Evidence for the existence of a stable association between nascent DNA and the nuclear membrane of HeLa cells.

Nascent DNA-nuclear membrane complexes isolated from HeLa cells and solubilized in a sodium dodecyl sulfate-urea solution were examined by gel electrophoresis, column chromatography, isopycnic centrifugation, and by extraction with chloroform/methanol. Radioactivity attributable to [3H]DNA co-migrated with three protein peaks during electrophoresis. This radioactivity was eliminated by prior treatment with DNAase. In addition, all of the radioactivity attributable to nascent DNA eluted with a specific protein on Sepharose 4B columns. This DNA - protein complex banded at a density of 1.58 gm/cm3 in sucrose-CsCl gradients. Treatment with DNAase, phospholipase A and C, and dilute alkali disrupted the complex. Moreover, 93% of the radioactivity attributable to protein and 70% of that attributable to DNA could be extracted from the complex with a chloroform/methanol solution. The results suggest that nascent DNA may be in a stable association with a proteolipid moiety of the nuclear membrane.     

Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy.

Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-delta, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin (mdx) or sarcoglycan-delta (sgcd-/-) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd-/-, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd-/- arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd-/- arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd-/- arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.     

Calcium Regulation of Hemorrhagic Fever Virus Budding: Mechanistic Implications for Host-Oriented Therapeutic Intervention

Hemorrhagic fever viruses, including the filoviruses (Ebola and Marburg) and arenaviruses (Lassa and Junín viruses), are serious human pathogens for which there are currently no FDA approved therapeutics or vaccines. Importantly, transmission of these viruses, and specifically late steps of budding, critically depend upon host cell machinery. Consequently, strategies which target these mechanisms represent potential targets for broad spectrum host oriented therapeutics. An important cellular signal implicated previously in EBOV budding is calcium. Indeed, host cell calcium signals are increasingly being recognized to play a role in steps of entry, replication, and transmission for a range of viruses, but if and how filoviruses and arenaviruses mobilize calcium and the precise stage of virus transmission regulated by calcium have not been defined. Here we demonstrate that expression of matrix proteins from both filoviruses and arenaviruses triggers an increase in host cytoplasmic Ca²⁺ concentration by a mechanism that requires host Orai1 channels. Furthermore, we demonstrate that Orai1 regulates both VLP and infectious filovirus and arenavirus production and spread. Notably, suppression of the protein that triggers Orai activation (Stromal Interaction Molecule 1, STIM1) and genetic inactivation or pharmacological blockade of Orai1 channels inhibits VLP and infectious virus egress. These findings are highly significant as they expand our understanding of host mechanisms that may broadly control enveloped RNA virus budding, and they establish Orai and STIM1 as novel targets for broad-spectrum host-oriented therapeutics to combat these emerging BSL-4 pathogens and potentially other enveloped RNA viruses that bud via similar mechanisms.     

Clinical Prognostic Value of RNA Viral Load and CD4 Cell Counts during Untreated HIV-1 Infection—A Quantitative Review

Background

The prognostic value of CD4 counts and RNA viral load for identifying treatment need in HIV-infected individuals depends on (a) variation within and among individuals, and (b) relative risks of clinical progression per unit CD4 or RNA difference.

Methodology/Principal Findings

We reviewed these measurements across (a) 30 studies, and (b) 16 cohorts of untreated seropositive adults. Median within-population interquartile ranges were 74,000 copies/mL for RNA with no significant change during the course of infection; and 330 cells/µL for CD4, with a slight proportional increase over infection. Applying measurement and physiological fluctuations observed on chronically infected patients, we estimate that 45% of population-level variation in RNA, and 25% of variation in CD4, were due to within-patient fluctuations. Comparing a patient with RNA at upper 75^th centile with a patient at median RNA, 5-year relative risks were 1.4 (95% CI 1.2–1.7) for AIDS and 1.5 (1.3–1.9) for death, without change over the course of infection. In contrast, for a patient with CD4 count at the lower 75^th centile, relative risks increased from 1.0 at seroconversion to maxima of 6.3 (4.4–8.9) for AIDS and 5.5 (2.7–10.1) for death by year 6, when the population median had fallen to 300 cells/µL. Below 300 cells/µL, prognostic power did not increase, due to a narrower CD4 range.

Conclusions

Findings support the current WHO recommendation (used with clinical criteria) to start antiretroviral treatment in low-income settings at CD4 thresholds of 200–350 cells/µL, without pre-treatment RNA monitoring – while not precluding earlier treatment based on clinical, socio-demographic or public health criteria.     

