Subsequent Passages Research Articles

Enrichment of Female Germline Stem Cells from Mouse Ovaries Using the Differential Adhesion Method

Background/Aims: The isolation and establishment of female germline stem cells (FGSCs) is controversial because of questions regarding the reliability and stability of the isolation method using antibody targeting mouse vasa homologue (MVH), and the molecular mechanism of FGSCs self-renewal remains unclear. Thus, there needs to be a simple and reliable method for sorting FGSCs to study them. Methods: We applied the differential adhesion method to enrich FGSCs (DA-FGSCs) from mouse ovaries. Through four rounds of purification and 7-9 subsequent passages, DA-FGSC lines were established. In addition, we assessed the role of the phosphoinositide-3 kinase (PI3K)-AKT pathway in regulating FGSC self-renewal. Results: The obtained DA-FGSCs spontaneously differentiated into oocyte-like cells in vitro and formed functional eggs in vivo that were fertilized and produced healthy offspring. AKT was rapidly phosphorylated when the proliferation rate of FGSCs increased after 10 passages, and the addition of a chemical PI3K inhibitor prevented FGSCs self-renewal. Furthermore, over-expression of AKT-induced proliferation and differentiation of FGSCs, c-Myc, Oct-4 and Gdf-9 levels were increased. Conclusions: The differential adhesion method provides a more feasible approach and is an easier procedure to establish FGSC lines than traditional methods. The AKT pathway plays an important role in regulation of the proliferation and maintenance of FGSCs. These findings could help promote stem cell studies and provide a better understanding of causes of ovarian infertility, thereby providing potential treatments for infertility.

Cognitive and Affective Probing for Neuroergonomics

Cognitive and Affective Probing for Neuroergonomics

Comparison of dissociation methods on pluripotency of stem cells

Induced pluripotent stem cells (iPSCs) can be generated from adult cells. Somatic cells can be reprogrammed to form iPSCs by overexpressing transcription factors such as Oct4, Sox2, cMyc, and Klf4. To maintain undifferentiated state of iPSCs in vitro, cells have traditionally been maintained on mouse embryonic fibroblast feeders and passaged by enzymatic or mechanical dissociation methods. In this study, we compared the morphology and pluripotency of porcine iPSCs (piPSCs) after subsequent passaging using enzymatic and mechanical dissociation methods. Enzymatically and mechanically passaged piPSCs showed embryonic stem cell-like morphologies with compact cell adhesion and clear colony borders. In addition, alkaline phosphatase staining was positive for both enzymatically and mechanically passaged piPSCs. However, visual observation revealed that some colonies of enzymatically passaged piPSCs were spontaneously differentiated more than those of piPSCs mechanically passaged from 5 passage. Quantitative real-time RT-PCR demonstrated that enzymatically and mechanically passaged piPSCs expressed pluripotent genes such as Oct4, Sox2 and Nanog well at early passage. Immunofluorescent staining also confirmed that pluripotent markers such as Oct4, Sox2, and Nanog were positively expressed at early passage. However, expression levels of pluripotent genes in mechanically passaged piPSCs were also higher than those in enzymatically passaged piPSCs at early passage. Collectively, we found that mechanical passage method was better than enzymatic passage in terms of morphology and pluripotency of piPSCs at early passage. Further studies are needed to compare these dissociation methods with those obtained after more passages of piPSCs.

Derivation of human iPSC line NCCSi002-A from umbilical cord blood (UCB) CD34 + cells of donor from Indian ethnicity

We discuss the reprogramming of CD34+ cells isolated from UCB of a healthy female child of Indian ethnicity. The CD34+cells were nucleofected using episomal vectors expressing Oct4, Sox2, L-Myc, Klf4, Lin28 and p53DD (negative mutation in p53). The colonies were stained for alkaline phosphatase and evaluated for pluripotency marker expression by PCR, immunofluorescence and flow-cytometry. The safety of cells was confirmed by absence of plasmid in subsequent passages by PCR. G-banded karyotype demonstrated a stable genome. The ability of tri-lineage differentiation was confirmed by specific marker expression by immunofluorescence invitro and teratoma formation invivo.

