Cryo-EM Structure of AAV2 Rep68 bound to integration site AAVS1: Insights into the mechanism of DNA melting.

The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions required for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin and catalyzes the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. Here, we report the Cryo-EM structures of Rep68 bound to double-stranded DNA (dsDNA) containing the sequence of the AAVS1 integration site in different nucleotide-bound states. In the apo state, Rep68 forms a heptameric complex around DNA, with three Origin Binding Domains (OBDs) bound to the Rep Binding Site (RBS) sequence and three other OBDs forming transient dimers with them. The AAA+ domains form an open ring with no interactions between subunits and with DNA. We hypothesize the heptameric quaternary structure is necessary to load onto dsDNA. In the ATPγS-bound state, a subset of three subunits binds the nucleotide, undergoing a large conformational change, inducing the formation of intersubunit interactions interaction and interaction with three consecutive DNA phosphate groups. Moreover, the induced conformational change positions three phenylalanine residues to come in close contact with the DNA backbone, producing a distortion in the DNA. We propose that the phenylalanine residues can potentially act as a hydrophobic wedge in the DNA melting process.

Enhancing the shelf life of chevon Seekh Kabab using chitosan edible film and Cinnamomum zeylanicum essential oil.

Chevon Seekh Kabab is a popular meat product of India. However, due to high protein and moisture content it undergoes quick microbial spoilage and oxidative reactions leading to lower shelf life. The combination of chitosan edible film and cinnamon essential oil (CEO) was chosen to remediate this problem because of its antimicrobial and antioxidative effect. Control and chitosan edible film with CEO coated chevon Seekh Kabab samples were stored at 4 °C. The physicochemical (pH, TBARS, TVBN, moisture, colour), microbiological (APC, psychrophilic, coliform and Staphylococcal count) and sensory attributes were evaluated over a 30 days period. The maximum shelf life of 27 days was observed when 2% chitosan edible film with 0.3% CEO was coated over samples. A reduction in moisture, L* value, a* value and sensory scores along with an increase in pH, TVBN, TBARS, b* value and microbiological parameters were observed during the storage period. Reaction kinetics for the physicochemical and microbiological parameters was also established. The physicochemical, microbiological and sensory parameters were within prescribed limits till spoilage in the treated sample. This investigation may aid researchers working on scaling up of processing and preservation of Seekh Kabab.

Pre-treatment delay and out of pocket expenses by notified new tuberculosis patients in an Indian mega city.

BACKGROUND: Study was carried out to find out delay from onset of symptoms and out of pocket expenditure (OOPE) until initiation of anti-TB treatment (ATT) by new Tuberculosis (TB) patients registered in public health facilities in Bengaluru. METHODS: Notified patients (N = 228) selected purposively were interviewed at initiation of ATT regarding number and type of facilities visited and delay in initiating ATT. OOPE was elicited separately for in- and out-patient visits, towards consultation, purchase of medicines, diagnostic tests, transportation, hospitalization and food. Dissaving or money borrowed was ascertained. RESULTS: Two-thirds of participants were 15-44 years of age and 56% were males, mean annual household income was $4357. About 75% first visited a private health facility; 68% and 87% respectively were diagnosed and started on ATT in public sector after visiting an average of three facilities and after a mean delay of 68 days; the median delay was 44 days. Of mean OOPE of $402, 54% was direct medical expenditure, 5% non-medical direct and 41% indirect. OOPE was higher for Extra-pulmonary TB compared to PTB and when number of health facilities visited before initiating treatment was >3 compared to those who visited ≤3 and when the time interval between onset of symptoms and treatment initiation (total delay) was >28 days compared to when this interval was ≤28 days. About 20% suffered catastrophic expenditure; 34% borrowed money and 37% sold assets. CONCLUSION: Concerted efforts are needed to reduce delay and OOPE in pre-treatment period and social protection to account for indirect expenditure.

