Serological evidence of Ebola virus infection in Indonesian orangutans

Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae and cause severe hemorrhagic fever in humans and nonhuman primates. Despite the discovery of EBOV (Reston virus) in nonhuman primates and domestic pigs in the Philippines and the serological evidence for its infection of humans and fruit bats, information on the reservoirs and potential amplifying hosts for filoviruses in Asia is lacking. In this study, serum samples collected from 353 healthy Bornean orangutans (Pongo pygmaeus) in Kalimantan Island, Indonesia, during the period from December 2005 to December 2006 were screened for filovirus-specific IgG antibodies using a highly sensitive enzyme-linked immunosorbent assay (ELISA) with recombinant viral surface glycoprotein (GP) antigens derived from multiple species of filoviruses (5 EBOV and 1 MARV species). Here we show that 18.4% (65/353) and 1.7% (6/353) of the samples were seropositive for EBOV and MARV, respectively, with little cross-reactivity among EBOV and MARV antigens. In these positive samples, IgG antibodies to viral internal proteins were also detected by immunoblotting. Interestingly, while the specificity for Reston virus, which has been recognized as an Asian filovirus, was the highest in only 1.4% (5/353) of the serum samples, the majority of EBOV-positive sera showed specificity to Zaire, Sudan, Cote d'Ivoire, or Bundibugyo viruses, all of which have been found so far only in Africa. These results suggest the existence of multiple species of filoviruses or unknown filovirus-related viruses in Indonesia, some of which are serologically similar to African EBOVs, and transmission of the viruses from yet unidentified reservoir hosts into the orangutan populations. Our findings point to the need for risk assessment and continued surveillance of filovirus infection of human and nonhuman primates, as well as wild and domestic animals, in Asia.     

Treatment with antibiotics that interfere with peptidoglycan biosynthesis inhibits chloroplast division in the desmid Closterium

Charophytes is a green algal group closely related to land plants. We investigated the effects of antibiotics that interfere with peptidoglycan biosynthesis on chloroplast division in the desmid Closterium peracerosum-strigosum-littorale complex. To detect cells just after division, we used colchicine, which inhibits Closterium cell elongation after division. Although normal Closterium cells had two chloroplasts before and after cell division, cells treated with ampicillin, D-cycloserine, or fosfomycin had only one chloroplast after cell division, suggesting that the cells divided without chloroplast division. The antibiotics bacitracin and vancomycin showed no obvious effect. Electron microscopic observation showed that irregular-shaped chloroplasts existed in ampicillin-treated Closterium cells. Because antibiotic treatments resulted in the appearance of long cells with irregular chloroplasts and cell death, we counted cell types in the culture. The results suggested that cells with one chloroplast appeared first and then a huge chloroplast was generated that inhibited cell division, causing elongation followed by cell death.     

Cell movements of the deep layer of non-neural ectoderm underlie complete neural tube closure in Xenopus

In developing vertebrates, the neural tube forms from a sheet of neural ectoderm by complex cell movements and morphogenesis. Convergent extension movements and the apical constriction along with apical-basal elongation of cells in the neural ectoderm are thought to be essential for the neural tube closure (NTC) process. In addition, it is known that non-neural ectoderm also plays a crucial role in this process, as the neural tube fails to close in the absence of this tissue in chick and axolotl. However, the cellular and molecular mechanisms by which it functions in NTC are as yet unclear. We demonstrate here that the non-neural superficial epithelium moves in the direction of tensile forces applied along the dorsal-ventral axis during NTC. We found that this force is partly attributable to the deep layer of non-neural ectoderm cells, which moved collectively towards the dorsal midline along with the superficial layer. Moreover, inhibition of this movement by deleting integrin β1 function resulted in incomplete NTC. Furthermore, we demonstrated that other proposed mechanisms, such as oriented cell division, cell rearrangement and cell-shape changes have no or only minor roles in the non-neural movement. This study is the first to demonstrate dorsally oriented deep-cell migration in non-neural ectoderm, and suggests that a global reorganization of embryo tissues is involved in NTC.     

Islet1 regulates establishment of the posterior hindlimb field upstream of the Hand2-Shh morphoregulatory gene network in mouse embryos

How divergent genetic systems regulate a common pathway during the development of two serial structures, forelimbs and hindlimbs, is not well understood. Specifically, HAND2 has been shown to regulate Shh directly to initiate its expression in the posterior margin of the limb mesenchyme. Although the Hand2-Shh morphoregulatory system operates in both the forelimb and hindlimb bud, a recent analysis suggested that its upstream regulation is different in the forelimb and hindlimb bud. A combination of all four Hox9 genes is required for Hand2 expression in the forelimb-forming region; however, it remains elusive what genetic system regulates the Hand2-Shh pathway in the hindlimb-forming region. By conditional inactivation of Islet1 in the hindlimb-forming region using the Hoxb6Cre transgene, we show that Islet1 is required for establishing the posterior hindlimb field, but not the forelimb field, upstream of the Hand2-Shh pathway. Inactivation of Islet1 caused the loss of posterior structures in the distal and proximal regions, specifically in the hindlimb. We found that Hand2 expression was downregulated in the hindlimb field and that Shh expression was severely impaired in the hindlimb bud. In the Hoxb6Cre; Islet1 mutant pelvis, the proximal element that is formed in a Shh-independent manner, displayed complementary defects in comparison with Pitx1(-/-) hindlimbs. This suggests that Islet1 and Pitx1 function in parallel during girdle development in hindlimbs, which is in contrast with the known requirement for Tbx5 in girdle development in forelimbs. Our studies have identified a role for Islet1 in hindlimb-specific development and have revealed Islet1 functions in two distinct processes: regulation upstream of the Hand2-Shh pathway and contributions to girdle development.     