First attempt on somatic cell cryopreservation of critically endangered Camelus bactrianus of India

The double-humped camel (Camelus bactrianus) of India is critically endangered since the population is around two hundred only. Immediate measures are required for its conservation so as to prevent loss of this precious germplasm forever. This is the first report on development of ear marginal tissue fibroblast cell line from Bactrian camels using primary explant technique followed by cryopreservation. The cells showed typical fusiform morphology with centrally located oval nuclei with radiating, flame like or whirlpool like migrating patterns. Four different culture media MEM, DMEM-Hi glucose, DMEM/Ham's F-12 and fibroblast specific media (HiFibroXL™) were tested with respect to the potential to support growth of camel fibroblasts. Fibroblast specific media was selected for the primary culture. Maximum cell multiplication during secondary culture was supported by the DMEM/Ham's F-12 as total cell count with same seeding (80,000 cells) and under similar culture conditions was 6,75,000, 3,05,000, 13,90,000 and 10,05,000cells/mL for MEM, DMEM-Hi glucose, DMEM/Ham's F-12 and fibroblast specific media (HiFibroXL™), respectively. Cells followed a typical sigmoid growth curve with population doubling time of 26.13h. Epithelial and fibroblast cell initially grew together. However, fibroblast cells outgrew their epithelial counterparts in subsequent passages. Reverse transcriptase PCR was performed for individual samples at different passages by designing primers specific to Cytokeratin19 (CK19) and Osteopontin (OPN). Osteocytes were absent from the very beginning whereas, epithelial cells were eliminated after the first passage. Thus, the cells were cryopreserved from 4th to 6th passages. Chromosome karyotyping showed normal diploid (2n=74) number and the cells were free from microbial contaminations. Cells had more than 90% viability in the culture after one month of cryopreservation. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Thus, genetic resource of Bactrian camel has been preserved at cellular level. The repository can also serves as a valuable resource for genome, post genome and somatic cell cloning research.

Pulsed cavitational therapy using high-frequency ultrasound for the treatment of deep vein thrombosis in an in vitro model of human blood clot

Post-thrombotic syndrome, a frequent complication of deep venous thrombosis, can be reduced with early vein recanalization. Pulsed cavitational therapy (PCT) using ultrasound is a recent non-invasive approach. We propose to test the efficacy and safety of high-frequency focused PCT for drug-free thrombolysis (thrombotripsy) in a realistic in vitro model of venous thrombosis.To reproduce venous thrombosis conditions, human whole blood was allowed to clot by stasis in silicone tubes (6 mm internal diameter) at a 30 cm H2O pressure, maintained during the whole experiment. We engineered an ultrasound device composed of dual 2.25 MHz transducers centered around a 6 MHz imaging probe. A therapeutic focus was generated at a 3.2 cm depth from the probe. Thrombotripsy was performed by longitudinally scanning the thrombus at three different speeds: 1 mm s−1 (n = 6); 2 mm s−1 (n = 6); 3 mm s−1 (n = 12). Restored outflow was measured every three passages. Filters were placed to evaluate the debris size.Twenty-four occlusive thrombi, of 2.5 cm mean length and 4.4 kPa mean stiffness, were studied. Flow restoration was systematically obtained by nine subsequent passages (4.5 min maximum). By varying the device’s speed, we found an optimal speed of 1 mm s−1 to be efficient for effective recanalization with 90 s (three passages). Within 90 s, flow restoration was of 80, 62 and 74% at respectively 1, 2 and 3 mm s−1. For all groups, cavitation cloud drilled a 1.7 mm mean diameter channel throughout the clot. Debris analysis showed 92% of debris <10 µm, with no fragment > 200 µm.

