In vitro Culture of Medicago arborea L. Anthers: Initial Response

High production of viable somatic embryos was obtained from cultured anthers in the second phase of meiosis, using microscopic level observations of tetrads. The medium with the greatest embryogenic efficiency was H6, composed of Murashige and Skoog (MS) medium with 2 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l⁻¹ of kinetin. All (100%) of the somatic embryos obtained germinated and produced 63% green and 37% albino seedlings. In general, embryogenic calli had a higher ion concentration than non-embryogenic calli, with the exception of calcium whose concentration was higher in non-embryogenic calli. The calli induced in the different media differed in their sucrose and starch compositions. The most embryogenic medium H6-induced calli with the highest sucrose concentration and the lowest starch concentration, before visible embryos were observed. In the leaves of the albino seedlings, sucrose concentrations were very high while those of starch were very low. Ion concentrations were also lower in albino plants than in the leaves of green seedlings, with the exception of calcium, whose concentration was higher. Most of the albino individuals were homozygous, even when their progenitors were heterozygous, thereby confirming their haploid nature.     

Aportaciones de los tests de supresión de cortisol al conocimiento de los trastornos psiquiátricos: revisión narrativa de la literatura

Activity of the hypothalamic-pituitary-adrenal axis had been studied for the past half century, when some researchers noted that some patients with Cushing's syndrome and severe mood disorders had high baseline cortisol levels, which resulted in an inhibited response in the 1 mg dexamethasone suppression test. Altered dexamethasone suppression test results were subsequently found in many psychiatric diseases, including anorexia nervosa, obsessive-compulsive disorder, degenerative dementia, bipolar disorders, and schizophrenia. The relationship between high baseline cortisol levels and stress has also been studied. Some researches on the genesis of borderline personality disorder focused on traumatic childhood backgrounds. Other investigations aimed at elucidating the relationship between traumatic backgrounds and some psychiatric disorders noted that patients with post-traumatic stress disorder and borderline personality disorder showed an enhanced cortisol suppression with low cortisol doses (0.5 mg). Recent studies showed that use of an ultra-low dose of cortisol during the dexamethasone suppression test may be helpful for deteting disorders with hyperactivity of the hypothalamic-pituitary-adrenal axis.Recent advances in neuroimaging support the existence of hyperactivity of the hypothalamic-pituitary-adrenal axis in patients with borderline personality disorder, relating a decreased pituitary gland volume to major traumatic backgrounds and suicidal attempts. The purpose of this paper is to make a narrative review of research using dexamethasone suppression test in psychiatric disorders, in order to ascertain its value as a supplemental diagnostic test or as a prognostic marker.     

Household Transmission of Influenza A(H1N1)pdm09 in the Pandemic and Post-Pandemic Seasons

Background

The transmission of influenza viruses occurs person to person and is facilitated by contacts within enclosed environments such as households. The aim of this study was to evaluate secondary attack rates and factors associated with household transmission of laboratory-confirmed influenza A(H1N1)pdm09 in the pandemic and post-pandemic seasons.

Methods

During the 2009–2010 and 2010–2011 influenza seasons, 76 sentinel physicians in Navarra, Spain, took nasopharyngeal and pharyngeal swabs from patients diagnosed with influenza-like illness. A trained nurse telephoned households of those patients who were laboratory-confirmed for influenza A(H1N1)pdm09 to ask about the symptoms, risk factors and vaccination status of each household member.

Results

In the 405 households with a patient laboratory-confirmed for influenza A(H1N1)pdm09, 977 susceptible contacts were identified; 16% of them (95% CI 14–19%) presented influenza-like illness and were considered as secondary cases. The secondary attack rate was 14% in 2009–2010 and 19% in the 2010–2011 season (p = 0.049), an increase that mainly affected persons with major chronic conditions. In the multivariate logistic regression analysis, the risk of being a secondary case was higher in the 2010–2011 season than in the 2009–2010 season (adjusted odds ratio: 1.72; 95% CI 1.17–2.54), and in children under 5 years, with a decreasing risk in older contacts. Influenza vaccination was associated with lesser incidence of influenza-like illness near to statistical significance (adjusted odds ratio: 0.29; 95% CI 0.08–1.03).

Conclusion

The secondary attack rate in households was higher in the second season than in the first pandemic season. Children had a greater risk of infection. Preventive measures should be maintained in the second pandemic season, especially in high-risk persons.     

