. Despite having a short half-life in plasma, GPE was detected in the cerebrospinal fluid up to 40 min after intraperitoneal administration. With present of peptidase inhibitors, GPE existed in the brain tissue up to 3 h after intracerebroventricular administration, suggesting a role for peptolysis in its stability. The endopeptidase inhibitors 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) reduced GPE metabolism in the brain tissue while acid peptidase inhibitor pepstatin-A decreased GPE metabolism in the plasma. GPE reduced neuronal loss in the CA1-2 sub-region of the hippocampus given (intraperitoneally) after 30 min of hypoxic-ischemic injury in adult rats, further suggested the effectiveness of GPE central uptake. These results indicated that GPE crosses the blood-CSF and the functional CSF-brain barriers. The longer half-life of GPE in the CNS may be due to its unique enzymatic stability.

Mitochondrial DNA signatures of restricted gene flow within divergent lineages of an atyid shrimp (Paratya australiensis).

We measured spatial genetic structure within three previously described mitochondrial lineages of the atyid shrimp, Paratya australiensis, occurring in upland streams of two major catchments within the Sydney Water Supply Catchment, New South Wales, Australia. In all three lineages, there was significant spatial structuring of genetic variation between catchments. In two lineages, recurrent but restricted maternal gene flow has apparently predominated in shaping within-catchment genetic structure, although this framework may be overlaid with episodic contiguous/long-distance expansion events. In the third lineage, there was no evidence of spatial genetic structuring within one of the catchments, because one haplotype was both common and widespread throughout the sampled area. High-frequency haplotypes were also shared among subcatchments in the other two lineages, and we discuss both historical and contemporary processes that may have left these genetic signatures. Our results are generally concordant with previous reports of significant population structuring in P. australiensis, occurring in upland river reaches elsewhere in eastern Australia. We propose that restricted dispersal and gene flow among upland populations of P. australiensis is linked to dramatic architectural structuring within and among mountain streams.

Patterns of spatial genetic structuring in a hydropsychid caddisfly (Cheumatopsyche sp. AV1) from southeastern Australia.

We assessed levels of mitochondrial genetic spatial structuring in the hydropsychid caddisfly Cheumatopsyche sp. AV1 in southeastern New South Wales, Australia. No significant spatial structuring was detected within or between catchments using analysis of molecular variance, and nested clade contingency analysis suggested no strong relationship between haplotypes and geographical location, at any clade level. However, tests for association among haplotypes incorporating geographical distance in the nested clade analysis, revealed patterns of historical range expansion and recent restricted gene flow. Most likely, population fragmentation preceded range expansion, although subsequent recontact and gene flow among the previously sundered populations has apparently obscured the geographical signature of the former fragmentation. Taken together, our analyses suggest that a number of populations fragmented during the Pleistocene evolved in isolation for a time and subsequently expanded into secondary contact. Since expansion, there has apparently been substantial (albeit somewhat restricted) dispersal and gene flow of adult female Cheumatopsyche sp. AV1, throughout the study area.

A novel hunting accident. Discharge of a firearm by a hunting dog.

The authors report the case of a 21-year-old man who was killed while duck hunting when a shotgun accidentally discharged, shooting him in the head. The loaded weapon, which had been lying on the ground with the safety off and the muzzle pointed toward a river a few feet away, discharged when a hunting dog stepped on the trigger. Scene investigation confirmed that the victim had been standing in the river, planting decoys, with his head approximately level with the adjacent bank. Autopsy examination and ballistic testing confirmed a range of fire consistent with the witness' statements. Examination of the weapon in question documented a light trigger pull but no mechanical defects. The authors review the epidemiology and causality of hunting accidents and discuss the various safety rules that were violated in this highly unusual case. The importance of a complete death investigation, including autopsy, when dealing with a firearm death is emphasized.

A Web-based diabetes care management support system.

BACKGROUND: Because of the often asymptomatic nature of diabetes and the long period between sustained hyperglycemia and observable complications, appropriate diabetes care relies on a long-term program of secondary prevention. Yet routine monitoring and screening among patients with diabetes is less than optimal. To support the provision of routine care to patients with diabetes, the Center for Health Services Research, Henry Ford Health System (Detroit), developed a Web-based Diabetes Care Management Support System (DCMSS). A nonrandomized, longitudinal study was conducted (January 1, 1998-October 31, 1999) with 13,325 health maintenance organization patients with diabetes who were aligned to 190 primary care providers practicing in 31 primary care clinics. RESULTS: Three DCMSS features--clinical practice guidelines, patient registries, and performance reports--were made available via a corporate intranet within an existing electronic medical record. The effect of DCMSS usage frequency was evaluated on the likelihood of a patient's receipt of glycated hemoglobin testing, lipid profile testing, and retinal examinations. Logistic regression models controlling for patient sociodemographic and clinical characteristics, and the testing history of the patient, the primary care physician, and the primary care clinic, were fit using generalized estimating equation methods. The more often a physician used DCMSS, the more likely his or her patients were to receive lipid profile testing (OR [odds ratio] = 1.01, 95% CI [confidence interval] = 1.01-1.02). Compared with patients of physicians who never used the system, patients of physicians who initiated 12 sessions were an estimated 19% more likely (95% CI = 7%-33%) to receive lipid profile testing. The analyses also suggested that the likelihood of a patient receiving a retinal exam was associated with system usage (OR = 1.01, 95% CI = 1.01-1.01). No relationship was found between system use and glycated hemoglobin testing. CONCLUSIONS: Computerized systems of clinical practice guidelines, patient registries, and performance feedback may help improve the rate of routine testing among patients with diabetes.

Polyclonal systemic immunoblast proliferation: an unusual hematologic entity presenting as a medical examiner case.

A 43-year-old woman who was receiving oral antibiotics for several days for a superficial foot infection developed a persistent rash, fever, and lymphadenopathy, despite discontinuation of the antibiotic and administration of steroids for a presumed drug reaction. Hours after a subsequent visit to the emergency room for worsening symptoms, she died at home. At autopsy, there was a florid, systemic proliferation of polyclonal plasma cells and immunoblasts infiltrating nearly every organ and tissue of the body, most notably the lymph nodes and spleen. The polyclonal nature of the process was confirmed by immunofixation electrophoresis and immunohistochemistry. Cases of fatal polyclonal systemic immunoblast proliferations are extremely rare, and the trigger for such proliferations is not always known. We review the literature on this unusual entity and discuss the clinical and pathologic findings.

Population genetic structure of Australian magpies: evidence for regional differences in juvenile dispersal behaviour.

Territorial group size in Australian magpies (Gymnorhina tibicen) ranges from monogamous pairs to groups of more than 20 individuals. It has been hypothesized that large territorial groups result from the retention of juveniles after a breeding effort. If this is true, local populations consisting of large groups are likely to exhibit the most genetic structure, because over time similar genotypes will tend to be confined to limited areas if juveniles are predominantly philopatric. The objective of the present study was to test this hypothesis using allozyme and mitochondrial DNA data to provide indirect estimates of regional gene flow (derived from hierarchical population subdivision analyses). These data were used in combination with estimates of group size to infer patterns of dispersal among magpie populations across mainland Australia. Territorial groups were significantly larger in the south-west compared to three eastern regions. Although inferred levels of gene flow were substantial for all four regions, a striking pattern emerged from both sets of genetic data: more differentiation was evident among populations in the south-western region than in any eastern region. We conclude that levels of juvenile dispersal influence group size in G. tibicen, because in the south-western region where groups were largest, populations were most genetically differentiated. Our results suggest that contrasting population genetic structures may develop within a single species as a result of differences in social system.