Effect of Threonine on the Proliferation and Viability of Porcine Granulosa Cells

The proliferation and viability of granulosa cells directly affects the development and function of follicles. The current study examined the effect of threonine on cell morphology and cell cycle of porcine granulosa cells. It would help to understand hormone secretion, reproductive health, and disease prevention and treatment in females. Granulosa cells were isolated from pig ovaries, and different passages of cells were stained and identified by FSHR antibody. Threonine was added to the culture medium, and cells were stained with HE to examine the changes in granulosa cell morphology. Cell viability was measured using the CCK-8 assay, and cell cycle phase was determined by flow cytometry. Immunofluorescence staining showed &gt;98% FSHR-positive cells demonstrating that the isolated granulosa cells were &gt;95% pure. HE-staining of different passages of granulosa cells showed that cells from the second to fourth passage have stable morphology, whereas those from the fifth and subsequent passages began to degenerate and degrade. CCK-8 results showed that =0.5 mM threonine can significantly enhance granulosa cell viability after 0.5, 1 and 2 h of treatment, and cells treated with 1 mM threonine for 4 h had the highest cell viability. In contrast, 5 and 10 mM threonine significantly reduced cell viability, indicating that only a certain amount of threonine can enhance granulosa cell viability. Flow cytometry results demonstrated that the percentage of G1 phase cells was higher in the 0.1, 0.5 and 1 mM groups than in the control group, but lower in the 10 mM group than in control group. While the 5 mM group had the highest percentage of S phase cells, the 10 mM group had no S phase cells. In contrast, the percentage of G2 phase cells was much higher in the 10 mM group than in the control group and other dose groups, indicating that an appropriate amount of threonine can promote granulosa cell proliferation, but excessive threonine can inhibit cell proliferation.

Abstract 5187: Potent radiosensitization of adenoid cystic carcinoma in a patient derived xenograft model using the MDM2 inhibitor AMG 232

Abstract Purpose: Adenoid cystic carcinoma (ACC) is a relatively rare cancer that typically arises in major or minor salivary gland tissues of the head and neck. There are currently no approved systemic agents for ACC and no established data supporting the delivery of chemoradiation for ACC patients. The scarcity of validated preclinical model systems has hampered research efforts. We report the successful establishment and propagation of an ACC patient derived xenograft (PDX), genomic evaluation of cancer associated mutations, and in vivo response to MDM2 inhibition combined with radiotherapy. Because TP53 mutations are seen in &amp;lt;5% of ACCs, we investigated the role of a small molecule MDM2 inhibitor, AMG 232. Methods: An ACC PDX was established and assessed for common cancer-associated mutations using the Illumina TruSeq Amplicon Cancer panel. Xenografts were treated with focal radiation with or without AMG 232. Focal radiation was delivered at 0, 2, 5, or 8 Gy x 8 fractions delivered twice weekly, with AMG 232 (50mg/kg) delivered daily by oral gavage. Tumor size was measured by caliper and comparisons between treatment groups made using a repeated measures ANOVA. A TCD50 was calculated by fitting log-transformed data to a dose-response curve and compared using the extra-sum-of-squares f test. Target inhibition and anti-cancer effect was confirmed via immuno-blotting of tumor lysates, and IHC staining or in situ hybridization of relevant targets within FFPE sections of tumors harvested 2 and 48 hrs post treatment. Results: The histologic characteristics of the primary human tumor are maintained in subsequent murine passages of this ACC PDX. Mutational profiling determined that the PDX was wildtype for TP53. AMG 232 alone resulted in modest slowing of tumor growth. Radiation alone produced a tumor growth delay in a dose dependent manner but did not result in sustained local tumor control. Strikingly, the combination of AMG 232 with RT (including low dose radiation of 2 Gy) produced dramatic tumor shrinkage and potent tumor control three months after the end of treatment. Target inhibition of MDM2 was confirmed by western blot of tumor lysates and IHC of FFPE samples taken 2 hrs post treatment. Antiproliferative and apoptotic effects were confirmed by western blot of tumor lysates and IHC of FFPE samples taken at 48 hrs post treatment. Conclusions: This study suggests that MDM2 inhibition may provide potent radiosensitization in TP53 WT ACC. While additional translational models are warranted, the powerful response profile observed suggests that phase I clinical trial evaluation of this combination is worthy for this challenging malignancy. Citation Format: Amal Javaid, Adam D. Swick, Lauryn Werner, Prashanth Prabakaran, Rong Hu, Kwangok P. Nickel, Irene Ong, Emmanuel Sampene, Justine Bruce, Gregory Hartig, Aaron Wieland, Jude Canon, Paul Harari, Randall Kimple. Potent radiosensitization of adenoid cystic carcinoma in a patient derived xenograft model using the MDM2 inhibitor AMG 232 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5187. doi:10.1158/1538-7445.AM2017-5187

