Biochemical and structural characterization of the intracellular mannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A

An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-beta-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 angstroms resolution showed that AaManA adopted a (beta/alpha)8-barrel fold. Two catalytic residues were identified: Glu151 at the C terminus of beta-stand beta4 and Glu231 at the C terminus of beta7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr95 and Cys150. We propose a model of substrate binding in AaManA in which Glu282 interacts with the axial OH-C(2) in-2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.     

How do we get a perfect complement of digits?

A crucial issue in limb development is how a correct set of precisely shaped digits forms in the digital plate. This process relies on patterning across the anterior-posterior axis of the limb bud, which is under the control of Sonic hedgehog emanating from the zone of polarizing activity. Recently, Sonic hedgehog function in the limb bud has been shown to have a dual character controlling both growth and patterning of the digital field. This finding has prompted the proposal of new models of how these two functions are achieved, and this will be discussed in this review.     

Methane emission from paddy soils as affected by wheat straw returning mode

To study influence of wheat straw returning mode on CH₄ emission from paddy soils, a field experiment was conducted at Jurong, Jiangsu Province, China in 2006. Five treatments, no wheat straw applied (CK), wheat straw evenly incorporated into the topsoil (WI), wheat straw buried in ditches (WD), wheat straw strip-mulched onto the field surface (WM) and wheat straw burned in-situ (WB), were laid out in a randomized block design. Results showed that seasonal CH₄ emissions ranged from 6.9 to 28.1 g CH₄ m^?2. Wheat straw incorporation significantly increased CH₄ emission. No significant difference was observed between Treatments WI and WD in total CH₄ emission. Compared with Treatment WI, Treatment WM reduced total CH₄ emission by 32% and Treatment WB by 42%. Treatment WM was about 27% higher than Treatment CK in grain yield. Based on the findings, the best management of wheat straw prior to rice cultivation is strip-mulching wheat straw onto the field surface, which largely reduced CH₄ emission from rice fields with no decrease in rice yield.     

Effect of Proline Analogues on Activity of Human Prolyl Hydroxylase and the Regulation of HIF Signal Transduction Pathway

Hypoxia inducible factor 1 (HIF-1) plays a pivotal role in cellular responses to hypoxia. Prolyl hydroxylase 3 (PHD3) degrades HIF-1α under normoxic conditions through the hydroxylation of HIF-1α for proteolysis. Inhibiting PHD3 activity is crucial for up-regulating HIF-1α, thereby acting as a potential target for treating hypoxia-related diseases. In this study, two proline analogues (PA1 and PA2) were screened as PHD3 inhibitors with apparent EC₅₀ values of 1.53 and 3.17 µM respectively, indicating good inhibition potency. Nine proteins, significantly regulated by PA1, were identified using 2-DE coupled with MALDI-TOF/TOF MS. Pyruvate kinase isozymes M1/M2 (PKM) and alpha-enolase 1 (ENO1), which are key modulators of glycolysis, are directly regulated by HIF-1α. Moreover, VEGF, a signal protein stimulating angiogenesis, was strongly promoted by PA1. Our findings suggest that PA1 stabilized HIF-1α as well as up-regulated glycolysis and angiogenesis proteins. Herein, for the first time, we systematically studied proline analogue PA1 as a PHD3 inhibitor, which provides innovative evidence for the treatment of HIF-related diseases.     

Impact of the Non-Contributory Social Pension Program 70 y más on Older Adults’ Mental Well-Being

Background

In 2007, a non-contributory pension program was launched in rural areas of Mexico. The program consisted in a non-conditional cash transfer of US$40 monthly to all older adults (OA) aged 70 and over. We evaluate the effect of the program on mental well-being of its beneficiaries.

Methods and Findings

Quantitative and qualitative methods were used. For the quantitative component, we used the selection criteria established by the program (age and locality size) to form the Intervention (OA aged 70–74 residing in rural localities, <2500 inhabitants) and Control groups (OA aged 70–74, in localities with 2501–2700 inhabitants). Baseline data collection was conducted in 2007 where 5,465 OA were interviewed. The follow-up survey was conducted in 2008, and it was possible to interview 5,270 OA, with a response rate of 96%. A difference-in-difference linear probability model with individual fixed effect was used to estimate the impact of the program on mental well-being indicators. In 2009 a qualitative component was designed to explore possible causal pathways of such effect.

