Age-related plumage differences of Dunlins along the East Asian-Australasian Flyway

ABSTRACT.   For some populations of Dunlins ( Calidris alpina ), determining the age of individuals on the nonbreeding grounds can be difficult. This difficulty arises in part because some populations undergo their primary molt during the boreal summer, leaving adult and first-year Dunlins with similar amounts of abrasion on their primaries. Ageing Dunlins is further complicated by the presence of adults with buff-fringed, inner median coverts in some populations, a feature often used to age juvenile Dunlins. We examined a number of characteristics helpful in the ageing Dunlins at nonbreeding areas along the East Asian-Australasian Flyway, including: (1) the pattern of white fringes on the inner primary coverts, (2) the color of fringes on the inner median wing coverts, (3) the color of the tip on the carpal covert, (4) the presence or absence of a dark subterminal band on tertial or tertial coverts, and (5) remnants of alternate or juvenile plumage on the belly. Our results demonstrate that the pattern of white fringes on the inner primary coverts is an important character for ageing Dunlin along the East Asian-Australasian Flyway. In addition, we found that all characteristics used for ageing fade with time, and that breeding adults along the East Asian-Australasian Flyway are more difficult to age than adults elsewhere, in part due to the presence of "adult buff" coverts (ABCs) and in part because of the early timing of primary molt. Finally, we suggest that the presence of ABCs might be useful for differentiating the two Dunlin subspecies ( C. a. arcticola and C. a. pacifica ) occurring in Alaska.     

Specific variants in the MLH1 gene region may drive DNA methylation, loss of protein expression, and MSI-H colorectal cancer

Background

We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability.

Methodology/Principal Findings

We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls) and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls) and from Seattle (591 cases, 629 controls). Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohisotchemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10⁻⁴ when the SNP was examined alone).

Conclusions/Significance

The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.     

Establishment of Protein Delivery Systems Targeting Podocytes

Background

Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.

Methodology/Principal Findings

Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as β-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95–100%. The delivered β-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.

Conclusions

Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes.     

Life cycle of the aquatic firefly Luciola ficta (Coleoptera: Lampyridae)

This research is the first to record the complete life history of the aquatic firefly Luciola ficta (Olivier) using a unique individual rearing method. Transparent containers (250 ml; height: 6 cm; bottom diameter: 8 cm; mouth diameter: 9.5 cm) were used to rear individuals from egg to adult in the laboratory, so that they could be observed throughout the whole life cycle. Larvae were fed on the meat of the water snail Cipangopaludina chinensis (Gray). Temperature ranged from 18 °C to 30 °C, relative humidity (RH) was 80 ± 5%, and the light:dark (L:D) ratio was 10:14. Of 80 eggs, 35 individuals completed their life cycle under these laboratory conditions in Jiji, Nantou County, Taiwan. The external morphological characteristics of each growing stage were described. Egg hatching rate was 95%. On average, one generation spanned 388.2 ± 25.7 days. The durations of egg, larva, climbing larva, cocoon, and adult stages were 19.1 ± 1.5 days, 328.9 ± 33.2 days, 10.9 ± 7.8 days, 14.7 ± 5.3 days, and 15.7 ± 5.2 days, respectively. The number of larval instars ranged from five to seven, with a modal value of six instars for males and seven instars for females. Female larval duration averaged 337.1 ± 31.2 days, which was higher than the 307.6 ± 34.1 days of the males. From January to December 2002, adult emergence peaked twice, with the main high peak appearing in April and the second peak occurring in August. The results of indoor rearing and of field investigations in Jiji, Nantou County, suggested that L. ficta is univoltine. Adult body length is negatively correlated with larval duration (P < 0.01). The life history traits of L. ficta show plasticity in adult occurrence, egg size, egg duration, larval duration, larval instars, and adult body length. Some variations were discussed in the context of survivorship in field habitats.     

PUF3 Acceleration of Deadenylation in Vivo Can Operate Independently of CCR4 Activity, Possibly Involving Effects on the PAB1-mRNP Structure

The evolutionarily conserved PUF proteins stimulate CCR4 mRNA deadenylation through binding to 3′ untranslated region sequences of specific mRNA. We have investigated the mechanisms by which PUF3 in Saccharomyces cerevisiae accelerates deadenylation of the COX17 mRNA. PUF3 was shown to affect PAN2 deadenylation of the COX17 mRNA independent of the presence of CCR4, suggesting that PUF3 acts through a general mechanism to affect deadenylation. Similarly, eIF4E, the cap-binding translation initiation factor known to control CCR4 deadenylation, was shown to affect PAN2 activity in vivo. PUF3 was found to be required for eIF4E effects on COX17 deadenylation. Both eIF4E and PUF3 effects on deadenylation were shown, in turn, to necessitate a functional poly(A)-binding protein (PAB1) in which removal of the RRM1 (RNA recognition motif 1) domain of PAB1 blocked both their effects on deadenylation. While removal of the proline-rich region (P domain) of PAB1 substantially reduces CCR4 deadenylation at non-PUF3-controlled mRNA and correspondingly blocked eIF4E effects on deadenylation, PUF3 essentially bypassed this P domain requirement. These results indicate that the PAB1-mRNP structure is critical for PUF3 action. We also found that multiple components of the CCR4-NOT deadenylase complex, but not PAN2, interacted with PUF3. PUF3 appears, therefore, both to act independently of CCR4 activity, possibly through effects on PAB1-mRNP structure, and to be capable of retaining the CCR4-NOT complex.     

The FRB domain of mTOR: NMR solution structure and inhibitor design

The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.     

NF-kappaB-mediated expression of iNOS promotes epithelial defense against infection by Cryptosporidium parvum in neonatal piglets

Cryptosporidium sp. parasitizes intestinal epithelium, resulting in enterocyte loss, villous atrophy, and malabsorptive diarrhea. We have shown that mucosal expression of inducible nitric oxide (NO) synthase (iNOS) is increased in infected piglets and that inhibition of iNOS in vitro has no short-term effect on barrier function. NO exerts inhibitory effects on a variety of pathogens; nevertheless, the specific sites of iNOS expression, pathways of iNOS induction, and mechanism of NO action in cryptosporidiosis remain unclear. Using an in vivo model of Cryptosporidium parvum infection, we have examined the location, mechanism of induction, specificity, and consequence of iNOS expression in neonatal piglets. In acute C. parvum infection, iNOS expression predominated in the villous epithelium, was NF-kappaB dependent, and was not restricted to infected enterocytes. Ongoing treatment of infected piglets with a selective iNOS inhibitor resulted in significant increases in villous epithelial parasitism and oocyst excretion but was not detrimental to maintenance of mucosal barrier function. Intensified parasitism could not be attributed to attenuated fluid loss or changes in epithelial proliferation or replacement rate, inasmuch as iNOS inhibition did not alter severity of diarrhea, piglet hydration, Cl- secretion, or kinetics of bromodeoxyuridine-labeled enterocytes. These findings suggest that induction of iNOS represents a nonspecific response of the epithelium that mediates enterocyte defense against C. parvum infection. iNOS did not contribute to the pathogenic sequelae of C. parvum infection.