Tuberculosis diagnostic and treatment practices in private sector: Implementation study in an Indian city.

SETTING: Implementation study in private health facilities in an Indian metropolis. OBJECTIVES: Improve Tuberculosis (TB) care by private practitioners (PPs). METHODS: PPs from a defined city area were imparted short training in TB care and linkages made with public facilities; subsequent practices were recorded. RESULTS: Of 364 presumptive TB patient records, 70 (19.3%) did not conform to its definition. Of the conforming, 174 (59.2%) had presumptive pulmonary TB (PTB), 53 (18%) presumptive extra-pulmonary (EPTB) and 67 (24%) had both. Of conforming presumptive PTB, most underwent Chest X-ray and sputum examination in private laboratories. Tissue based diagnostics were not advised for most presumptive EPTB patients. Of 101 cases diagnosed with TB, 82% were new, 23% known diabetic and 4.7% human immune deficiency virus (HIV) reactive out of 64 tested. Most were notified and initiated treatment within 15 days of diagnosis. One-fourth was prescribed standard treatment regimen and treatment was not directly observed for most. One third was initial defaulters or lost during treatment; 62% of PTB and 46% EPTB cases initiated on treatment in private were successfully treated. Of successfully treated PTB cases, 61% had undergone follow-up sputum examination. CONCLUSION: Much intensified support mechanisms are needed to improve TB care in private sector.

Polymorphism distribution of RYR1, PRKAG3, HFABP, MYF-5 and MC4R genes in crossbred pigs.

This study was designed to screen the crossbred pigs for SNPs in five candidate genes, associated with pork quality traits and to differentiate their genotypes by PCR-RFLP. The results indicated that genotypes of crossbred pigs were NN (90%) and Nn (10%) for RYR1; RR (83%) and QR (17%) for PRKAG3; HH (98%), Hh (1%) and hh (1%) for HFABP; DD (99%) and CD (1%) for MYF-5; and AG (57%), GG (26%) and AA (17%) for MC4R SNPs, respectively. Allelic frequencies for five SNPs {RYR1 (1843C>T), PRKAG3 (c.599G>A), HFABP (c.1322C>T), MYF-5 (c.1205A>C) and MC4R (c.1426A>G)} were 0.95 and 0.05 (N/n), 0.08 and 0.92 (Q/R), 0.99 and 0.01 (H/h), 0.00 and 1.00 (C/D) and 0.45 and 0.55 (A/G), respectively. The effect of RYR1 (1843C>T) SNP was significant on pH45 (P < 0.05), pH24 (P < 0.05) and protein % (P < 0.05). The PRKAG3 (c.599G>A) and MC4R (c.1426A>G) SNP had significant association with dressing percentages. The results revealed that RYR1, PRKAG3 and MC4R SNPs may be used in marker associated selection for pork quality traits in crossbred pigs.

Acute liver failure with amiodarone infusion: A case report and systematic review.

WHAT IS KNOWN AND OBJECTIVE: Amiodarone, a commonly used class III antiarrhythmic agent notable for a relatively long half-life of up to 6 months and its pronounced adverse effect profile, is used for both acute and chronic management of cardiac arrhythmias. Chronic use of amiodarone has been associated with asymptomatic hepatotoxicity; however, acute toxicity is thought to be uncommon. There are only six reported cases of acute liver failure (ALF) secondary to amiodarone. In all these cases the outcome of death during the same hospitalization resulted. We aimed to report the only case of acute liver failure secondary to amiodarone infusion in the existing literature where the patient survived. CASE SUMMARY: A 79-year-old woman admitted with atrial flutter was being treated with intravenous (IV) amiodarone when she abruptly developed coagulopathy, altered mental status and liver enzyme derangement. She was diagnosed with acute liver failure (ALF) secondary to an amiodarone adverse drug reaction, with a calculated score of seven on the Naranjo adverse drug reaction probability scale. Amiodarone was immediately withheld, and N-acetylcysteine (NAC) was initiated. Clinical improvement was seen within 48 hours of holding the drug and within 24 hours of initiating NAC. On post-hospital follow-up visit she was reported to have complete recovery. WHAT IS NEW AND CONCLUSION: This report emphasizes the importance of monitoring liver enzymes and mental status while a patient is being administered IV amiodarone. N-acetylcysteine administration may have possibly contributed to the early and successful recovery from ALF in our patient. To date, she is the only patient in the existing literature who has been reported to survive ALF secondary to amiodarone administration.