Results

After a year of exposure, the program had a significant effect on reduction of depressive symptoms (β = −0.06, CI_95% −0.12; −0.01) and an increase in empowerment indicators: OA participated in important household decisions (β = 0.09, CI_95% 0.03;0.15); and OA participated in household decisions pertaining to expenses (β = 0.11, CI_95% 0.05;0.18). Qualitative analysis found a strong trend showing a reduction of sadness, and feeling of increasing empowerment.

Conclusions

These results suggest that a non-conditional transfer in older ages have an impact beyond the economic sphere, impacting even the mental well-being. This effect could be explained because the pension produces feelings of safety and welfare. It is recommendable that governments should invest efforts towards universalizing the non-contributory pension programs in order to ensure a basic income for the elderly.     

Depletion of intermediate filament protein Nestin,a target of microRNA-940, suppresses tumorigenesis by inducing spontaneous DNA damage accumulation in human nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a major malignant tumor of the head and neck region in southern China. The understanding of its underlying etiology is essential for the development of novel effective therapies. We report for the first time that microRNA-940 (miR-940) significantly suppresses the proliferation of a variety of cancer cell lines, arrests cells cycle, induces caspase-3/7-dependent apoptosis and inhibits the formation of NPC xenograft tumors in mice. We further show that miR-940 directly binds to the 3′-untranslated regions of Nestin mRNA and promotes its degradation. Likewise, depletion of Nestin inhibits tumor cell proliferation, arrest cells at G2/M, induces apoptosis and suppresses xenograft tumor formation in vivo. These functions of miR-940 can be reversed by ectopic expression of Nestin, suggesting that miR-940 regulates cell proliferation and survival through Nestin. Notably, we observed reduced miR-940 and increased Nestin levels in NPC patient samples. Protein microarray revealed that knockdown of Nestin in 5-8F NPC cells alters the phosphorylation of proteins involved in the DNA damage response, suggesting a mechanism for the miR-940/Nestin axis. Consistently, depletion of Nestin induced spontaneous DNA damage accumulation, delayed the DNA damage repair process and increased the sensitivity to irradiation and the chemotherapeutic agent doxorubicin. Collectively, our findings indicate that Nestin, which is downregulated by miR-940, can promote tumorigenesis in NPC cells through involvement in the DNA damage response. The levels of microRNA-940 and Nestin may serve as indicators of cancer status and prognosis.Nasopharyngeal carcinoma (NPC), a major malignant tumor of the head and neck region, is endemic to Southeast Asia, southern China, the Arctic, the Middle East and North Africa.¹ Low differentiation and high metastatic potential and recurrence rates are major pathologic features of NPC. The incidence of NPC in southern China has remained very high, with a 5-year overall survival rate of approximately 70%.² Within 4 years after radiation therapy, about 30–40% of NPC patients develop distant metastasis, which is associated with poor prognosis.³ Therefore, an understanding of the underlying etiology is essential for the development of novel effective therapies for NPC.MicroRNAs (miRNAs), a class of small (∼22 nucleotides) noncoding RNAs, reduce mRNA stability and/or suppress translation by binding to the 3′-untranslated regions (3′-UTRs) or coding sequences of target mRNAs.⁴ As such, miRNAs are involved in the majority of basic biologic processes, including cell proliferation, apoptosis, differentiation and development.⁵ Cumulative evidence also suggests that miRNAs can function as potential oncogenes or tumor suppressor genes.^{6, 7} Abnormal expression of miRNAs and mutations of their genes have been documented in various types of tumors.⁸ Recently, a growing number of miRNAs have been implicated in the development of NPC. For instance, the decreased expression of miR-100 has been reported to cause Plk1 overexpression, which in turn contributes to NPC progression.⁹ MiR-200a upregulation in the undifferentiated cell line C666-1 inhibits cell growth, migration and invasion by targeting ZEB2 and CTNNB1.¹⁰ Inhibition of miR-141, which is upregulated in NPC specimens, may affect cell cycle, apoptosis, cell growth, migration and invasion through targeting of BRD3, UBAP1 and PTEN.¹¹ In addition, reduced levels of let-7 in NPC might have a role in the proliferation through DNA methylation.¹² In view of the roles of miRNAs in tumorigenesis, identification of key miRNAs and their targets that contribute to NPC progression may provide novel targets for NPC diagnosis and treatment.Nestin, a member of the type VI intermediate filament protein family, is widely expressed in mammalian nervous tissue, some immortalized mammalian stem cell lines¹³ and precursor cells of some tissues, for which expression is decreased with differentiation.^{14, 15, 16} As a stem cell/progenitor cell marker,¹⁷ Nestin is essential for mitogen-stimulated proliferation of neural progenitor cells,¹⁸ and loss of Nestin leads to apoptosis of neural progenitor cells in zebrafish.¹⁹ Recently, Nestin has been detected in various cell lines established from human solid tumors²⁰ and has been associated with aggressive nervous system tumors.²¹ All of these findings suggest that Nestin is associated with tumorigenesis; however, the precise role of Nestin and the relationship between Nestin and NPC progression are still unknown.In this study, we screen 350 different miRNAs and determined that miR-940 inhibits the proliferation of the NPC cell lines 5-8F and CNE2. Furthermore, miR-940 expression induces G2/M arrest, promotes apoptosis and suppresses xenograft tumor growth. Bioinformatic and luciferase reporter assays revealed that miR-940 targets two putative binding sites in the Nestin 3′-UTR region. A physiologic role for miR-940 was suggested by its common downregulation in NPC tissues, whereas Nestin showed a converse pattern of upregulation. Knockdown of Nestin in 5-8F and CNE2 cells induces G2/M arrest and apoptosis and inhibits cell proliferation and xenograft tumor growth; conversely, ectopic expression of Nestin partially reverses the effects of miR-940 on cell proliferation, cell cycle and apoptosis. Interestingly, knockdown of Nestin induces spontaneous DNA damage accumulation, delays DNA damage repair and enhances sensitivity to ionizing radiation (IR) of 5-8F cells both in vitro and in vivo. These results elucidate a pathway by which miR-940 regulates tumor progression in NPC by targeting Nestin.     

