An intronic G run within HIV-1 intron 2 is critical for splicing regulation of vif mRNA

Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.

Flexible selection of a single treatment incorporating short-term endpoint information in a phase II/III clinical trial

Seamless phase II/III clinical trials in which an experimental treatment is selected at an interim analysis have been the focus of much recent research interest. Many of the methods proposed are based on the group sequential approach. This paper considers designs of this type in which the treatment selection can be based on short-term endpoint information for more patients than have primary endpoint data available. We show that in such a case, the familywise type I error rate may be inflated if previously proposed group sequential methods are used and the treatment selection rule is not specified in advance. A method is proposed to avoid this inflation by considering the treatment selection that maximises the conditional error given the data available at the interim analysis. A simulation study is reported that illustrates the type I error rate inflation and compares the power of the new approach with two other methods: a combination testing approach and a group sequential method that does not use the short-term endpoint data, both of which also strongly control the type I error rate. The new method is also illustrated through application to a study in Alzheimer's disease. © 2015 The Authors. Statistics in Medicine Published by John Wiley & Sons Ltd.

Effects of forage source and extruded linseed supplementation on methane emissions from growing dairy cattle of differing body weights

Changes in diet carbohydrate amount and type (i.e., starch vs. fiber) and dietary oil supplements can affect ruminant methane emissions. Our objectives were to measure methane emissions, whole-tract digestibility, and energy and nitrogen utilization from growing dairy cattle at 2 body weight (BW) ranges, fed diets containing either high maize silage (MS) or high grass silage (GS), without or with supplemental oil from extruded linseed (ELS). Four Holstein-Friesian heifers aged 13 mo (BW range from start to finish of 382 to 526 kg) were used in experiment 1, whereas 4 lighter heifers aged 12 mo (BW range from start to finish of 292 to 419 kg) were used in experiment 2. Diets were fed as total mixed rations with forage dry matter (DM) containing high MS or high GS and concentrates in proportions (forage:concentrate, DM basis) of either 75:25 (experiment 1) or 60:40 (experiment 2), respectively. Diets were supplemented without or with ELS (Lintec[AU1: Add manufacturer name and location.]; 260 g of oil/ kg of DM) at 6% of ration DM. Each experiment was a 4 × 4 Latin square design with 33-d periods, with measurements during d 29 to 33 while animals were housed in respiration chambers. Heifers fed MS at a heavier BW (experiment 1) emitted 20% less methane per unit of DM intake (yield) compared with GS (21.4 vs. 26.6, respectively). However, when repeated with heifers of a lower BW (experiment 2), methane yield did not differ between the 2 diets (26.6 g/kg of DM intake). Differences in heifer BW had no overall effect on methane emissions, except when expressed as grams per kilogram of digestible organic matter (OMD) intake (32.4 vs. 36.6, heavy vs. light heifers). Heavier heifers fed MS in experiment 1 had a greater DM intake (9.4 kg/d) and lower OMD (755 g/kg), but no difference in N utilization (31% of N intake) compared with heifers fed GS (7.9 kg/d and 799 g/kg, respectively). Tissue energy retention was nearly double for heifers fed MS compared with GS in experiment 1 (15 vs. 8% of energy intake, respectively). Heifers fed MS in experiment 2 had similar DM intake (7.2 kg/d) and retention of energy (5% of intake energy) and N (28% of N intake), compared with GS-fed heifers, but OMD was lower (741 vs. 765 g/kg, respectively). No effect of ELS was noted on any of the variables measured, irrespective of animal BW, and this was likely due to the relatively low amount of supplemental oil provided. Differences in heifer BW did not markedly influence dietary effects on methane emissions. Differences in methane yield were attributable to differences in dietary starch and fiber composition associated with forage type and source.

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.

Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.