Molecular epidemiology of Crimean-Congo haemorrhagic fever virus in India.

Crimean-Congo haemorrhagic fever (CCHF) is an emerging zoonotic disease in India which is prevalent in neighbouring countries. CCHF virus (CCHFV) is a widespread tick-borne virus which is endemic in Africa, Asia, Eastern Europe and the Middle East. In the present study, samples of clinically suspected human cases from different areas of northern-western India were tested for the presence of CCHFV by RT-PCR through amplification of nucleocapsid (N) gene of CCHFV. Positive samples were sequenced to reveal the prevailing CCHFV genotype(s) and phylogenetic relatedness. A phylogenetic tree revealed the emergence of diverse strains in the study region showing maximum identity with the Pakistan, Afghanistan and Iran strains, which was different from earlier reported Indian strains. Our findings reveal for the first time the emergence of the Asia 1 group in India; while earlier reported CCHFV strains belong to the Asia 2 group.

Microparticles and their emerging role in cancer multidrug resistance.

Drug resistance is a major obstacle to the successful treatment of cancer as tumor cells either fail to reduce in size following chemotherapy or the cancer recurs after an initial response. The phenomenon of multidrug resistance (MDR) is particularly problematic as it involves the simultaneous resistance to numerous chemotherapeutics of different classes. MDR is predominantly attributed to the overexpression of efflux transporters such as P-glycoprotein (P-gp) and the Multidrug Resistance-Associated Protein 1 (MRP1). P-gp and MRP1 are members of the ATP Binding Cassette (ABC) superfamily of transporters and are capable of effluxing many chemotherapeutics out of cancer cells, allowing them to survive the toxic insult. Numerous strategies have been developed over the years to circumvent MDR. Of these, the discovery and implementation of P-gp and MRP1 inhibitors have been most extensively studied. However, these inhibitors have not been able to be used clinically. While research continues in this area, it must also be acknowledged that other avenues must be explored. Recently, the novel 'non-genetic' acquisition of P-gp-mediated MDR by microparticles (MPs) has been reported. MPs are vesicles 0.1-1µm in diameter that are released via plasma membrane blebbing. They are important mediators of inflammation, coagulation and vascular homeostasis. In addition to surface P-gp protein, MPs also carry various nucleic acid species as cargo. This 'non-genetic' intercellular transfer provides an alternative pathway for the cellular acquisition and dissemination of traits and implicates MPs as important mediators in the spread of MDR and provides a novel pathway for the circumvention of MDR.

Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

Repeatability of 2-wave and 3-wave patterns of ovarian follicular development during the bovine estrous cycle.