Sequence analysis and homology modeling of laccase from Pycnoporus cinnabarinus

Industrial effluents of textile, paper, and leather industries contain various toxic dyes as one of the waste material. It imparts major impact on human health as well as environment. The white rot fungus Pycnoporus cinnabarinus Laccase is generally used to degrade these toxic dyes. In order to decipher the mechanism of process by which Laccase degrade dyes, it is essential to know its 3D structure. Homology modeling was performed in presented work, by satisfying Spatial restrains using Modeller Program, which is considered as standard in this field, to generate 3D structure of Laccase in unison, SWISSMODEL web server was also utilized to generate and verify the alternative models. We observed that models created using Modeller stands better on structure evaluation tests. This study can further be used in molecular docking techniques, to understand the interaction of enzyme with its mediators like 2, 2-azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) and Vanillin that are known to enhance the Laccase activity.     

Comparison of the Therapeutic Effects of Yeast-incorporated Gallium with those of Inorganic Gallium on Ovariectomized Osteopenic Rats

The purpose of this study was to verify the effect of organic gallium on ovariectomized osteopenic rats. Thirty Wistar female rats used were divided into three groups: (1) sham-operation rats (control), (2) ovariectomized (OVX) rats with osteopenia, and (3) OVX rats with osteopenia treated with organic gallium. Treatments were performed over an 8-week period. At sacrifice, the fifth lumbar vertebral body, one tibia, one femur, and the fourth lumbar vertebrae were removed, subjected to micro-CT for determination of trabecular bone structure, and then processed for histomorphometry to assess bone turnover. The femoral neck was used for mechanical compression testing. Treatment with organic gallium increased bone volume in OVX animals. Organic gallium-treated animals had significant increases in trabecular and cortical thickness and bone strength. The plasma total calcium and inorganic phosphate concentrations in OVX rats decreased and bone mineral content in the lumbar vertebrae and femur increased after treatment with organic gallium. These data provide an important proof of concept that organic gallium may represent a powerful approach to treating or reversing severe osteoporosis in humans.