A retrospective study of zygomatico-orbital complex and/or zygomatic arch fractures over a 71-month period

Background/Aim: The aim of this retrospective study was to evaluate the epidemiology, treatment, and complications of zygomatico-orbital complex (ZOC) and/or zygomatic arch (ZA) fractures either associated with other facial fractures or not over a 71-month period. Material and methods: This survey was performed in three hospitals of Ribeirao Preto in Sao Paulo, Brazil, from August 2002 to July 2008. The records of 1575 patients with facial trauma were reviewed. There were 140 cases of ZOC and ZA fractures either associated with other facial fractures or not. Data regarding gender, age, race, addictions, day of trauma, etiology, signs and symptoms, oral hygiene condition, day of initial evaluation, hospital admission, day of surgery, surgery approach, pattern of fractures, treatment performed, post-operative antibiotic therapy, day of hospital discharge, and post-operative complications were collected. The data were subjected to descriptive statistical analyses. Results: The most frequent fractures affected Caucasian men and occurred during the fourth decade of life. The most frequent etiology was traffic accident, and symptoms and signs included pain and edema. Type I fractures were the main injury observed, and the treatment of choice was always rigid internal fixation. Post-operative antibiotic therapy was solely employed when there was an indication. Complications were observed in 13.1% of the cases. Conclusions: The treatment protocol yielded suitable post-operative results and also showed success rates comparable to published data around the world.

Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: calvarial bone

Objectives Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa beta ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P < 0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. To cite this article:Pedrosa Jr WF, Okamoto R, Faria PEP, Arnez MFM, Xavier SP, Salata LA. Immunohistochemical, tomographic and histological study on onlay bone grafts remodeling. Part II: calvarial bone.Clin. Oral Impl. Res. 20, 2009; 1254-1264.doi: 10.1111/j.1600-0501.2009.01747.x.

Immunohistochemical, tomographic and histological study on onlay iliac grafts remodeling

The information concerning the molecular events taking place in onlay bone grafts are still incipient. The objective of the present study is to correlate the effects of perforation of resident bone bed on (1) the timing of onlay autogenous graft revascularization; (2) the maintenance of volume/density of the graft (assessed through tomography); and (3) the occurrence of bone remodeling proteins (using immunohistochemistry technique) delivered in the graft. Thirty-six New Zealand White rabbits were subjected to iliac crest onlay bone grafting on both sides of the mandible. The bone bed was drill-perforated on one side aiming at accelerating revascularization, whereas on the other side it was kept intact. After grafts fixation and flaps suture all animals were submitted to tomography on both mandible sites. Six animals were sacrificed, respectively, at 3, 5, 7, 10, 20 and 60 days after surgery. A second tomography was taken just before sacrifice. Histological slides were prepared from each grafted site for both immunohistochemistry analysis [osteopontin, osteocalcin, type I collagen and vascular endothelial growth factor (VEGF) anti-bodies] and histometric analysis. The values on bone volume measured on tomography showed no statistic significance (P >= 0.05) between perforated and intact sites. Grafts placed on perforated beds showed higher bone density values compared with non-perforated ones at 3 days (P <= 0.05). This correlation was inverted at 60 days postoperatively. The findings from VEGF labeling revealed a tendency for earlier revascularization in the perforated group. The early revascularization of bone grafts accelerated the bone remodeling process (osteocalcin, type I collagen and osteopontin) that led to an increased bone deposition at 10 days. The extended osteoblast differentiation process at intermediate stages in the perforated group cooperated for a denser bone at 60 days.

Diabetes mellitus-related morphoquantitative changes in the celiac ganglion neurons of the dog

Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus. (C) 2007 Elsevier Ltd. All rights reserved.

Lifetime Overproduction of Circulating Angiotensin-(1-7) Attenuates Deoxycorticosterone Acetate-Salt Hypertension-Induced Cardiac Dysfunction and Remodeling