Characteristics predictive of a 2-wave versus 3-wave pattern of ovarian follicular development during the interovulatory interval (IOI) were examined by ultrasonographic monitoring of 91 IOIs from 31 beef heifers. Repeatability of the wave pattern within individuals and the effects of season and age were determined using a subset of 75 IOIs from 15 heifers examined for multiple IOIs. The 2-wave pattern was detected in 62 of 91 (68%) IOIs, and the 3-wave pattern was detected in 29 of 91 (32%) IOIs. The preponderance of the 2-wave versus 3-wave pattern (P<0.05) was not influenced by season (P=0.61) but was even greater in the more mature age group (P=0.02). The majority of IOIs or=22 d was of the 3-wave pattern (78%; P<0.05). The proportion of nonalternating patterns (repeatability) was more than twofold greater than the proportion of alternating patterns (70% vs. 30%; P<0.01). This relationship was consistent among seasons (P<0.01) and even more marked in the more mature age group (P=0.01). Emergence and follicular dominance of Wave 2 were delayed (P<0.01), and the onset of corpus luteum regression was earlier (P<0.01) in 2-wave versus 3-wave IOI. In conclusion, the duration of the IOI was predictive of the wave pattern, and the pattern was repeatable within individuals. Factors influencing the period of follicular dominance of Wave 1 in 2-wave versus 3-wave IOI may be responsible for regulating the wave pattern and may be associated with heifer maturity or relative nutritional demand during the postpubertal period. The impact of greater follicular attrition recorded in 3-wave versus 2-wave IOI on ovarian depletion and reproductive senescence is worthy of critical evaluation.

HELLP syndrome--a pregnancy disorder with poor prognosis.

HELLP syndrome is a pregnancy-specific disorder defined by hemolysis, elevated liver enzymes and low platelet count that is found in parturients, more frequent in older multiparas. It is frequently associated with severe preeclampsia or eclampsia, but can also be diagnosed in the absence of these disorders. The etiology of HELLP syndrome is unknown, and the pathogenesis of this disorder (including the hepatological manifestations) is not fully understood. The most widely accepted hypotheses are: a change in the immune feto-maternal balance, platelet aggregation, endothelial dysfunction, arterial hypertension and an inborn error of the fatty acid oxidative metabolism. Hepatic involvement occurs by intravascular fibrin deposition and hypovolemia. Serum LDH and platelet count are the two most important clinical tools for disease assessment. LDH reflects both the extent of hemolysis and hepatic dysfunction. Maternofetal complications cause a 7.0-70.0% perinatal mortality rate and a 1.0-24.0% maternal mortality rate. The recognition of HELLP syndrome and an aggressive multidisciplinary approach and prompt transfer of these women to obstetric centers with expertise in this field are required for the improvement of materno-fetal prognosis.

Progress in understanding ovarian follicular dynamics in cattle.

The study of follicular dynamics began in the mid-20th century, but progress has been particularly rapid in the last two decades through the use of tools that have enabled serial, non-invasive examination. A brief overview of early oogenesis and folliculogenesis is provided as a backdrop to the evolution of our understanding of follicular dynamics during the bovine estrous cycle. Studies to date support the concept that the pair of ovaries acts as a single unit and influences follicular development primarily via systemic endocrine routes involving ovarian and uterine products, the gonadotropins, and their receptors. Dominant and subordinate follicles pass through growing, static and regressing phases that have distinct morphologic and biochemical characteristics; these changes are the basis of efforts focused on diagnosing and manipulating follicular status. An update of research progress highlights recent findings on the repeatability (predictability) within individuals of follicle recruitment and wave pattern (two- versus three-wave cycles), the relationship between oocyte competence and follicular status, and the dynamics of small follicles. Recent studies documented that wave emergence and follicular dominance are apparent earlier than previously reported, and on the basis of periodic endogenous FSH surges and the presence of FSH receptors, the hypothesis that follicles become progressively entrained to waves from the earliest stages of development is introduced. Lastly, recent studies comparing old cows and their young daughters provide a new understanding of the effects of aging on gonadotropins and ovarian steroids, follicular dynamics, ovarian response to synchronization, superstimulation, and oocyte competence.

Development and evaluation of an in vitro isolation of street rabies virus in mouse neuroblastoma cells as compared to conventional tests used for diagnosis of rabies.

