A novel testis-enriched gene Spata33 is expressed during spermatogenesis.

With an increasing incidence of male idiopathic infertility, identification of novel genes involved in spermatogenesis is an important aspect for the understanding of human testicular failure. In the present study, we have identified a novel gene Spata33, also called as 4732415M23Rik or C16orf55, which is conserved in mammalian species. Spata33 was predominantly expressed in the postpartum and adult mouse testes at mRNA and protein levels. Its expression was increased during the first wave of the spermatogenesis, indicating that Spata33 may be associated with the meiotic process. Further immunohistochemistry analysis revealed that Spata33 was mainly expressed in the spermatocytes, spermatogonia and round spermatids. Its expression was uniformly distributed in the nucleus and cytosol in these germ cells, which was further confirmed by Spata33-tagged with GFP staining in the GC-1 and TM4 cells. These results indicated that Spata33 was predominantly expressed in the mouse testis and associated with spermatogenesis. Identification and characterization of the novel testis-enriched gene Spata33 may provide a new route for understanding of spermatogenesis failure

RNAi-based targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica

Microalgae are promising feedstock for renewable fuels such as biodiesel, yet development of industrial oleaginous strains has been hindered by the paucity and inefficiency of reverse genetics tools. Here we established an efficient RNAi-based targeted gene-knockdown method for Nannochloropsis spp., which are emerging model organisms for industrial microalgal oil production. The method achieved a 40-80% success rate in Nannochloropsis oceanica strain IMET1. When transcript level of one carbonic anhydrase (CA) was inhibited by 6283% via RNAi, mutant cells exhibited photosynthetic oxygen evolution (POE) rates that were 68-100% higher than wild-type (WT) at pH 6.0, equivalent to WT at pH 8.2, yet 39-45% lower than WT at pH 9.0. Moreover, the mutant POE rates were negatively correlated with the increase of culture pH, an exact opposite of WT. Thus, a dynamic carbon concentration mechanism (CCM) that is highly sensitive to pH homeostasis was revealed, where the CA inhibition likely partially abrogated the mechanism that normally deactivates CCM under a high level of dissolved CO2. Extension of the method to another sequenced N. oceanica strain of CCMP 1779 demonstrated comparable performance. Finally, McrBC-PCR followed by bisulfite sequencing revealed that the gene knockdown is mediated by the CG, CHG and CHH types of DNA methylation at the coding region of the targeted gene. The efficiency, robustness and general applicability of this reverse genetics approach suggested the possibility of large-scale RNAi-based gene function screening in industrial microalgae

Soil Quality Changes and Quality Status: A Case Study of the Subtropical China Region Ultisol