We evaluated the development of arterial hypertension, cardiac function, and collagen deposition, as well as the level of components of the renin-angiotensin system in the heart of transgenic rats that overexpress an angiotensin (Ang)-(1-7)-producing fusion protein, TGR(A1-7)3292 (TG), which induces a lifetime increase in circulating levels of this peptide. After 30 days of the induction of the deoxycorticosterone acetate (DOCA)-salt hypertension model, DOCA-TG rats were hypertensive but presented a lower systolic arterial pressure in comparison with DOCA-Sprague-Dawley (SD) rats. In contrast to DOCA-SD rats that presented left ventricle (LV) hypertrophy and diastolic dysfunction, DOCA-TG rats did not develop cardiac hypertrophy or changes in ventricular function. In addition, DOCA-TG rats showed attenuation in mRNA expression for collagen type I and III compared with the increased levels of DOCA-SD rats. Ang II plasma and LV levels were reduced in SD and TG hypertensive rats in comparison with normotensive animals. DOCA-TG rats presented a reduction in plasma Ang-(1-7) levels; however, there was a great increase in Ang-(1-7) (approximate to 3-fold) accompanied by a decrease in mRNA expression of both angiotensin-converting enzyme and angiotensin-converting enzyme 2 in the LV. The mRNA expression of Mas and Ang II type 1 receptors in the LV was not significantly changed in DOCA-SD or DOCA-TG rats. This study showed that TG rats with increased circulating levels of Ang-(1-7) are protected against cardiac dysfunction and fibrosis and also present an attenuated increase in blood pressure after DOCA-salt hypertension. In addition, DOCA-TG rats showed an important local increase in Ang-(1-7) levels in the LV, which might have contributed to the attenuation of cardiac dysfunction and prefibrotic lesions. (Hypertension. 2010;55:889-896.)

The connective tissue of the adductor canal - a morphological study in fetal and adult specimens

The adductor canal is a conical or pyramid-shaped pathway that contains the femoral vessels, saphenous nerve and a varying amount of fibrous tissue. It is involved in adductor canal syndrome, a claudication syndrome involving young individuals. Our objective was to study modifications induced by aging on the connective tissue and to correlate them to the proposed pathophysiological mechanism. The bilateral adductor canals and femoral vessels of four adult and five fetal specimens were removed en bloc and analyzed. Sections 12 mu m thick were obtained and the connective tissue studied with Sirius Red, Verhoeff, Weigert and Azo stains. Scanning electron microscopy (SEM) photomicrographs of the surfaces of each adductor canal were also analyzed. Findings were homogeneous inside each group. The connective tissue of the canal was continuous with the outer layer of the vessels in both groups. The pattern of concentric, thick collagen type I bundles in fetal specimens was replaced by a diffuse network of compact collagen bundles with several transversal fibers and an impressive content of collagen III fibers. Elastic fibers in adults were not concentrated in the thick bundles but dispersed in line with the transversal fiber system. A dynamic compression mechanism with or without an evident constricting fibrous band has been proposed previously for adductor canal syndrome, possibly involving the connective tissue inside the canal. The vessels may not slide freely during movement. These age-related modifications in normal individuals may represent necessary conditions for this syndrome to develop.

Morphological and quantitative study of ganglionated plexus of Calomys callosus trachea

Calomys callosus is a wild, native forest rodent found in South America. In Brazil, this species has been reported to harbour the parasitic protozoan Trypanosoma cruzi. The ganglionated plexus of this species was studied using whole-mount preparations of trachea that were stained using histological and histochemical methods. The histological methods were used to determine the position of the ganglia with respect to the trachea muscle and to determine the presence of elastic and collagen fibers. The histochemical method of NADH-diaphorase was used for morphometric evaluations of the plexus. The tracheal plexus lies exclusively over the muscular part of the organ, dorsal to the muscle itself. It varies in pattern and extent between animals. The average number of neurons was 279 and the cellular profile area ranged from 38.37 mu m(2) to 805.89 mu m(2). Acetylcholinesterase (AChE) histochemistry verified that both ganglia and single neurons lie along nerve trunks and are reciprocally interconnected with the plexus. Intensely AChE-reactive neurons were found to be intermingled with poorly reactive ones. Two longitudinal AChE-positive nerve trunks were also observed and there was a diverse number of ganglia along the intricate network of nerves interconnecting the trunks. A ganglion capsule of collagen and elastic fibers surrounding the neurons was observed. Under polarized light, the capsule appeared to be formed by Type I collagen fibers. (C) 2008 Elsevier B.V. All rights reserved.