Enamino-oxindole HIV protease inhibitors

We have designed and synthesized a series of HIV protease inhibitors (PIs) with enamino-oxindole substituents optimized to interact with the S2' subsite of the HIV protease binding pocket. Several of these inhibitors have sub-nanomolar K(i) and antiviral IC(50) in the low nM range against WT HIV and against a panel of multi-drug resistant (MDR) strains.     

A structural mechanism for dimeric to tetrameric oligomer conversion in Halomonas sp. nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X‐ray crystallography. The molecular weight of HaNDK is 33,660, and the X‐ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1–65.3 kDa, and the oligomerization state was investigated by X‐ray crystallography to 2.3 and 2.5 Å resolution with space groups of P2₁ and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild‐type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild‐type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.     

MRI Characterization of Paranodal Junction Failure and Related Spinal Cord Changes in Mice

The paranodal junction is a specialized axon-glia contact zone that is important for normal neuronal activity and behavioral locomotor function in the central nervous system (CNS). Histological examination has been the only method for detecting pathological paranodal junction conditions. Recently, diffusion tensor MRI (DTI) has been used to detect microstructural changes in various CNS diseases. This study was conducted to determine whether MRI and DTI could detect structural changes in the paranodal junctions of the spinal cord in cerebroside sulfotransferase knock-out (CST-KO) mice. Here, we showed that high-resolution MRI and DTI characteristics can reflect paranodal junction failure in CST-KO mice. We found significantly lower T1 times and significantly higher T2 times in the spinal cord MRIs of CST-KO mice as compared to wild-type (WT) mice. Spinal cord DTI showed significantly lower axial diffusivity and significantly higher radial diffusivity in CST-KO mice as compared to WT mice. In contrast, the histological differences in the paranodal junctions of WT and CST-KO mice were so subtle that electron microscopy or immunohistological analyses were necessary to detect them. We also measured gait disturbance in the CST-KO mice, and determined the conduction latency by electrophysiology. These findings demonstrate the potential of using MRI and DTI to evaluate white matter disorders that involve paranodal junction failure.     

Nucleolin Participates in DNA Double-Strand Break-Induced Damage Response through MDC1-Dependent Pathway

H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.     

Individual Variation of the Genetic Response to Bisphenol A in Human Foreskin Fibroblast Cells Derived from Cryptorchidism and Hypospadias Patients

Background/Purpose

We hypothesized that polymorphic differences among individuals might cause variations in the effect that environmental endocrine disruptors (EEDs) have on male genital malformations (MGMs). In this study, individual variation in the genetic response to low-dose bisphenol A (BPA) was investigated in human foreskin fibroblast cells (hFFCs) derived from child cryptorchidism (CO) and hypospadias (HS) patients.

Methodology/Principal Findings

hFFCs were collected from control children without MGMs (n = 5) and child CO and HS patients (n = 8 and 21, respectively). BPA exposure (10 nM) was found to inhibit matrix metalloproteinase-11 (MMP11) expression in the HS group (0.74-fold, P = 0.0034) but not in the control group (0.93-fold, P = 0.84) and CO group (0.94-fold, P = 0.70). Significantly lower levels of MMP11 expression were observed in the HS group compared with the control group (0.80-fold, P = 0.0088) and CO group (0.79-fold, P = 0.039) in response to 10 nM BPA. The effect of single-nucleotide polymorphism rs5000770 (G>A), located within the aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) locus, on individual sensitivity to low-dose BPA was investigated in the HS group. A significant difference in neurotensin receptor 1 (NTSR1) expression in response to 10 nM BPA was observed between AA and AG/GG groups (n = 6 and 15, respectively. P = 0.031). However, no significant difference in ARNT2 expression was observed (P = 0.18).

Conclusions/Significance

This study advances our understanding of the specificity of low-dose BPA effects on human reproductive health. Our results suggest that genetic variability among individuals affects susceptibility to the effects of EEDs exposure as a potential cause of HS.     

A new class of non-thiazolidinedione, non-carboxylic-acid-based highly selective peroxisome proliferator-activated receptor (PPAR) γ agonists: design and synthesis of benzylpyrazole acylsulfonamides

Herein, we describe the design, synthesis, and structure-activity relationships of novel benzylpyrazole acylsulfonamides as non-thiazolidinedione (TZD), non-carboxylic-acid-based peroxisome proliferator-activated receptor (PPAR) γ agonists. Docking model analysis of in-house weak agonist 2 bound to the reported PPARγ ligand binding domain suggested that modification of the carboxylic acid of 2 would help strengthen the interaction of 2 with the TZD pocket and afford non-carboxylic-acid-based agonists. In this study, we used an acylsulfonamide group as the ring-opening analog of TZD as an isosteric replacement of carboxylic acid moiety of 2; further, preliminary modification of the terminal alkyl chain on the sulfonyl group gave the lead compound 3c. Subsequent optimization of the resulting compound gave the potent agonists 25c, 30b, and 30c with high metabolic stability and significant antidiabetic activity. Further, we have described the difference in binding mode of the carboxylic-acid-based agonist 1 and acylsulfonamide 3d.