DNA barcoding and the differentiation between North American and West European Phormia regina (Diptera,?Calliphoridae,Chrysomyinae)

Phormia regina (the black fly) is a common Holarctic blow fly species which serves as a primary indicator taxon to estimate minimal post mortem intervals. It is also a major research model in physiological and neurological studies on insect feeding. Previous studies have shown a sequence divergence of up to 4.3% in the mitochondrial COI gene between W European and N American P. regina populations. Here, we DNA barcoded P. regina specimens from six N American and 17 W European populations and confirmed a mean sequence divergence of ca. 4% between the populations of the two continents, while sequence divergence within each continent was a ten-fold lower. Comparable mean mtDNA sequence divergences were observed for COII (3.7%) and cyt b (5.3%), but mean divergence was lower for 16S (0.4–0.6%). Intercontinental divergence at nuclear DNA was very low (≤ 0.1% for both 28S and ITS2), and we did not detect any morphological differentiation between N American and W European specimens. Therefore, we consider the strong differentiation at COI, COII and cyt b as intraspecific mtDNA sequence divergence that should be taken into account when using P. regina in forensic casework or experimental research.     

Deleted in liver cancer 1 (DLC1) negatively regulates Rho/ROCK/MLC pathway in hepatocellular carcinoma

Aims

Deleted in liver cancer 1 (DLC1), a member of RhoGTPase activating protein (GAP) family, is known to have suppressive activities in tumorigenicity and cancer metastasis. However, the underlying molecular mechanisms of how DLC1 suppresses cell motility have not been fully elucidated. Rho-kinase (ROCK) is an immediate down-stream effector of RhoA in mediating cellular cytoskeletal events and cell motility. In the present study, we aimed to investigate the effects of DLC1 on Rho/ROCK signaling pathway in hepatocellular carcinoma (HCC).

Methodology/Principal Findings

We demonstrated that DLC1 negatively regulated ROCK-dependent actomyosin contractility. From immumofluorescence study, we found that ectopic expression of DLC1 abrogated Rho/ROCK-mediated cytoskeletal reorganization including formation of stress fibers and focal adhesions. It also downregulated cortical phosphorylation of myosin light chain 2 (MLC2). These inhibitory events by DLC1 were RhoGAP-dependent, as RhoGAP-deficient mutant of DLC1 (DLC1 K714E) abolished these inhibitory events. In addition, from western study, DLC1 inhibited ROCK-related myosin light chain phosphatase targeting unit 1 (MYPT1) phosphorylation at Threonine 853. By examining cell morphology under microscope, we found that ectopic expression of dominant-active ROCK released cells from DLC1-induced cytoskeletal collapse and cell shrinkage.

Conclusion

Our data suggest that DLC1 negatively regulates Rho/ROCK/MLC2. This implicates a ROCK-mediated pathway of DLC1 in suppressing metastasis of HCC cells and enriches our understanding in the molecular mechanisms involved in the progression of hepatocellular carcinoma.     

A novel thioredoxin-like protein encoded by the C. elegans dpy-11 gene is required for body and sensory organ morphogenesis

Sensory ray morphogenesis in C. elegans requires active cellular interaction regulated by multiple genetic activities. We report here the cloning of one of these genes, dpy-11, which encodes a membrane-associated thioredoxin-like protein. The DPY-11 protein is made exclusively in the hypodermis and resides in the cytoplasmic compartment. Whereas the TRX domain of DPY-11 displays a catalytic activity in vitro, mapping of lesions in different mutant alleles and functional analysis of deletion transgenes reveal that both this enzymatic activity and transmembrane topology are essential for determining body shape and ray morphology. Based on the abnormal features in both the expressing and non-expressing ray cells, we propose that the DPY-11 is required in the hypodermis for modification of its substrates. In turn, ray cell interaction and the whole morphogenetic process can be modulated by these substrate molecules.     

Diabetic ketoacidosis induces in vivo activation of human T-lymphocytes

Diabetic ketoacidosis (DKA) is an inflammatory state associated with immune responses in polymorphonuclear cells (PMN). Activation of subgroup of T-lymphocytes in PMN of DKA patients, however, is not known. We studied in vivo activation of CD4 and CD8 lymphocytes by measuring de novo growth factor receptor for insulin, IGF-1, and IL-2 in eight patients on admission and at resolution of DKA, and compared them with matched controls. The presence of these receptors was demonstrated in all patients' lymphocytes on admission, but not in control subjects. This event was associated with increased levels of thiobarbituric acid-reacting material and dichlorofluorescien, as markers of oxidative stress. Based on these new findings and works in the literature, we hypothesize that hyperglycemia/ketosis results in increased reactive oxygen species, leading to increased levels of cytokines and emergence of growth factor receptors. We propose DKA changes the T-lymphocytes to insulin sensitive tissues as a compensatory mechanism.     

Transposon tagging in diploid strawberry

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T(1) progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T(0) launch pads, putative transposants in the T(1) generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T(1) plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T(1) plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T(0) generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T(2) transposon-tagged plants. The mutant collection has been catalogued in an on-line database.     