Aims: To provide a soil quality assessment frame work and threshold limits for assessing soil quality in Ultisol of subtropical China region. Study Design: Selected minimum data set for soil quality assessment and threshold limits for the study were total carbon, nitrogen, soil pH and phosphorus, biomass carbon, nitrogen and phosphorus, maize grain and fresh potato tuber yields. Soil data (2000-2010), maize grain and fresh potato yield data (2000-2009) from a long term experiment under the Institute of subtropical Agriculture, China were analyzed using the SAS statistical package and means were graphically compared to determine threshold limits for selected data set and fitted into a soil quality model. Place and Duration of Study: The key Laboratory for Agro-ecological Processes in Subtropical Regions, Chinese Academy of Sciences; Institute of Subtropical Agriculture, Changsha, Hunan China long-term experimental site in Taoyuan county, conducted from the year 2000 to 2010. Methodology: Soils samples at the experimental fields were obtained from depths 0-20 cm using an auger at each replicate in triplicates and homogenized to obtain a composite sub sample, air-dried, sieved through 2.0 mm to obtain samples for analysis in the Laboratory. Parameters analyzed for were organic carbon concentration, measured by the combustion method using an automated C/N analyzer (Vario MAX CN, Elemental Co., Germany) while total nitrogen was by the Kjeldahl method of ISSCAS (1978). Microbial carbon, nitrogen and phosphorus levels were determined using the chloroform-fumigationextraction method (Jenkinson and Powlson, 1976; Vance et al., 1987; Brookes et al., 1982) and adopting the conversion factors 0.45 (Wu et al., 1990), 0.45 (Brookes et al., 1985), and 0.29 (Wu et al., 2000) respectively for the C, N and P. Extractable N and Olson P were taken from values obtained from the non fumigated soil samples. Data obtained were statistically analyzed using the SAS package for ANOVA and significant means were separated using the Duncan’s New Multiple Range Test (DNMRT). Treatment means were also matched graphically to delineate critical threshold limits between classes for each parameter. Soil quality was assessed by using the Parr et al. (1992) equation; SQ =ƒ(SP,P,E,H, ER,BD,FQ, MI); where SQ= soil quality, SP= soil properties, P = potential productivity, E=environmental factor, H= health (human/animal), ER= erodibility, BD= biodiversity, FQ= food quality and MI= management input. A score scale of 1 to 5 was used in the assessment of parameters in the model; where 1 is best and 5 is the worst condition. However, E, H, ER, FQ and MI were each scored 1.0 because the long-term experiment has an environmental component, health factor, biodiversity, food quality and management input components that are being optimally managed. Therefore SQ= f(SP, P) was used to assess quality of the Ultisol at the uplands and slope land locations. Results: At the uplands, the practice of maize-rape/marsh residue+NK (8.54gkg-1 C, 1.0 gkg-1 N and 5.67 mgkg-1 P) treatments could be rotated with Maize-rape/nil fertilizer (7.51 gkg-1 C, 0.87 gkg-1 and 0.39 mgkg-1 P) to encourage improved soil quality by allowing for more years with soil carbon sequestration, nitrogen and phosphorus credit than years of depletion and discourage soil degradation. At the slope lands, treatments that combined application of organic and inorganic fertilizer materials [Sweet potato-rape/NP+straw (7.18 gkg-1 C, 0.88 gkg-1 N and 0.38 mgkg-1 P) and Peanut-broadbean/NP+straw (6.81 gkg-1 C, 0.86 gkg-1 N and 0.38 mgkg-1 P)] improved soil quality significantly over time by sequestering significantly higher total carbon, nitrogen and phosphorus better than sole inorganic fertilizer [Sweet potato-rape/NPK (6.52 gkg-1 C, 0.81 gkg-1 N and 0.38 mgkg-1 P)]. Conclusion: Ultisol at the upland positions had better quality (SQ1) than those at the slope (SQ2) positions. Threshold limits for nutrients, pH and yield of maize and Fresh Potato tubers in the subtropical China region Ultisol was developed

Soil Quality Changes and Quality Status: A Case Study of the Subtropical China Region Ultisol