Chorioallantoic membranes of embryonated chicken eggs as an alternative system for isolation of equine influenza virus

BackgroundInfluenza virus isolation in embryonated chicken eggs (ECEs) is not applicable for rapid diagnosis, however it allows the recovery and propagation of the viable virus. A low number of infectious virus particles in the swabs, poor quality of samples or individual strain properties can lead to difficulties during the virus isolation process. We propose to utilize chorioallantoic membranes (CAM) of ECEs with the assistance of real-time RT PCR to facilitate equine influenza virus isolation.MethodsReal-time RT PCR was used to detect influenza virus genetic material in amniotic/allantoic fluids (AF) and CAM of ECEs. Haemagglutination assay was used for AF. We used highly diluted virus as a substitute of clinical specimen for ECEs inoculation.ResultsOur study demonstrated that real-time RT PCR testing of CAM homogenates was more useful than testing of AF for EIV detection in ECEs. Positive results from CAM allowed to select the embryos from those with haemagglutination assay (HA) - and real-time RT PCR-negative AF for further passages. Using homogenates of CAM for subsequent passages, we finally obtained HA-positive AF, which confirmed virus replication.ConclusionWe postulate that real-time RT PCR testing of CAM homogenates and their subsequent passages may facilitate the isolation of equine influenza viruses.

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells.

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population.

Tidally Induced Bars in Dwarf Galaxies on Different Orbits around a Milky Way-like Host

Abstract Bars in galaxies may develop through a global instability or as a result of an interaction with another system. We study bar formation in disky dwarf galaxies orbiting a Milky Way-like galaxy. We employ N-body simulations to study the impact of the initial orbital parameters: the size of the dwarf galaxy orbit, and the inclination of its disk with respect to the orbital plane. In all cases, a bar develops in the center of the dwarf during the first pericenter on its orbit around the host. Between subsequent pericenter passages, the bars are stable, but at the pericenters, they are usually weakened and shortened. The initial properties and details of the further evolution of the bars depend heavily on the orbital configuration. We find that for the exactly prograde orientation, the strongest bar is formed for the intermediate-sized orbit. On the tighter orbit, the disk is too disturbed and stripped to form a strong bar. On the wider orbit, the tidal interaction is too weak. The dependence on the disk inclination is such that weaker bars form in more inclined disks. The bars experience either a very weak buckling or none at all. We do not observe any secular evolution, possibly because the dwarfs are perturbed at each pericenter passage. The rotation speed of the bars can be classified as slow (R CR/l bar ∼ 2–3). We attribute this to the loss of a significant fraction of the disk rotation during the encounter with the host galaxy.

Establishment of MDCK/FX Cell for Efficient Replication of Influenza Viruses

Background: Replication of influenza virus to high titer is a prerequisite for successful cell-based vaccine production. Entry of virus into the cell depends on the cleavage of the hemagglutinin precursor (HA0) protein mediated by trypsin. Objectives: The aim of the present study was to apply a technique to establish MDCK/FX manipulated cell, which may provide a new platform for developing influenza vaccine based on the cell culture approaches. Methods: Chicken embryo FX expressed into the pCDNA3.1 vector was transfected into the MDCK cell line. The longevity of the generated cell and the viable cell density were evaluated for 17 passages prior to virus inoculation. Then, the ability of MDCK/FX cell for efficient replication of H9N2 influenza virus was evaluated by viral titration and quantitative RT-PCR. Results: RT-PCR data revealed that FX was stably expressed in the cell after the subsequent passages without any change in the rate of culture’s confluency. Growth kinetic of H9N2 virus analysis demonstrated that MDCK/FX cell supported high-titer growth of the virus in which the viral titer is comparable to the virus grown in MDCK cells supplemented with TPCK-trypsin. Quantification of influenza infectious particles in the cell culture revealed the equivalents viral RNA copies and viral titers. Conclusions: The results indicated a potential application for the MDCK/FX in influenza virus replication procedure and related studies.