In vitro isolation of rabies virus using mouse neuroblastoma cells (MNA) was evaluated. The sensitivity and reliability of in vitro procedure was performed in comparison with mouse inoculation test (MIT), the in vivo method of virus isolation, direct fluorescent antibody test (FAT) and Sellers staining. Of the 33 animal brain samples tested, 24 (72.72%) were positive by MIT. Sensitivity of Sellers stain, FAT and rapid tissue culture infection test (RTCIT) was found to be 54.16, 100 and 91.6% respectively. Concordance of Sellers stain, FAT, RTCIT with MIT was found to be 66.6, 100 and 93.93% respectively. Two samples which were positive by FAT and MIT showed gross contamination in cell lines, which is one of the drawbacks of RTCIT. However, rabies virus could be isolated in MNA cells from two of the eight human cerebrospinal fluid (CSF) samples from clinico-epidemiologically suspected cases of rabies. Both MIT and FAT showed negative results in the two CSF samples. RTCIT appears to be a fast and reliable alternative to MIT and holds promise in antemortem diagnosis of rabies, which is otherwise, a challenging task for a reference laboratory.

Effect of Fertilizers and Neem Cake Amendment in Soil on Spore Germination of Arthrobotrys dactyloides.

Application of fertilizers such as urea, diammonium phosphate (DAP) and muriate of potash in soil adversely affected the spore germination of Arthrobotrys dactyloides. Amendment of soil with urea at the concentrations of 1.0%, 0.5% and 0.1% completely inhibited spore germination and direct trap formation on the conidium, whereas muriate of potash delayed and reduced the spore germination even at the lowest concentration. DAP also inhibited spore germination at 1.0% concentration, while at lower concentration the percentage of spore germination was reduced. Application of neem cake at the concentration of 0.5% also inhibited spore germination after 24 h of amendment. The inhibitory effect of neem cake was reduced after 15 days of amendment, while after 30 days after amendment the inhibitory effect was completely lost and the spore germinated by direct trap as in unamended soil. Nematodes were not attracted to ungerminated spores after 24 h of amendment. After 15 days of amendment nematodes were attracted to agar blocks containing fewer germinated spores after 24 h of incubation but after 48 h of incubation large number of nematodes were attracted and trapped by the germinated spores with direct traps. After 30 days of amendment, larger number of nematodes were attracted and trapped by direct traps.

Screening of Different Media and Substrates for Cultural Variability and Mass Culture of Arthrobotrys dactyloides Drechsler.

Variability in growth and sporulation of five isolates of Arthrobotrys dactyloides was studied on five agar, 6 bran and 5 grain media. Potato dextrose agar (PDA) supported maximum growth of isolate A, C and E, while growth of isolate B and D was significantly lower on this medium. On Czapek's agar and yeast glucose agar media the differentiation in the isolates in relation to growth was poor than PDA. The other two media showed much poorer differentiation. On Czapek's agar medium, sporulation was recorded in isolate B only, whereas other isolates showed rare sporulation. Among the bran media, pea bran agar medium supported maximum growth of all the isolates except isolate B. Gram and rice bran agar media were next best. However, the growth of isolate B on the gram bran agar medium was more or less equal as other isolates. On pigeon pea bran agar medium, isolate E failed to grow while other isolates recorded poor growth. On lentil bran agar medium, only isolate B and D recorded little growth, whereas other isolates failed to grow. All the isolates recorded good sporulation on bran agar media except pigeon pea and lentil bran agar media. The grain agar media supported moderate to very good growth of all the isolates. In general isolate B remained slow growing on these media except gram grain and sorghum grain agar media on which growth of this isolate was comparable to other isolates. Sporulation in general, was good on all the grain agar media. Among different substrates screened, barley grain and pea bran were found superior to others for mass culture of isolate A of A. dactyloides.

Developmental pattern of small antral follicles in the bovine ovary.