Aims: To provide a soil quality assessment frame work and threshold limits for assessing soil quality in Ultisol of subtropical China region. Study Design: Selected minimum data set for soil quality assessment and threshold limits for the study were total carbon, nitrogen, soil pH and phosphorus, biomass carbon, nitrogen and phosphorus, maize grain and fresh potato tuber yields. Soil data (2000-2010), maize grain and fresh potato yield data (2000-2009) from a long term experiment under the Institute of subtropical Agriculture, China were analyzed using the SAS statistical package and means were graphically compared to determine threshold limits for selected data set and fitted into a soil quality model. Place and Duration of Study: The key Laboratory for Agro-ecological Processes in Subtropical Regions, Chinese Academy of Sciences; Institute of Subtropical Agriculture, Changsha, Hunan China long-term experimental site in Taoyuan county, conducted from the year 2000 to 2010. Methodology: Soils samples at the experimental fields were obtained from depths 0-20 cm using an auger at each replicate in triplicates and homogenized to obtain a composite sub sample, air-dried, sieved through 2.0 mm to obtain samples for analysis in the Laboratory. Parameters analyzed for were organic carbon concentration, measured by the combustion method using an automated C/N analyzer (Vario MAX CN, Elemental Co., Germany) while total nitrogen was by the Kjeldahl method of ISSCAS (1978). Microbial carbon, nitrogen and phosphorus levels were determined using the chloroform-fumigation-extraction method (Jenkinson and Powlson, 1976; Vance et al., 1987; Brookes et al., 1982) and adopting the conversion factors 0.45 (Wu et al., 1990), 0.45 (Brookes et al., 1985), and 0.29 (Wu et al., 2000) respectively for the C, N and P. Extractable N and Olson P were taken from values obtained from the non fumigated soil samples. Data obtained were statistically analyzed using the SAS package for ANOVA and significant means were separated using the Duncan’s New Multiple Range Test (DNMRT). Treatment means were also matched graphically to delineate critical threshold limits between classes for each parameter. Soil quality was assessed by using the Parr et al. (1992) equation; SQ =ƒ(SP,P,E,H, ER,BD,FQ, MI); where SQ= soil quality, SP= soil properties, P = potential productivity, E=environmental factor, H= health (human/animal), ER= erodibility, BD= biodiversity, FQ= food quality and MI= management input. A score scale of 1 to 5 was used in the assessment of parameters in the model; where 1 is best and 5 is the worst condition. However, E, H, ER, FQ and MI were each scored 1.0 because the long-term experiment has an environmental component, health factor, biodiversity, food quality and management input components that are being optimally managed. Therefore SQ= f(SP, P) was used to assess quality of the Ultisol at the uplands and slope land locations. Results: At the uplands, the practice of maize-rape/marsh residue+NK (8.54gkg-1 C, 1.0 gkg-1 N and 5.67 mgkg-1 P) treatments could be rotated with Maize-rape/nil fertilizer (7.51 gkg-1 C, 0.87 gkg-1 and 0.39 mgkg-1 P) to encourage improved soil quality by allowing for more years with soil carbon sequestration, nitrogen and phosphorus credit than years of depletion and discourage soil degradation. At the slope lands, treatments that combined application of organic and inorganic fertilizer materials [Sweet potato-rape/NP+straw (7.18 gkg-1 C, 0.88 gkg-1 N and 0.38 mgkg-1 P) and Peanut-broadbean/NP+straw (6.81 gkg-1 C, 0.86 gkg-1 N and 0.38 mgkg-1 P)] improved soil quality significantly over time by sequestering significantly higher total carbon, nitrogen and phosphorus better than sole inorganic fertilizer [Sweet potato-rape/NPK (6.52 gkg-1 C, 0.81 gkg-1 N and 0.38 mgkg-1 P)]. Conclusion: Ultisol at the upland positions had better quality (SQ1) than those at the slope (SQ2) positions. Threshold limits for nutrients, pH and yield of maize and Fresh Potato tubers in the subtropical China region Ultisol was developed

Analysis of distribution method of designed air quantity in coal mine ventilation—a case study

Abstract In a coal mine, air leakage exists in some roadways through doors and other ventilation structures inevitably. Based on this opinion, there are different views on whether these roadways must be assigned airflow in coal mine ventilation design. This paper analyses some relevant regulations and criteria on the designed air quantity of coal mines. Then, based on the ventilation design of the Guizhou Yizhong Coal Mine, through the study of the calculation of needed air quantity of every working place and its distribution method in coal mine ventilation design, this paper puts forward that explosion-proof door, safety exit, and other short distance roadways with ventilation structures need not assign airflow in coal mine ventilation design, while some long-distance roadways need. Additionally, it presents the main reason to support this opinion, gives the distribution method of inner air leakage quantity, which comes with the calculation of the designed mine total air quantity, puts forward the remedy method for the air leakage through ventilation structures in a coal mine ventilation system, then offers the mine operator with the basic opinions for the day-to-day planning and effective operation of a coal mine ventilation system

Identification of a testis-enriched heat shock protein and fourteen members of Hsp70 family in the swamp eel.

BACKGROUND: Gonad differentiation is one of the most important developmental events in vertebrates. Some heat shock proteins are associated with gonad development. Heat shock protein 70 (Hsp70) in the teleost fish and its roles in sex differentiation are poorly understood. METHODS AND FINDINGS: We have identified a testis-enriched heat shock protein Hspa8b2 in the swamp eel using Western blot analysis and Mass Spectrometry (MS). Fourteen Hsp70 family genes were further identified in this species based on transcriptome information. The phylogenetic tree of Hsp70 family was constructed using the Maximum Likelihood method and their expression patterns in the swamp eel gonads were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). CONCLUSION: There are fourteen gene members in the Hsp70 family in the swamp eel genome. Hsp70 family, particularly Hspa8, has expanded in the species. One of the family members Hspa8b2 is predominantly expressed in testis of the swamp eel

Phylogenetic tree, amino acid alignments, domains and modification sites of Spata33.

