Production and characterization of a monoclonal antibody able to discriminate galectin-1 from galectin-2 and galectin-3

Antisera raised against galectin-1 exhibit crossreactivities with other galectins or related molecules. In order to overcome this problem, a monoclonal antibody to human brain galectin-1 was obtained by selecting clones without reactivity toward galectin-3. This mAb specifically bound galectin-1 of various animal origins but neither galectin-2 nor galectin-3. Western-blotting analysis of soluble human brain extracts after 2D gel electrophoresis revealed only the two most acidic isoforms of galectin-1. The ability of this mAb to bind galectin-1/asialofetuin complexes indicates that its epitope is not localized in the carbohydrate recognition domain of galectin-1. This particularity induces with efficiency its monospecificity.      

An anchored restriction-mapping approach applied to the genetic analysis of the Anopheles gambiae malaria vector complex 1

We introduce here a simple approach for rapidly determining restriction maps for a number of regions of a genome; this involves "anchoring" a map with a rare restriction site (in this case the seldom-cutting EagI) followed by partial digestion of a frequent-cutting enzyme (e.g., Sau 3A). We applied this technology to five species of the Anopheles gambiae complex. In a single Southern blot we obtained about a 15-kb restriction map each for the mtDNA, rRNA gene, and a scnDNA region for each of five species. Phylogenetic analyses of these regions yield trees at odds with the more traditional chromosome inversion-based trees. The value of the approach for systematic purposes is the ease with which several large, independent regions of the genome can be quickly assayed for molecular variation.      

Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate

The present study was carried out to evaluate the effects of different activation protocols, enucleation methods, and culture media on the development of parthenogenetic and nuclear transfer (NT) rabbit embryos. Electroporation of 25 mM inositol 1,4, 5-trisphosphate (IP3) in calcium- and magnesium-free PBS immediately induced a single intracellular calcium transient in 6 out of 14 metaphase II-stage rabbit oocytes evaluated during a 10-min recording period. The percentage of oocytes treated with IP3 followed by 6-dimethylaminopurine (IP3 + DMAP) that cleaved (83.9%) and reached the blastocyst stage (50%) was significantly higher (p < 0.05) than those activated with multiple pulses (61.6% and 30.1%, respectively) or treated with ionomycin + DMAP (52.9% and 5.7%, respectively). Development of IP3 + DMAP-activated rabbit oocytes and in vivo-fertilized zygotes in different culture media was studied. Development of activated oocytes to the blastocyst stage in Earle's balanced salt solution (EBSS) supplemented with MEM nonessential amino acids, basal medium Eagle amino acids, 1 mM L-glutamine, 0.4 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (EBSS-complete) (40.6%) was significantly higher (p < 0.05) than those that developed in either Dulbecco's Modified Eagle's medium (DMEM)/RPMI + 10% FBS (15.5%) or CR1aa + 10% FBS (4%) medium. In addition, 100% of in vivo-fertilized rabbit zygotes developed to the blastocyst stage in EBSS-complete. A third set of experiments was carried out to study the efficiency of blind versus stained (Hoechst 33342) enucleation of oocytes. Twenty-nine of 48 blind enucleated and IP3 + DMAP-activated oocytes cleaved (60.4%), and 15 (31.2%) subsequently reached the blastocyst stage, whereas 9 of 52 oocytes enucleated using epifluorescence (17.3%) cleaved, and none of these reached the blastocyst stage. When the above parameters that yielded the highest blastocysts were combined in an NT experiment using adult rabbit fibroblast nuclei, 72.2% (39 of 54) of the fused nuclear transplant embryos cleaved and 29.6% (16 of 54) reached the blastocyst stage.     

The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts

Arthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra-terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA-seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT-qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFβ1-induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA-mediated depletion of BRD4. Together, these data reveal that BRD4-mediated epigenetic events support TGFβ1-mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.     

Gait characteristics of patients with knee osteoarthritis.

The knee kinematics and kinetics of 139 patients (47 males and 92 females) with Grade II knee osteoarthritis (OA) were measured during level walking, stair ascent and stair descent. There was no significant difference in knee motion between the patients and normal subjects. The patients with knee OA had a significantly reduced internal knee extensor moment compared to normal subjects. This difference reflects the patient's compensation to reduce the knee joint loading. Further, subjects with OA and a higher body mass index have a lower knee extensor moment. The female subjects had significantly greater knee flexion and a greater knee extensor moment. This gender difference may partially explain the increased prevalence of OA in females. Most tests of OA treatments are assessed by criteria that do not reflect functional activities. This study demonstrates that objective gait analysis can be used to document gait adaptations used by patients with knee OA.     

The genetic architecture of fitness in a seed beetle: assessing the potential for indirect genetic benefits of female choice

Background  

Quantifying the amount of standing genetic variation in fitness represents an empirical challenge. Unfortunately, the shortage of detailed studies of the genetic architecture of fitness has hampered progress in several domains of evolutionary biology. One such area is the study of sexual selection. In particular, the evolution of adaptive female choice by indirect genetic benefits relies on the presence of genetic variation for fitness. Female choice by genetic benefits fall broadly into good genes (additive) models and compatibility (non-additive) models where the strength of selection is dictated by the genetic architecture of fitness. To characterize the genetic architecture of fitness, we employed a quantitative genetic design (the diallel cross) in a population of the seed beetle Callosobruchus maculatus, which is known to exhibit post-copulatory female choice. From reciprocal crosses of inbred lines, we assayed egg production, egg-to-adult survival, and lifetime offspring production of the outbred F1 daughters (F1 productivity).