The study was designed to characterize the developmental pattern of 1- to 3-mm follicles and to determine the stage at which the future dominant follicle first attains a size advantage among its cohorts. In experiment 1, heifers (n = 18) were examined every 24 h by transrectal ultrasonography for one interovulatory interval (IOI). In experiment 2, cows (n = 9) were examined every 6 h from 5 to 13 days after ovulation to monitor precisely the diameter changes of individual follicles >/=1 mm during emergence of wave 2. Results revealed a change over days (P < 0.05) in the number of 1- to 3-mm follicles, with a maximum (P < 0.05) 1 or 2 days before wave emergence (conventionally defined as the time when the dominant follicle is first detected at 4 mm), followed 3-4 days later by a maximum (P < 0.05) in the number of >/=4-mm follicles. The profiles of small (1-3 mm) and large (>/=4-mm) follicles were inversely proportional (r = -0.79; P = 0.01). The profile of the number of 1- to 3-mm follicles during wave emergence was similar (P = 0.63) between waves in two-wave IOI, but differed (P < 0.01) among waves in three-wave IOI as a result of a greater number of follicles in the ovulatory wave (P < 0.04). As well, the number of follicles in the ovulatory wave tended to be greater (P < 0.06) in three-wave IOI than in two-wave IOI. The future dominant follicle was first identified at a diameter of 1 mm and emerged 6-12 h earlier than the first subordinate follicle (P < 0.01). After detection of the dominant follicle at 1 mm (0 h), its diameter differed from that of the first and second subordinate follicles at 24 h (P = 0.04) and 12 h (P = 0.01), when the dominant follicle was 2.4 +/- 0.17 mm and 1.7 +/- 0.14 mm, respectively. The growth rate of the dominant follicle differed from that of the first and second subordinate follicles at 120 h (P = 0.03) and 108 h (P = 0.02), when the dominant follicle was 9.5 +/- 0.30 mm and 8.8 +/- 0.49 mm, respectively. Emergence of the future dominant (r = 0.71), first (r = 0.73), and second (r = 0.76) subordinate follicles was temporally associated (P < 0.01) with a rise in circulating concentrations of FSH. Transient, nocturnal elevations in plasma FSH concentration were followed within 6 h by an increase in the growth rate of 1- to 3-mm follicles. We conclude that 1) 1- to 3-mm follicles develop in a wave-like manner in association with surges in plasma concentrations of FSH, 2) 1- to 3-mm follicles are exquisitely responsive to transient elevations in FSH, and 3) selection of the dominant follicle is manifest earlier than previously documented and is characterized by a hierarchical progression over a period encompassing the entire FSH surge (5 days).

Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells.

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.

Activation of peroxisome proliferator-activated receptor-gamma pathway inhibits osteoclast differentiation.

The nuclear receptor and transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), regulates the activity of other transcription factors in the adipogenic differentiation and inflammatory response pathways. We examined the possible function of the PPAR-gamma pathway in osteoclast (Ocl) formation from CD34(+) hematopoietic stem cells (CD34(+) HSCs), using a co-culture system comprised of human mesenchymal stem cells (hMSCs) and CD34(+) HSCs, both derived from bone marrow. Ocl formation in this co-culture system is enhanced by the addition of exogenous osteoprotegerin ligand (OPGL), an essential Ocl differentiation factor, and macrophage-colony stimulating factor (M-CSF). The data indicate that soluble OPGL (sOPGL) and M-CSF stimulate Ocl formation in the co-cultures up to 4-fold compared with CD34(+) HSCs alone treated with sOPGL and M-CSF. CD34(+) HSCs, but not hMSCs, express PPAR-gamma, and 15-deoxy-Delta(12, 14)-prostaglandin-J2 (15d-PG-J2), a PPAR-gamma agonist, completely blocked the effects of sOPGL and M-CSF on Ocl formation and activity. The inhibitory effect of 15d-PG-J2 is specific to the Ocl lineage in both human and mouse models of osteoclastogenesis. Accordingly, parallel experiments demonstrate that sOPGL activates the NF-kappaB pathway within mouse Ocl progenitors, and this effect was abolished by 15d-PG-J2. These data establish a link between PPAR-gamma and OPGL signaling within Ocl progenitors, and support a role for PPAR-gamma pathway in the modulation of osteoclastogenesis.

Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase.

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.