<p>A). Phylogenetic tree of Spata33 in mammals. Phylogenetic analysis was performed with Phylip. Numbers on the branches represent the bootstrap values from 1000 replicates obtained using the Neighbor-Joining method. The scale bar corresponds to the estimated evolutionary distance units. GenBank accession numbers are as follows: <i>Callithrix jacchus</i>, XP_002761326.1; <i>Canis lupus familiaris</i>, XP_003434741.1; <i>Cricetulus griseus</i>, XP_003495126.1; <i>Gorilla gorilla gorilla</i>, XP_004058209.1; <i>Homo sapiens,</i> BAG64150.1; <i>Macaca mulatta,</i> XP_001104069.2; <i>Mus musculus</i>, NP_796253.2; <i>Nomascus leucogenys</i>, XP_003280677.1; <i>Otolemur garnettii,</i> XP_003800884.1; <i>Pan paniscus</i>, XP_003805830.1; <i>Pan troglodytes</i>, XP_511172.4; <i>Papio anubis,</i> P_003917381.1; <i>Rattus norvegicus</i>, NP_001099665.1; <i>Saimiri boliviensis boliviensis</i>, XP_003944616.1; <i>Tupaia_chinensis</i>, ELW62868.1. B). Alignment of amino acid sequences of the Spata33 proteins. Amino acids that are identical in all these species are shown in white letters on black background. The DUF4609 domain predicted by Pfam is boxed. C). Schematic mapping of potential protein domains and post-translational modification sties. The predicted sites for N-myristoylation, N-glycosylation and phosphorylations in Spata33 were indicated (N-Myr, N-myristoylation site; N-Glyc, N-glycosylation site; S/T, Serine/Threonine phosphorylation sites).</p

Immunostaining of Spata33 protein in the juvenile (aged 10–20 days) and mature testes (aged 35 days) using anti-Spata33 antibody (A), and subcellular localization of Spata33 protein in the GC-1 cells and TM4 cells (B).

<p>A). Spata33 was expressed mainly in the spermatogonia (white arrow heads), spermatocytes (red arrows) and round spermatids (red arrow heads). The signals were observed in both the cytosol and nuclei from P12-P35, whereas no signals were detected in P10. In controls, no positive signals were observed in P35 testis sections when Spata33 antibody was replaced by 1% normal rabbit serum. Nuclei were re-dyed with Hematoxylin (blue). Bars, 20 µm. B). GC-1 and TM4 cells were transfected with Spata33-GFP (green, Excitation 488 nm, Emission 507 nm) and stained with Hoechst (blue, Excitation 352 nm, Emission 461 nm) respectively. Spata33 is localized both in the nuclei and cytoplasm within GC-1 cells or TM4 cells. Bar, 10 µm.</p

Expression of <i>Spata33</i> mRNA and protein in the postnatal testes of mice.

<p>A). RT-PCR showed expression pattern of <i>Spata33</i> mRNA in testes from P2 to P20 (postnatal days). Total RNAs were isolated from mouse testes and then cDNAs were synthesized. <i>Hprt</i> was used as an internal control. Molecular weight is shown on the right. <i>Spata33</i> mRNA was increased markedly after P12. B). Real-time fluorescent quantitative PCR of the <i>Spata33</i> in testes from P2 to P20. Error bars indicate the standard deviation (SD) of the mean (n = 3). Y-axis represents relative expression levels of <i>Spata33</i> and X-axis shows different development stages P2-P20. C). Expression of Spata33 protein in testes from P2 to P20. Mouse testes were individually collected from aged 2 to 20 days and were subjected to Western blot analysis with antibody against Spata33. The protein level was markedly increased from P12. Actin was used as an internal control. Molecular weight is shown on the right. All experiments were performed three times with independent individuals.</p