Effect of serial pig passages on the adaptation of an avian H9N2 influenza virus to swine.

H9N2 avian influenza viruses are endemic in poultry in Asia and the Middle East. These viruses sporadically cause dead-end infections in pigs and humans raising concerns about their potential to adapt to mammals or reassort with human or swine influenza viruses. We performed ten serial passages with an avian H9N2 virus (A/quail/Hong Kong/G1/1997) in influenza naïve pigs to assess the potential of this virus to adapt to swine. Virus replication in the entire respiratory tract and nasal virus excretion were examined after each passage and we deep sequenced viral genomic RNA of the parental and passage four H9N2 virus isolated from the nasal mucosa and lung. The parental H9N2 virus caused a productive infection in pigs with a predominant tropism for the nasal mucosa, whereas only 50% lung samples were virus-positive. In contrast, inoculation of pigs with passage four virus resulted in viral replication in the entire respiratory tract. Subsequent passages were associated with reduced virus replication in the lungs and infectious virus was no longer detectable in the upper and lower respiratory tract of inoculated pigs at passage ten. The broader tissue tropism after four passages was associated with an amino acid residue substitution at position 225, within the receptor-binding site of the hemagglutinin. We also compared the parental H9N2, passage four H9N2 and the 2009 pandemic H1N1 (pH1N1) virus in a direct contact transmission experiment. Whereas only one out of six contact pigs showed nasal virus excretion of the wild-type H9N2 for more than four days, all six contact animals shed the passage four H9N2 virus. Nevertheless, the amount of excreted virus was significantly lower when compared to that of the pH1N1, which readily transmitted and replicated in all six contact animals. Our data demonstrate that serial passaging of H9N2 virus in pigs enhances its replication and transmissibility. However, full adaptation of an avian H9N2 virus to pigs likely requires an extensive set of mutations.

An in vivo system for directed experimental evolution of rabbit haemorrhagic disease virus.

The calicivirus Rabbit haemorrhagic disease virus (RHDV) is widely used in Australia as a biocontrol agent to manage wild European rabbit (Oryctolagus cuniculus) populations. However, widespread herd immunity limits the effectiveness of the currently used strain, CAPM V-351. To overcome this, we developed an experimental platform for the selection and characterisation of novel RHDV strains. As RHDV does not replicate in cell culture, variant viruses were selected by serially passaging a highly virulent RHDV field isolate in immunologically naïve laboratory rabbits that were passively immunised 18–24 hours post-challenge with a neutralising monoclonal antibody. After seven passages, two amino acid substitutions in the P2 domain of the capsid protein became fixed within the virus population. Furthermore, a synonymous substitution within the coding sequence of the viral polymerase appeared and was also maintained in all subsequent passages. These findings demonstrate proof-of-concept that RHDV evolution can be experimentally manipulated to select for virus variants with altered phenotypes, in this case partial immune escape.

Fatty Acid Comprising Lysine Conjugates: Anti-MRSA Agents That Display In Vivo Efficacy by Disrupting Biofilms with No Resistance Development.