Populus CEN/TFL1 regulates first onset of flowering,axillary meristem identity and dormancy release in Populus

Members of the CENTRORADIALIS (CEN)/TERMINAL FLOWER 1 (TFL1) subfamily control shoot meristem identity, and loss‐of‐function mutations in both monopodial and sympodial herbaceous plants result in dramatic changes in plant architecture. We studied the degree of conservation between herbaceous and woody perennial plants in shoot system regulation by overexpression and RNA interference (RNAi)‐mediated suppression of poplar orthologs of CEN, and the related gene MOTHER OF FT AND TFL 1 (MFT). Field study of transgenic poplars (Populus spp.) for over 6 years showed that downregulation of PopCEN1 and its close paralog, PopCEN2, accelerated the onset of mature tree characteristics, including age of first flowering, number of inflorescences and proportion of short shoots. Surprisingly, terminal vegetative meristems remained indeterminate in PopCEN1‐RNAi trees, suggesting the possibility that florigen signals are transported to axillary mersitems rather than the shoot apex. However, the axillary inflorescences (catkins) of PopCEN1‐RNAi trees contained fewer flowers than did wild‐type catkins, suggesting a possible role in maintaining the indeterminacy of the inflorescence apex. Expression of PopCEN1 was significantly correlated with delayed spring bud flush in multiple years, and in controlled environment experiments, 35S::PopCEN1 and RNAi transgenics required different chilling times to release dormancy. Considered together, these results indicate that PopCEN1/PopCEN2 help to integrate shoot developmental transitions that recur during each seasonal cycle with the age‐related changes that occur over years of growth.     

Mutations in the slow skeletal muscle fiber myosin heavy chain gene (MYH7) cause laing early-onset distal myopathy (MPD1)

We previously linked Laing-type early-onset autosomal dominant distal myopathy (MPD1) to a 22-cM region of chromosome 14. One candidate gene in the region, MYH7, which is mutated in cardiomyopathy and myosin storage myopathy, codes for the myosin heavy chain of type I skeletal muscle fibers and cardiac ventricles. We have identified five novel heterozygous mutations--Arg1500Pro, Lys1617del, Ala1663Pro, Leu1706Pro, and Lys1729del in exons 32, 34, 35, and 36 of MYH7--in six families with early-onset distal myopathy. All five mutations are predicted, by in silico analysis, to locally disrupt the ability of the myosin tail to form the coiled coil, which is its normal structure. These findings demonstrate that heterozygous mutations toward the 3' end of MYH7 cause Laing-type early-onset distal myopathy. MYH7 is the fourth distal-myopathy gene to have been identified.     

Modulation of the endocannabinoid system in viable and non-viable first trimester pregnancies by pregnancy-related hormones

Background

In early pregnancy, increased plasma levels of the endocannabinoid anandamide (AEA) are associated with miscarriage through mechanisms that might affect the developing placenta or maternal decidua.

Methods

In this study, we compare AEA levels in failed and viable pregnancies with the levels of the trophoblastic hormones (beta-human chorionic gonadotrophin (beta-hCG), progesterone (P4) and (pregnancy-associated placental protein-A (PAPP-A)) essential for early pregnancy success and relate that to the expression of the cannabinoid receptors and enzymes that modulate AEA levels.

Results

The median plasma AEA level in non-viable pregnancies (1.48 nM; n = 20) was higher than in viable pregnancies (1.21 nM; n = 25; P = 0.013), as were progesterone and beta-hCG levels (41.0 vs 51.5 ng/mL; P = 0.052 for P4 and 28,650 vs 6,560 mIU/L; P = 0.144 for beta-hCG, respectively, but were not statistically significant). Serum PAPP-A levels in the viable group were approximately 6.8 times lower than those in the non-viable group (1.82 vs 12.25 mg/L; P = 0.071), but again these differences were statistically insignificant. In the spontaneous miscarriage group, significant correlations between P4 and beta-hCG, P4 and PAPP-A and AEA and PAPP-A levels were observed. Simultaneously, immunohistochemical distributions of the two main cannabinoid receptors and the AEA-modifying enzymes, fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD), changed within both the decidua and trophoblast.

Conclusions

The association of higher AEA levels with early pregnancy failure and with beta-hCG and PAPP-A, but not with progesterone concentrations suggest that plasma AEA levels and pregnancy failure are linked via a mechanism that may involve trophoblastic beta-hCG, and PAPP-A, but not, progesterone production. Although the trophoblast, decidua and embryo contain receptors for AEA, the main AEA target in early pregnancy failure remains unknown.     

Evidence that bears are induced ovulators

The objective of this study was to determine if black bears are induced ovulators. We conducted a single experiment with two replicates; each replicate was divided into two arms: females exposed to male bears and females without male exposure. We used laparoscopy to examine ovaries for corpora lutea and measured serum progesterone concentrations. Six of the seven isolated females failed to ovulate, while seven of the eight females exposed to males produced one to four corpora lutea. Furthermore, isolated females had significantly lower progesterone concentrations than females exposed to males. Thus, our data suggest that the American black bear is an induced ovulator. These results may aid biologists in their efforts to reproduce ursids in controlled environments.