Understanding human cancer using Drosophila: Tid47, a cytosolic product of the DnaJ-like tumor suppressor gene l2Tid,is a novel molecular partner of patched related to skin cancer

Recessive mutations of the Drosophila gene lethal(2)-tumorous imaginal discs (l(2)tid) cause neoplastic growth of the anlagen of the adult organs, the imaginal discs. Here we report that the three proteins encoded by this evolutionarily conserved gene, Tid50, Tid47, and Tid40, identified as members of the DnaJ cochaperone family, are destined for different cellular compartments, build complexes with many proteins in a developmental stage-specific manner, and are likely to be involved in different cellular processes. We show that the cytosolic Tid47 molecule is a novel component of the Hedgehog (Hh)-Patched (Ptc) signaling regulating cell/tissue polarity and spatial patterning during development and is associated with human tumors such as basal cell carcinoma (BCC) and medulloblastoma. We provide functional evidence for its direct in vivo interaction with the Hh-bound Ptc receptor during signal transmission. Because loss of l(2)tid causes neoplastic transformation of Hh-responsive cells, we suggest that Tid47 may at least act as a guardian of the Hh signaling gradient by regulating Ptc homeostasis in the tissue. Finally, we show that the expression of htid-1, the human counterpart of l(2)tid, is altered in human BCCs. We demonstrate that in BCCs loss of htid expression correlates with loss of differentiation capacity of the neoplastic cells similar to that found in the Drosophila tumor model.     

Aerobic chromate reduction by Bacillus subtilis

We have studied the reduction of hexavalent chromium (chromate) to the less toxic trivalent form by using cell suspensions and cell-free extracts from the common soil bacterium, Bacillus subtilis. B. subtilis was able to grow and reduce chromate at concentrations ranging from 0.1 to 1 mM K₂CrO₄. Chromate reduction was not affected by a 20-fold excess of nitrate-compound that serves as alternate electron acceptor and antagonizes chromate reduction by anaerobic bacteria. Metabolic poisons including sodium azide and sodium cyanide inhibited chromate reduction. Reduction was effected by a constitutive system associated with the soluble protein fraction and not with the membrane fraction. The reducing activity was heat labile and showed a K_m of 188 m CrO₄ ^2-. The reductase can mediate the transfer of electrons from NAD(P)H to chromate. The results suggest that chromate is reduced via a detoxification system rather than dissimilatory electron transport.     

Semliki Forest Virus Expressing Interleukin-12 Induces Antiviral and Antitumoral Responses in Woodchucks with Chronic Viral Hepatitis and Hepatocellular Carcinoma