Methicillin-resistant Staphylococcus aureus (MRSA) has developed resistance to antibiotics of last resort such as vancomycin, linezolid, and daptomycin. Additionally, their biofilm forming capability has set an alarming situation in the treatment of bacterial infections. Herein we report the potency of fatty acid comprising lysine conjugates as novel anti-MRSA agents, which were not only capable of killing growing planktonic MRSA at low concentration (MIC = 3.1-6.3 μg/mL), but also displayed potent activity against nondividing stationary phase cells. Furthermore, the conjugates eradicated established biofilms of MRSA. The bactericidal activity of d-lysine conjugated tetradecanoyl analogue (D-LANA-14) is attributed to its membrane disruption against these metabolically distinct cells. In a mouse model of superficial skin infection, D-LANA-14 displayed potent in vivo anti-MRSA activity (2.7 and 3.9 Log reduction at 20 mg/kg and 40 mg/kg, respectively) without showing any skin toxicity even at 200 mg/kg of the compound exposure. Additionally, MRSA could not develop resistance against D-LANA-14 even after 18 subsequent passages, whereas the topical anti-MRSA antibiotic fusidic acid succumbed to rapid resistance development. Collectively, the results suggested that this new class of membrane targeting conjugates bear immense potential to treat MRSA infections over conventional antibiotic therapy.

EFFICACY AND VIRULENCE OF Spodoptera littoralis NUCLEOPOLYHEDROVIRUS on S. littoralis LARVAL FEEDING AND SUSCEPTIBILITY

Entomopathogenic baculoviruses have long been recognized as vital environmentally safe alternatives to chemical pesticides. A study for investigating the virulence and susceptibility of Spodoptera littoralis larvae to S. littoralis nucleopolyhedrovirus (SpliNPV) isolate was carried out. Bioassay experiments on the 1st and 2nd larval instars using droplet feeding method revealed that polyhedral for 50% or 90% mortality of the 2nd larval instar of S. littoralis was significantly lower compared with the 1st instar. While, the 1st instar was killed faster than the 2nd one especially with the higher concentration. The susceptibility of the infected 2nd instar when feeding on different hosts showed that larvae were more susceptible when feeding on eggplant leaves followed by lettuce leaves, artificial diet and tomato leaves as shown by larval weight and reduction in feeding. The reduction in both larval weight and feeding were significantly higher with 1x107 PIB/ml than 1x104 PIB/ml in the majority of cases. S. littoralis larvae showed more than 2 times of tolerant to the 3rd passage of SpliNPV on the level of LC50 and more than 18 times tolerant on the level of LC90. The tolerant levels of S. littoralis larvae for the 3rd passage of virus was associated with more than 20% increase in time taken for kill with no changes in DNA patterns regarding subsequent passages in S. littoralis larvae using a restriction enzyme EcoRI.

Culture and expansion of Adipose derived Mesenchymal stem cells in Ovine

Culture and expansion of Adipose derived Mesenchymal stem cells (ADMSCs) in Ovine was carried out in this study. Ovine adipose tissue samples were collected from Chennai Corporation slaughter house under sterile condition in normal saline with five per cent povidone iodine, antibiotic and antimycotic solutions. Collected tissue samples were weighed and digested using collagenase enzyme to isolate adipose tissue derived mesenchymal stem cells (ADMSCs). Cell yield and viability of the cells were calculated by using trypan blue exclusion test. The cells were seeded at the density of one million cells in one T25 culture flask in Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG). On the day of seeding, the cells showed spherical morphology. Plastic adherence was noticed 24 hrs after seeding. Cell expansion was observed after 3 days. At P0 level, 70% confluency was attained on day 14 and the time taken to reach 70% confluency was reduced to 3-4 days in subsequent passages.