Nest taphonomy of common terns (Sterna hirundo) on Poplar Island,Chesapeake Bay,Maryland

Mesozoic non-avian theropod dinosaurs displayed a diverse range of egg types and clutch forms, suggesting a variety of nesting behaviours, some of which may be shared with birds. More accurate inferences of these behaviours require taphonomic studies of modern nesting sites. Here, we document common tern (Sterna hirundo) nesting sites on Poplar Island in Chesapeake Bay, Maryland. Nests were surveyed on multiple occasions, documenting nest composition, density and distribution, as well as eggshell concentration and orientation. Three colonies yielded 79 tern nests with 193 eggs. Twelve nests hatched, 7 were predated, 30 failed and the fates of 30 remain unknown. Abundant eggshells occurred at the centre of the nests. Concave-up eggshell in or on the nest surface characterised hatched and predated nests, whereas eggshell forced into the subsurface seemed to favour concave-down orientation and may reflect post-hatching extended use of the nest. Eggs buried deep within the nest materials and/or substrate suggest adult abandonment. Quantitative data on eggshell orientation and observations regarding controls on nest distribution, egg predation and incorporation of intact eggs into the substrate at modern nesting sites provide physical guidelines for the improved interpretation of fossil nesting localities.     

Cyclin A/cdk2 Regulates Adenomatous Polyposis Coli-dependent Mitotic Spindle Anchoring

Mutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-β-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis.Adenomatous polyposis coli (APC)⁵ was initially identified as a tumor suppressor in familial colon cancers. It is a regulator of Wnt-β-catenin signaling and thereby regulates progression into the cell cycle, but also has Wnt-independent mitotic roles in spindle anchoring and kinetochore function (1–3). These latter functions of APC are mediated through its ability to bind microtubules and the end-binding protein, EB1 (2). Loss or mutation of APC has been demonstrated to increase chromosomal instability, although whether this is through its Wnt-dependent or independent functions is unclear (3). The mitotic defects caused by APC mutation and depletion are characterized by an inability to locate the center of the cell and failure of chromosomal alignment (4). It was also associated with a loss of normal spindle orientation in small intestinal crypts of APC^Min/+ mice (5), suggesting that disruption of the normal mitotic functions of APC are likely to be major contributors to the chromosomal instability observed.APC interaction with EB1 is regulated by phosphorylation of its C-terminal domain by cyclin B/cdk1 in mitosis (6, 7). The majority of APC mutations occurs in a region from codons 1,000 to 1,500 called the mutation cluster region (MCR) and result in truncations of the C-terminal half of the protein, which includes the β-catenin, microtubule, and EB1 binding sites of APC (1, 2). Depletion of either APC or EB1 produce almost identical mitotic defects, indicating their interaction is critical to normal spindle formation (4, 8). However, expression of various truncation mutants across the MCR revealed interesting differences the spindle defects observed, suggesting that this role of APC in spindle function is not solely due to interaction with EB1 (4). Progression into mitosis is regulated by cyclin B/cdk1, but the timing of its activation is regulated by cyclin A/cdk2 (9–12), which in turn is regulated by the dual specificity phosphatase cdc25B in G2 phase (13). Cyclin A is destroyed at prometaphase (14) suggesting that its activity is required for not only entry into mitosis but during the early part of mitosis itself. The majority of substrates identified for cyclin A/cdk2 are nuclear, where the majority of cyclin A/cdk2 is localized in G2, but reports also suggest that cyclin A is capable of localizing to both the cytoplasm and centrosomes (9, 14, 15), thus there are likely to be additional substrates for this complex in the cytoplasmic compartment. In vitro studies using Xenopus extracts have demonstrated that cyclin A/cdk is capable of increasing microtubule nucleation at the centrosomes (16). Thus it is likely that cyclin A in association with its cdk partner has roles in not only promoting entry into mitosis but also in establishing mitosis, possibly by influencing the mitotic machinery.We have used siRNA to knockdown cyclin A2, the major cyclin A isoform in somatic cells, and cdk2 inhibitors to examine the role of the G2 phase cyclin A/cdk2 complex in cell cycle progression. We demonstrate that knockdown of cyclin A delayed progression through mitosis and activation of the spindle assembly checkpoint. Spindle anchoring was also defective, a phenotype identical to APC-truncating mutants. We demonstrate that cyclin A/cdk2 binds to APC in late G2 phase/early mitosis and phosphorylates Ser-1360, and that the lack of this phosphorylation of APC results in identical mitotic defects to the absence of cyclin A/cdk2.     

Self-fitting shape memory polymer foam inducing bone regeneration: A rabbit femoral defect study

Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.     

The synthesis of 17-substituted 2α-chloro-5α-androstan-3-ols

This paper describes the synthesis of 2α-chloro-3α-hydroxy-5α-androstan-17-one and 2α-chloro-5α-androstane-3α,17β-diol and their 3-epimers. The epimers were characterized by nmr spectroscopy.