A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with ≥1.5-log₁₀ reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.Hepatocellular carcinoma (HCC) is a major public health problem worldwide, representing the fifth most common type of cancer. HCC is also the third leading cause of cancer-related death, mainly because only surgical and local ablative therapeutic options have shown efficacy in patients with this type of cancer (21). Approximately 80% of all HCC cases are attributed to chronic infection with hepatitis C virus and/or hepatitis B virus (HBV). Chronic carriers of HBV have a greater than 100-fold-increased relative risk of developing HCC compared to HBV-uninfected humans, with an annual incidence rate of 2 to 6% in cirrhotic patients. The high incidence of HCC, together with its poor prognosis and limited therapeutic options, warrants the development of new treatment strategies for this disease.There is increasing evidence that stimulation of the immune system for subsequent recognition and killing of tumor cells may be a valuable treatment option for liver cancer. In general, HCC appears to be an attractive target for immunotherapy because cases of spontaneous tumor regression have been reported, HCC is often infiltrated with lymphocytes, and HCC-associated proteins such as alpha-fetoprotein may be used as targets for immune-mediated killing of tumors (5, 49).A promising strategy to stimulate the deficient antitumoral immune response is based on the transfer and subsequent expression of immunostimulatory genes in tumor cells using viral or nonviral delivery vectors. One of the most effective immunostimulatory cytokines is interleukin-12 (IL-12), a protein usually expressed by macrophages and dendritic cells. IL-12 has been demonstrated to induce strong antitumoral effects that are mediated by the stimulation of T-helper cell type 1 (Th1) responses, including the activation of cytolytic T lymphocytes (CTL) and natural killer cells, and by the inhibition of angiognesis (48, 50). All of these effects are dependent on the production of gamma interferon (IFN-γ). Viral vectors that are based on adenovirus have been used to deliver IL-12 into several animal models with transplantable HCC, resulting in a localized expression of this cytokine and usually leading to antitumoral effects (3, 14, 37). However, and despite successful treatment of HCC in preclinical studies, a phase I clinical trial with a first-generation adenoviral vector for delivery and expression of IL-12 in patients with primary and metastatic liver cancer produced only a modest antitumoral effect (41). This poor response was probably due to the low and transient IL-12 expression in tumors. These results in humans indicated a need for vectors with higher potency and for preclinical testing in relevant models of HCC (i.e., large animals with spontaneous tumors).Vectors based on Semliki Forest virus (SFV), a member of the alphavirus group, are highly efficient in inducing antitumoral responses in a variety of animal models (2, 9, 10, 39, 44, 53). The SFV vector used in the present study is based on a viral RNA genome in which the region coding for the structural proteins has been replaced by a heterologous gene (24). Recombinant SFV RNA can be transcribed in vitro and transfected into cells, resulting in viral replication and subsequent production of a subgenomic RNA from which the heterologous protein is expressed at very high levels. Recombinant SFV RNA can be packaged into viral particles (vp) by cotransfecting it into cells together with two helper RNAs coding for the capsid and the envelope proteins (43). Compared to adenoviral vectors expressing IL-12, tumor treatment with SFV vectors expressing the same cytokine resulted in greater antitumoral effects in a murine colon adenocarcinoma model and also in a rat orthotopic HCC model (16, 39). The greater antitumoral effect mediated by SFV vectors has been attributed to the higher expression of IL-12 and to the induction of apoptosis caused by SFV replication within tumor cells. Apoptosis leads to the release of tumor antigens that can be taken up by antigen-presenting cells, thereby potentiating the antitumoral response induced by IL-12 (54). Furthermore, SFV vectors have low immunogenicity when delivered intratumorally, allowing repetitive administrations into the same tumor, which is not possible with adenoviral vectors (38).In the present study, the antitumoral efficacy of an SFV vector expressing IL-12 (SFV-enhIL-12) was investigated in woodchucks with HCC. The Eastern woodchuck (Marmota monax) is frequently infected with the woodchuck hepatitis virus (WHV), which is closely related to the human HBV in its structure, genomic organization, mechanism of replication, and course of infection (29). The woodchuck has been used as a mammalian model for research on HBV, including the pathogenesis of acute and chronic HBV infection, and for preclinical evaluation of the safety and efficacy of candidate antiviral drugs and therapeutic immunomodulators for the treatment of chronic HBV infection (29) and prevention of HCC (47).All woodchucks chronically infected with WHV as neonates develop HCC, and the median time for tumor appearance is 24 months of age (34, 47). After identification of HCC, the median survival time of woodchucks is 6 months, a situation similar to that for patients with HCC. In addition, WHV-induced hepatocarcinogenesis shows strong similarity to HBV-induced carcinogenesis in humans (34, 47). These features of HCC that are associated with persistent hepatitis virus infection make the woodchuck model unique compared to other animal models, in which HCC is induced by a chemical carcinogen or by transplantation of established tumor cell lines into immune-deficient or immune-compatible hosts. Woodchucks with large liver tumors that acquire malignant characteristics in a stepwise process similar to HCC in humans are an attractive and suitable model for the preclinical evaluation of new treatment strategies for HBV-induced HCC in humans (47).The antitumoral efficacy of a SFV vector expressing high levels of IL-12 (SFV-enhIL-12) was investigated in six woodchucks with established chronic WHV infection and primary HCC. The results demonstrate that SFV-delivered IL-12 expression produced a dose-dependent, partial tumor remission that was associated with a general activation of cellular immune responses against HCC. The antitumoral activity, in addition to an antiviral activity against WHV, and the favorable safety profile in woodchucks suggest that a therapeutic approach based on SFV-enhIL-12 may represent a treatment strategy for HCC in patients with chronic HBV infection, but the overall results also indicate that this approach needs further improvement for inducing a complete tumor remission.     

Viscosity decrease of pectin and fruit juices catalyzed by pectin lyase from Penicillium italicum in batch and continuous-flow membrane reactors

Summary The extracellular pectin lyase (PNL, E.C. 4.2.2.10) from Penicillium italicum was utilized in batch and confined in a continuous-flow ultrafiltration membrane reactor. The enzyme catalyzed the decrease in viscosity of pectin solutions at pH 6.0 as well as of different fruit juices at their respective pH. PNL decreased the viscosity of pectin solutions in the membrane (60% after 30 min) more than in the batch (46% after 70 min) reactors, but similar viscosity reduction of fruit juices was achieved in both reactors. The enzyme decreased the viscosity of grape, peach and melon juices to different extents, but failed to do so with apple or pear juices. It can be concluded that the utilization of PNL in a membrane reactor appears of interest for the clarification of fruit juices.