Hosea by Bo H. Lim And Daniel Castelo

Reviewed by: Hosea by Bo H. Lim And Daniel Castelo David Penchansky bo h. lim and daniel castelo, Hosea (Two Horizons Old Testament Commentary; Grand Rapids: Eerdmans, 2015). Pp. xii + 260. Paper $25. This book is a hybrid, eleven chapters of commentary by a biblical scholar (Lim), with four lengthy theological reflections on Hosea interspersed (Castelo). The collaboration works because these two authors, who both teach at Seattle Pacific University, see eye to eye regarding Hosea. They have together provided a fairly comprehensive overview of Hosea scholarship in the twenty-first century. Although allowing some later editing, they claim that the Book of Hosea came substantially from a single hand and from one person's design, the eighth-century prophet Hosea. This is a contested position in biblical scholarship, where many see Hosea as loosely related fragments and alternative versions, having a diversity of centers and conflicting themes. The authors stake out the more conservative position, claiming that Hosea functions as a single harmonious work. The authors see covenant as the idea that unifies the Book of Hosea. The marriage and sexual references (thought by others to dominate) serve, they say, only to give content to Israel's covenant obligations and their violation. Hosea's God creates problems for the authors. God commands the prophet to marry a promiscuous woman. In subsequent passages, God abrogates the eternal covenant made with Israel. God compares Godself to violent and repellant things—a lion rending its victim, rot, and pestilence. The authors explain and justify this divine behavior by drawing a distinction. On one side, God's inner self, God's true being, never changes, needs nothing, and is benevolent, constant. On the other side is God's reactive self. When Hosea depicts God as vindictive, violent, or conflicted, he is not describing God's inner core. Another problem L. and C. address is the glaring misogyny in Hosea. For example, in Hosea 2 the woman is punished by stripping her naked in front of her former lovers: "Now I will uncover her shame in the sight of her lovers" (2:10). In chap. 3 she is imprisoned for a season and not allowed to see anybody but her husband, who denies sexual intimacy to her (3:3). Finally, "their little ones shall be dashed to pieces, and their pregnant women ripped open" (13:16). Castelo describes the pattern: [S]ince women are exclusively depicted as the offending sex … [t]he text [of Hosea] gives a preference for and an affirmation of the male gender while offering a negative image of the female gender. … [Such a text] could legitimate and support misogyny, both ancient and modern. …[W]omanhood is continually depicted as the image of choice for infidelity, impurity, and sexual lasciviousness; manhood simply is not. (p. 233) [End Page 510] The authors minimize the impact of Hosea's misogyny because the words come as metaphors that refer to Israel. A metaphor has two parts—in this case, Israel and the promiscuous woman. The authors claim that Hosea is concerned about Israel and not about the condition of women. Therefore, the misogyny in the metaphor is irrelevant to the meaning of the passage. The nature of metaphor, however, is such that each side in the metaphor participates in the other, and so when the author uses misogynistic language to make a point, the misogyny spills over into all aspects of the text. No contextualization can normalize it. Surprisingly, L.'s and C.'s writing takes a postmodern turn. They recognize their own approach as "heuristic," that is, a means by which to investigate Hosea and not the only truth. Meaning is fluid, they say, largely determined by the reading audience. A text has multiple meanings that depend on the questions addressed and who has interest in the answers. They therefore acknowledge that statements about God (both Hosea's and theirs) are provisional and metaphorical. Their own evangelical community desires a unified and coherent text. Yet the authors recognize that this remains one interpretation out of many possibilities. They therefore manage to remain objective about their own subjectivity, recognizing their bias and the filter that shapes their reading. This recognition opens up their text...

Valproic acid assisted reprogramming of fibroblasts for generation of pluripotent stem cells in buffalo (Bubalus bubalis).

Generation of pluripotent stem cells by reprogramming somatic cells of quality animals has numerous potential applications in agricultural and biomedical sciences. Unfortunately, till now, reprogramming of buffalo fetal fibroblast cells (bFFs) has been very ineffient despite intensive efforts. Here, we attempted to enhance reprogramming efficiency by using the HDAC inhibitor valproic acid (VPA) in bFFs transfected with pLentG-KOSM pseudo virus carrying mouse specific pluripotent genes. FACS analysis revealed that VPA treatment significantly increased (p < 0.05) GFP+ cells in comparison to VPA untreated control. Further, among different concentrations, 1.5 mM VPA was found to be optimal, increasing about 5 fold GFP+ cells and 2.5-fold GFP+ colonies with significantly (P < 0.05) larger size as compared to control. These colonies were further propagated and characterised. The colonies displayed embryonic stem cell (ESC)-like morphology, normal karyotype, and were positive for alkaline phosphatase staining as well as immune-positive for the ESC specific markers Oct4, Nanog, SSEA1, TRA-1-60 and TRA-1-81. The primary colonies revealed significantly higher (P < 0.05) expression of pluripotent genes than control, which declined gradually on subsequent passages. The reprogrammed cells readily formed embryoid bodies in vitro and cells of all three germ layers. These results indicated that VPA treatment of viral transducted cells can improve the generation of induced pluripotent stem cells and help their long term maintenance in buffalo.

Reformation Commentary on Scripture: Old Testament V. 1–2 Samuel, 1–2 Kings, 1–2 Chronicles ed. by Derek Cooper and Martin J. Lohrmann

Reviewed by: Reformation Commentary on Scripture: Old Testament V. 1–2 Samuel, 1–2 Kings, 1–2 Chronicles ed. by Derek Cooper and Martin J. Lohrmann Mark A. Throntveit Reformation Commentary on Scripture: Old Testament V. 1–2 Samuel, 1–2 Kings, 1–2 Chronicles. Edited by Derek Cooper and Martin J. Lohrmann. Downers Grove: InterVarsity Press Academic, 2016. 799 pp. Readers of Lutheran Quarterly will be familiar with the tedious process of retrieving commentary on Bible passages by that intrepid band of reformers in the sixteenth century. Even more irksome is the tracking down of the context in which the passage appeared. Nevertheless, what the various reformers thought about the Bible is extremely important for a proper evaluation of their contribution and impact. But how is this achieved? According to the publishers, these Reformation Commentaries on Scripture were produced with four goals in mind: (1) to renew contemporary biblical interpretation through exposure to Reformation-era biblical interpretation; (2) to renew contemporary preaching through exposure to the biblical insights of the Reformation writers; (3) to contribute to a deeper understanding of the Reformation and the breadth of perspectives represented within it; and (4) to advance Christian scholarship in the fields of historical, biblical, theological, and pastoral studies. Ambitious as these goals might seem, this volume succeeds admirably on all four fronts. In a day when reader response trumps the history of interpretation in biblical studies, the present volume provides a fascinating take on these historical books of the Old Testament. Unsullied by the frequent excesses of the post-Enlightenment hermeneutics, Reformation exegetes frequently interpret the histories as harbingers of subsequent New Testament passages. As for proclamation, the reformers tend to see models and negative examples for contemporary Christian behavior in the wide-ranging cast of characters that we encounter in these pages. The breadth of Reformation perspectives is amply attested as well. We do hear from Luther, Melanchthon, and Calvin, but the bulk of citations hark back to Luther’s pastor, Bugenhagen, Lucas Osiander and Viktorin Strigel. Reformed representatives include Konrad Pellikan and Johannes Piscator. The English reformation is well represented by Andrew Willet and John Mayer. Although the Radicals and [End Page 100] Anabaptists eschewed publishing commentaries on these particular books, we are treated to an occasional remark from Menno Simons, Hans Denck, Balthasar Hubmaier, and Dirk Philips with regard to God’s promise to David of an eternal dynasty. One might expect only Protestants to appear yet commentary from Catholic scholars Alonso Tostado and Cardinal Cajetan pepper the presentation with their rather different point of view. All of this serves to advance scholarship in the fields of history, Bible, theology, and pastoral care, not least by the unearthing and translation of little-known works. When these goals are coupled with an absolutely first-rate introduction to the historical context of the Protestant Reformation, the Reformers and how they read Scripture, and the books of Samuel, Kings, and Chronicles, the result is a delightful view of these books through the eyes of the reformers. The explicit format is a welcome guide on our journey: Following Reformation-era practice, the text is divided into pericopes or passages drawn from the English Standard Version of the Bible. These passages are then followed by very useful overviews of the subsequent comments pointing out exegetical, theological, or pastoral concerns by the editors. Finally, headings identify the nature of the comments gathered together under each pericope. The reviewer’s copy was curiously flawed in that pages 475–522 were inserted upside down and backwards right after 2 Kings 17, the description of the destruction of the Northern Kingdom by Assyria, rendering the so-called “ten lost tribes of Israel” a textual as well as a historical occurrence! Mark A. Throntveit Luther Seminary Saint Paul, Minnesota Copyright © 2017 Johns Hopkins University Press and Lutheran Quarterly, Inc.