Maternal diabetes affects specific extracellular matrix components during placentation

During embryo implantation, invasive trophoblast cells mediate embryo invasion into the decidualized stroma, forming a rich network of lacunae that connect the embryonic tissues to the maternal blood vessels. Placentation is probably guided by the composition and organization of the endometrial extracellular matrix. Certain pathological conditions that occur during pregnancy, including diabetes, have been linked to abnormal placental morphology and consequent fetal morbidity. We used immunoperoxidase techniques to identify members of the collagen, proteoglycan and glycoprotein families in the various compartments of the rat placenta and to determine whether experimentally induced diabetes affects placental morphology and alters the distribution of these molecules during pregnancy. Single injections of alloxan (40 mg kg(-1) i.v.) were used to induce diabetes on day 2 of pregnancy in Wistar rats. Placentas were collected on days 14, 17, and 20. Type I and III collagen, as well as the proteoglycans decorin and biglycan, were found to be distributed throughout the placentas of control and diabetic rats. In both groups, laminin expression decreased at the end of pregnancy. In contrast, fibronectin was detected in the labyrinth region of diabetic rats at all gestational stages studied, whereas it was detected only at term pregnancy in the placentas of control rats. These results show for the first time that some extracellular matrix molecules are modulated during placental development. However, as diabetic rats presented increased fibronectin deposition exclusively in the labyrinth region, we speculate that diabetes alters the microenvironment at the maternal-fetal interface, leading to developmental abnormalities in the offspring.

Mesenchymal Stem Cells Attenuate Renal Fibrosis Through Immune Modulation and Remodeling Properties in a Rat Remnant Kidney Model

Mesenchymal stem cells (MSCs) have regenerative properties in acute kidney injury, but their role in chronic kidney diseases is still unknown. More specifically, it is not known whether MSCs halt fibrosis. The purpose of this work was to investigate the role of MSCs in fibrogenesis using a model of chronic renal failure. MSCs were obtained from the tibias and femurs of male Wistar-EPM rats. Female Wistar rats were subjected to the remnant model, and 2 vertical bar x vertical bar 10(5) MSCs were intravenously administrated to each rat every other week for 8 weeks or only once and followed for 12 weeks. SRY gene expression was observed in female rats treated with male MSCs, and immune localization of CD73(+)CD90(+) cells at 8 weeks was also assessed. Serum and urine analyses showed an amelioration of functional parameters in MSC-treated animals at 8 weeks, but not at 12 weeks. Masson`s trichrome and Sirius red staining demonstrated reduced levels of fibrosis in MSC-treated animals. These results were corroborated by reduced vimentin, type I collagen, transforming growth factor beta, fibroblast specific protein 1 (FSP-1), monocyte chemoattractant protein 1, and Smad3 mRNA expression and alpha smooth muscle actin and FSP-1 protein expression. Renal interleukin (IL)-6 and tumor necrosis factor alpha mRNA expression levels were significantly decreased after MSC treatment, whereas IL-4 and IL-10 expression levels were increased. All serum cytokine expression levels were decreased in MSC-treated animals. Taken together, these results suggested that MSC therapy can indeed modulate the inflammatory response that follows the initial phase of a chronic renal injury. The immunosuppressive and remodeling properties of MSCs may be involved in the decreased fibrosis in the kidney. STEM CELLS 2009;27:3063-3073

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To Study this, pregnant once were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved Lip to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocyres, in addition to a slight delay in endochondral and intramembranous ossification. This Suggests that LIP to E 16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (similar to 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast. D2 mRNA expression increased significantly by E18.5 and markedly (similar to 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time Suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development. (c) 2008 Elsevier Inc. All rights reserved.

Role of MMP9 on Invadopodia Formation in Cells From Adenoid Cystic Carcinoma. Study by Laser Scanning Confocal Microscopy

Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type I matrix metalloproteinase (MT1-MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent-substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel-gelatin-FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1-MMR Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1-MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After I h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1-MMR Furthermore, MMP9 would be required in the initial steps of invadopodia formation. Microsc. Res. Tech. 73:99-108, 2010. (C) 2009 Wiley-Liss, Inc.