Murine progesterone receptor exists predominantly as the 83-kilodalton 'A' form

Progesterone receptors (PgR) are known to exist in two molecular forms commonly designated as 'A' and 'B' forms, and the relative ratio of these two forms has been shown to vary among species. Although the rodent systems were some of the earliest experimental systems used to examine the regulation of PgR, as yet very little is known concerning the molecular composition of PgR in this species. Accordingly, to define the relative ratio of 'A' and 'B' forms in murine PgR, we have analyzed tissue extracts from normal, ovariectomized, and estradiol treated animals by photoaffinity labeling and immunoblotting techniques using a variety of anti-PgR antibodies. Under all experimental conditions, two forms of PgR with approximate molecular weights of 115 kDa ('B' form) and 83 kDa ('A' form) were found. In all tissues examined, the 83 kDa 'A' form was predominant, and this was independent of the hormonal status of the animal and different buffers used to prepare tissue extracts. In uterus the ratio of 'A' to 'B' was 3:1, in vagina it was 2:1, and in mammary glands it more closely resembled the uterus. This leads us to conclude that murine PgR exists predominantly as the 83-kDa 'A' form which may represent a general characteristic of rodent PgR. In this species there may also be some tissue specificity with regard to the absolute ratio of the two forms of PgR.     

Physical Activity Energy Expenditure of Adolescents in India

Physical activity (PA) has rarely been quantified in adolescent populations undergoing economic transition; therefore relationships with disease still remain uncertain. This study assessed whether absolute PA energy expenditure (PAEE), PAEE/kg, and PAEE/kg_FFM could be accurately estimated using accelerometry and a questionnaire in Indian adolescents and how these values compared to those of other populations. PAEE was assessed using doubly labeled water (DLW) in 30 adolescents from Chennai, India, over seven consecutive days, simultaneous with the measurement of PA using accelerometry and a previous‐week recall questionnaire. Accelerometry counts (regression analysis) and questionnaire data were used to estimate PAEE; estimates were cross‐validated using the Bland‐Altman method. Accelerometry data and DLW‐derived PAEE were visually compared to values from four North American and European populations. For boys, 49% of the variance in DLW‐derived PAEE was explained with an equation including accelerometry counts and fat‐free mass (FFM). Questionnaire‐derived estimates did not contribute to the explained variance in DLW derived PAEE. The group‐level PA of these Indian adolescents was successfully assessed using accelerometry, but not questionnaire. DLW‐derived PAEE/kg_FFM (mean (s.d.): 53.0 (27.5) kJ/kg_FFM/day) was lower in this group than other adolescent populations in Europe and similar to those in North America. Additionally, four boys and none of the girls accumulated ≥60 min/day of accelerometry‐derived moderate intensity activity, indicating low levels of PAEE and PA in these adolescents. Further research is necessary to investigate the association between PA and health outcomes in Indian adolescents.     

Development of a Tiered Multilocus Sequence Typing Scheme for Members of the Lactobacillus acidophilus Complex

Members of the Lactobacillus acidophilus complex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp60 gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of the L. acidophilus complex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection of Lactobacillus species.     

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells

In this study we used differentiated adult human upcyte^® cells for the in vitro generation of liver organoids. Upcyte^® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte^® process). Proliferating upcyte^® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte^® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.     

The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study

IntroductionWe designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis.MethodsTwenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed.ResultsThe OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites.ConclusionThe peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0753-8) contains supplementary material, which is available to authorized users.     

Computational approaches to understanding protein aggregation in neurodegeneration

The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer＇s disease, Parkinson＇s disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggre- gation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute signifi- cantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both aU-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders.