"The politics of vaccination. A global history" is a multi-faceted study of the connections between vaccination, identity (constructions and politics), political systems and resistance across time and place. The edited volume is based on the proceedings of two conferences in Berlin and Norwich, with an impressive total of twelve contributions from across the social sciences including anthropology, history of medicine, philosophy and local cultural studies. The volume is divided into three parts: Vaccination and national identity; Nationality, vaccine production and the end of sovereign manufacture; and Vaccination, the individual and the society. Each contribution situates the practice of vaccination and immunization as well as the production of vaccines in a different political regime and geographic locale. The authors cover the periods of colonialism and post-colonialism, post-WWII Communism and "ruthless" neo-liberalism, in four of the five continents.
The first chapter, by Paul Greenough, looks at the beginnings of the global campaign to eradicate smallpox, which surprisingly originated in Pakistan. Greenough's description of physicians and epidemiologists in Cold War Pakistan and their attempts to control the smallpox epidemic serves as an engaging entry into the complex and sometimes painful topic of the politics of vaccinations. The next contribution, by Nils Brimnes, depicts local resistance to immunizations in India from the late colonial period to the early years of Independence. He links this resistance to anti-colonialism and the creation of an Indian national identity: in other words, in contrast to their unifying role elsewhere, immunization campaigns here were seen as a threat to the nation. Chapter 3, by Dora Vargha, examines how Hungary became the model for the global WHO strategy of polio eradication. Providing a wealth of fascinating historical details, the author explores the ways in which the image of the body (in this case the productive, laborious body) coheres with a political ideology underlying public health initiatives. In the fourth and final chapter of Part I, Eun Kyung Choi and Young-Gyung Paik examine the production of modernity through the skillful introduction of Hepatitis B vaccines in a multi-decade study of 20th century Korea. Here it becomes clear that the notion of "backwardness" plays an important role in political decisions, particularly those concerning vaccinations and other public health issues.
The second part of the volume opens with a piece by Ana Maria Carrillo on the health institutions which produced vaccines and national political strategies on immunization in 20th century Mexico. These involved a great deal of toing and froing on matters of safety and efficacy. Chapter 6, by Stuart Blume, discusses the history of the Dutch Institute of Public Health in the Netherlands from the 1960s to the present day. This well-argued piece delves into globalization and the privatization of vaccine production in Europe, providing reflections on the ownership of scientific knowledge beyond the specific place and time covered in the chapter. In Chapter 7, Jaime Benchimol examines the production of yellow fever vaccines in Rio de Janeiro from the mid 19th to the mid 20th century. This chapter stands out for the compelling way it traces etiologies of treatment for diseases such as malaria, dengue, and yellow fever, showing how these came to be understood through modern theories of disease. Finally, in the last chapter of this section, Julia Yongue reviews Japan's unique approach to global health and immunization policy from the mid 19th century onwards. Here, the notion of modernity played a central role in driving discussions about public health, as the Japanese state put into place explicit policies to "modernize" the country in line with western nations.
The third part of the volume begins with a chapter by Andrea Stöckl and Anna Smajdor on the public controversy surrounding the combined MMR (measles, mumps and rubella) vaccine in the UK. This controversy, which played out in British media some twenty years ago, became a national political issue when it called into question the personal health decisions of then-prime minister Tony Blair. Chapter 10, by Britta Lundgren and Martin Holmberg, discusses influenza immunization in contemporary Sweden. Here, vaccination is seen as an act of solidarity with the state, which was nevertheless shaken by the influenza pandemic in 2009. In the last chapter of this section, Elisha P. Renne examines the obstacles faced by the Nigerian state (including cultural challenges, trust, resistance, difficulties with water and electricity) in its immunization campaigns. Her research also points to the creative ways in which the state faced up to these challenges, for instance by giving gifts as incentives to get vaccinations.
In the afterword, William Muraskin critiques the global humanitarian approach to eradicating disease, which often operates through a willful ignorance of local factors or a downright colonial mindset. Muraskin's piece is a compelling argument for why public health interventions should take social and cultural factors into account, as these have a direct effect on the success of initiatives and how they are received in different national, regional and/or cultural contexts.
Overall, the volume is a dense, diverse and well-written collection, rich in facts, which covers a significant part of the global history of immunization. The case studies take the reader through scientific error, media manipulation, the inter-cultural transmission of values and concepts, and the quest for modernity through public health. The strength of the volume lies in the diversity of its material, but this is also a weakness at times: for instance, the two competing polio vaccines invented by Sabin and Salk are mentioned in multiple historical and geographic contexts without being brought together in a cohesive analysis. The volume as a whole would have benefited from greater attention to the details that recur in the different histories (like the development of polio vaccines) and how these are connected across time and place.
Readers who are not already specialists in the history of immunization may be distressed to learn that it is also a history of violence and death. The chapters shed light on numerous vaccine tests with "volunteers" such as the 1.5 million people who were unwitting test subjects for vaccines in the USSR, in Brazilian orphanages, or even prisons in 'democratic' countries. There were also sacrifices on the part of researchers, like the early and sudden deaths of scientists in the field of yellow fever. These "collateral damages," as Muraskin puts it in the afterword, contributed to the development of many safer vaccines which are now in common use.
While the volume covers a great deal of historical material, it leaves open a number of questions on immunization today. For instance, how are European states responding to homogeneous EU guidelines for immunization strategies? How does the face of civil resistance change if nearly all global vaccine production is now in the hands of multinational pharmaceutical companies instead of national institutes?
Despite such minor shortcomings, however, this volume is an overdue benchmark in the study of immunization, providing a basis for the analysis of current and historical political struggles and public health strategies on a national, transnational and global level. It is to be hoped that the volume will encourage further inquiry into this ever relevant field.
Eva Jansen is a medical anthropologist and Fritz Thyssen postdoctoral fellow at the University of Tübingen. Currently, she is working on cancer and complementary medicine in Germany. She is also interested in migration, health policies, and Science and Technology Studies. Eva is the author of "Naturopathy in South India: Clinics between Professionalization and Empowerment", published by Brill in 2016.


WFTDA Playoffs Announced – So does Derby Math Rankings work???
So here we are, did Derby Math Rankings actually work? Could you accurately produce rankings on a weekly (or close to weekly) basis to show how teams are really setting up for the upcoming playoffs. Im going to go with a big YES on that one. My goal was to never be perfect, those who have been here thru the whole year know that my goal was to be close and give teams a good idea of what their rankings will be come playoffs. I've discussed there will be a margin of error involved due to my methods, and it proved to be slightly higher than I anticipated from a WFTDA Score perspective, but I think you will see that not only was I able to come pretty close with just the Top 40 (which is easier due to the wide range between teams) but you will also see that I was able to be pretty close with most of the Top 100. For those looking for a full breakdown of the playoffs check out the WFTDA website.
As you will see below 24 of the 40 D1 teams rankings were perfect and the other 16 were only off by one placing. That isnt too shabby. Better yet is that I correctly picked the correct seed for 31 of the 40 teams. Again that shows just how good the rankings were at being able to give a good idea (again not perfect) of where a team will end up.
D2 was a little harder as there are half as many teams, but I still accurately picked 10 of the 20 seeds correctly. There were a couple of bigger misses as Santa Cruz, Sacred City, Emerald City and Chicago Outfit all came in at 2 away from their projections. With the incredibly tight race that happened at the top of D2 this year that wasnt that unexpected.
And then going down the rest of the Top 100 you will see that a majority of the teams were pretty close to their projections, with the biggest exceptions being teams that played unranked teams right at the end of the season. Without being able to see an initial ranking (or knowing the WFTDA Strength Factor Challenge score) it was a bit hard to get them down just right.
I'm not going to make a huge post tonight as I know you all are incredibly excited for the actual playoffs coming up, but just remember for 2016 that Derby Math can help you get a good projection of where in the overall playoff spectrum your team may fall.


This Giving Tuesday, consider making a donation to help us help others. We are in need of repairs to help start some new programs to help the community. We want to be a place for more than just worship, for more than just members of Saint Luke's, but we need your help to reach our goals.


The EPR o-ring is the standard material of construction fro the o-ring as it is supplied on the 8880361 adapter assembly that connects the syringe to the controller and delivers the air supply to the piston inside the syringe.
Is your material eating the o-ring? Try one of our 8880379 Viton O-ring packs!


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Annual Bioethics Conference
Harvard Clinical Bioethics Course
Contemporary Authors in Bioethics Series
Weekly Consortia
Public Bioethics Forums
Behind Bars 2017
Mobile Utility Navigation
MyHMS
Clinical Affiliates and Ethics Leaders Group
Community Ethics Committee
Opportunities & Resources
Master of Bioethics
Fellowship in Bioethics
Responsible Conduct of Research (RCR)
Medical Ethics & Professionalism (MA 750)
Annual Beecher Prize in Medical Ethics
Behind Bars: Ethics and Human Rights in U.S. Prisons
A two-day conference examining human rights and health issues in U.S. prisons.
Harvard Medical School Campus, Boston, MA
Danielle Allen, James Bryant Conant University Professor
Keynote Speaker:
Danielle Allen, PhD
Director, Edmond J. Safra Center for Ethics, Harvard University
James Bryant Conant University Professor
The United States leads the world in incarceration. The "War on Drugs" and prioritizing punishment over rehabilitation has led to mass imprisonment, mainly of the nation's most vulnerable populations: people of color, the economically disadvantaged and undereducated, and those suffering from mental illness. Although these social disparities are striking, the health discrepancies are even more pronounced. What can be done to address this health and human rights crisis?
This conference examined various aspects of human rights and health issues in our prisons. In collaboration with educators, health professionals, and those involved in the criminal justice system—including formerly incarcerated people, advocates, and law enforcement—the conference clarified the issues, explored possible policy and educational responses, and established avenues for action.
2017 Prison Conference Presenters
2017 Prison Conference Schedule
13th Documentary & Discussion
Behind Bars Conference Keynote Address by Danielle Allen
The Behind Bars: Ethics and Human Rights in U.S. Prisons conference was open to the public. Registration was required to attend. There was a $50 refreshment fee to offset costs for meals during the conference, and to reduce waste. If this fee would prevent you from attending, please contact the Center for Bioethics at 617-432-2570 for assistance with your registration.
The Conference was co-sponsored by the Center for Bioethics at Harvard Medical School, the Human Rights Program at Harvard Law School, the Edmond J. Safra Center for Ethics at Harvard University, and the Petrie-Flom Center for Health Law Policy, Biotechnology, and Bioethics at Harvard Law School, with support from the Oswald DeN. Cammann Fund.

redpajama_set_name: RedPajamaCommonCrawl


text:
Frontiers in Plant Science
Maoteng Li
Huazhong University of Science and Technology, China
Chunyu Zhang
Huazhong Agricultural University, China
Liezhao Liu
College of Agronomy and Biotechnology, Southwest University, China
Front. Plant Sci., 02 September 2016 | https://doi.org/10.3389/fpls.2016.01313
Morphological Structure and Transcriptome Comparison of the Cytoplasmic Male Sterility Line in Brassica napus (SaNa-1A) Derived from Somatic Hybridization and Its Maintainer Line SaNa-1B
Kun Du1, Qier Liu1, Xinyue Wu1, Jinjin Jiang1, Jian Wu1, Yujie Fang1, Aimin Li2 and Youping Wang1*
1Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou, China
2Institute of Agricultural Sciences for Lixiahe Region in Jiangsu, Jiangsu Academy of Agricultural Sciences, Yangzhou, China
SaNa-1A is a novel cytoplasmic male sterility (CMS) line in Brassica napus derived from progenies of somatic hybrids between B.napus and Sinapis alba, and SaNa-1B is the corresponding maintainer line. In this study, phenotypic differences of floral organs between CMS and the maintainer lines were observed. By microscope observation in different anther developmental stages of two lines, we found the anther development in SaNa-1A was abnormal since the tetrad stage, and microspore development was ceased during the uninucleate stage. Transcriptomic sequencing for floral buds of sterile and fertile plants were conducted to elucidate gene expression and regulation caused by the alien chromosome and cytoplasm from S. alba. Clean tags obtained were assembled into 195,568 unigenes, and 7811 unigenes distributed in the metabolic and protein synthesis pathways were identified with significant expression differences between two libraries. We also observed that genes participating in carbon metabolism, tricarboxylic acid cycle, oxidative phosphorylation, oxidation–reduction system, pentatricopeptide repeat, and anther development were downregulated in the sterile line. Some of them are candidates for researches on the sterility mechanism in the CMS material, fertility restoration, and improvement of economic traits in the maintainer line. Further research on the tags with expressional specificity in the fertile line would be helpful to explore desirable agronomic traits from wild species of rapeseed.
Heterosis is important to improve rapeseed yield, and cytoplasmic male sterility (CMS), as one of the ideal systems for pollination control, has significantly contributed to increase rapeseed production. According to the origin of the cytoplasm, CMS in rapeseed was classified into two types (Yamagishi and Bhat, 2014). The first type was derived from mutation or intergeneric hybridization during natural reproduction, including pol CMS, Shan2ACMS, and nap CMS. To date, 70% of the rapeseed hybrids in China were bred using CMS lines as a pollination control system, of which pol CMS and Shan2A CMS were extensively used (Shen et al., 2008). The second type was obtained by nucleus substitution or mitochondrial gene recombination during wide hybridization or protoplast fusion between different species, including Ogura/kos CMS and tour CMS (Leino et al., 2003). As reported, CMS is a maternally inherited trait resulting from the interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene (Yamagishi and Bhat, 2014). For instance, the mitochondrial genome of pol CMS contains a causal gene named orf224, a chimeric gene located at upstream of and co-transcribed with the atp6 gene. In the presence of Rf gene, processing of orf224/atp6 is affected and the transcripts are specifically altered (L'Homme and Brown, 1993).
Somatic hybridization can effectively create new CMS lines by potential mitochondrial genome recombination between two parent lines. The recombination results in new open reading frames and forms chimeric genes, which affect the function of mitochondria and finally lead to the formation of CMS (Hanson and Bentolila, 2004; Carlsson et al., 2008). Previous studies reported that the mitochondrial structure of kos CMS and tour-Stiewe CMS bred from somatic hybrids differed from that of the CMS lines (kos and tour CMS) derived from the natural mutation of radish and mustard (Dieterich et al., 2003; Oshima et al., 2010). A sterile line (SaNa-1A) containing 38 chromosomes was previously selected from the BC3 progenies of Brassica napus–Sinapis alba somatic hybrids, using B. napus cv. "Yangyou6" as recurrent parent (Wang et al., 2005). The corresponding maintainer line (SaNa-1B), which could ensure sterility by hybridization with the CMS line, was selected by crossing SaNa-1A with different varieties of B. napus (Zheng et al., 2012).
Next-generation sequencing (NGS), including RNA-seq and digital gene expression (DGE), has been extensively applied in transcriptomic studies on plants. Platforms, such as Illumina HiSeq2500, Ion Torrent, and Roche 454 GS FLX+, ensure more proficient research on transcriptomes and genomes than DGEs and microarrays by improving the sequencing efficiency and reducing the costs (Liu et al., 2013; Wu et al., 2016). Recently, NGS has also been used in analyzing plant mitochondria genomes and gene expression in CMS lines. An et al. (2014) created the NIL line of pol CMS and analyzed the gene expression differences between floral buds of the sterile and fertile lines using RNA-seq. They identified that energy deficiency controlled by orf224/atp6 led to the down regulation of genes regulating anther development, thus resulting in failure of sporogenous cell differentiation and pollen abortion (An et al., 2014). Yan et al. (2013) compared the gene expression differences between the young bud (<2 mm) of Nsa CMS and its restorer line NR1 using DGE analysis, and 11 differentially expressed genes (DEGs) uniquely expressed in the restorer line were related to the synthesis of pollen wall, including chalcone synthase gene, β-1,3-glucanase gene, and glycosyl hydrolase gene. Of these DEGs, 40% were annotated with catalytic activity, 4.4% were annotated with transporter activity, and 2.2% were annotated with antioxidant activity (Yan et al., 2013).
In the present study, we aim to compare the morphological and cytological differences between the CMS line and its maintainer line to better understand the mechanism of abortion in the CMS line. Subsequently, RNA-seq analysis was conducted on the floral bud of two rapeseed lines at the abortion stage in accordance with the cytological result. This work could be the basis for molecular dissection of the CMS mechanism in SaNa-1A.
Plant Materials
The novel CMS line (SaNa-1A) generated from progenies of somatic hybrids between B. napus and S. alba and the maintainer line (SaNa-1B) were used in this study. Plant materials were cultivated in the experimental fields of Jiangsu Institute of Agricultural Science in the Lixiahe District (Yangzhou, Jiangsu Province). Different sizes of sterile and fertile anthers were fixed for morphological analysis, aiming to acknowledge the abortion stage. In accordance with the abortion stage (tetrad stage), floral buds (0.6–1.3 mm in length) were collected and frozen at 80°C prior to semithin sections and RNA-seq.
Semithin Sections and Light Microscopy
Different sizes of sterile and fertile anthers were fixed in 2.5% glutaraldehyde. After 24 h, the fixative solution was replaced with a fresh solution. After 1 day, the anther was washed three times with phosphate buffer (PB; pH 7.2; 15 min for each wash) and postfixed with 1% osmium tetroxide for 4 h. The postfixed tissue was again washed three times with PB. The anther was dehydrated with a graded ethanol series (50, 70, 80, 90, 95, and 100%) for 15 min each, infiltrated with acetone, and embedded in 812 resin. Semithin sections (1 μm) were cut from the polymerized blocks and stained with 1% toluidine blue for 3 min for light microscope observations (Olympus CX51).
RNA Extraction and Illumina/Solexa Sequencing
Total RNA from buds (0.6–1.3 mm in length, at abortion stage) of four sterile and fertile individual plants was extracted using RNAiso Plus (Takara, China) in accordance with the protocol of the manufacturer (Wang et al., 2014). The quality of total RNA was analyzed using the Agilent 2100 BioAnalyzer with threshold values of RIN ≥ 8 and 28S:18S RNA≥ 1.5:1. Then, RNA (0.1–4 μg) was precisely quantified using the QUBIT RNA Assay Kit. mRNA was purified with oligo(dT) and fragmented into 120–210 bp. cDNA was synthesized using the SuperScript II Kit (Invitrogen), following end-filling and adding A in the 3′-end. After ligation with the Illumina paired-end adapter, cDNAs were purified twice with AMPure XP Beads (Beckman) to eliminate redundant adapters and amplified with PCR. Finally, a gel purification procedure was conducted to select the fragments ranging from 300 to 350 bp to produce the paired-end library. Fragment size was controlled by the High-Sensitivity ChIP Kit, and the precise concentration of each library was tested with the KAPA qPCR Kit. A 10 μL library (2 nM) was fixed onto cBot and sequenced with Illumina HiSeq 2000.
Processing of Sequencing Tags and Gene Expression Annotation
According to Cox et al. (2010), clean reads were obtained by filtering the adapter sequences and low-quality sequences in the raw data using the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit), which were then assembled into contigs by the Velvet and Oases software (Zerbino and Birney, 2008). Afterwards, the clean reads were mapped onto the unigenes using Bowtie (Langmead et al., 2009). Function of unigenes was annotated by BLASTX searches against the NR, Swiss-Prot, and COG databases (E < 1e−5). The GO annotations for them were determined using Blast2GO (Conesa et al., 2005), which were then submitted to WEGO for the classification graph (Ye et al., 2006; An et al., 2014). For GO enrichment analysis, a corrected p ≤ 0.05 was chosen as the threshold value. The GO term (p ≤ 0.05) is defined as significantly enriched terms of DEGs. Pathway annotations of differential gene expression were conducted to understand gene function through BLASTX of the KEGG database. The raw reads were deposited in the NCBI Short Read Archive with the accession number SRP075203. The assembled unigenes are shown in Additional File 1.
Analysis of DEGs
After filtering the low-quality data, all the tags were aligned to the unigenes. Similar to credibility interval approaches reported for the analysis of SAGE data (Vêncio et al., 2003), we employed IDEG6 (Romualdi et al., 2003) to identify mRNAs showing statistically significant differences based on their relative abundance (as reflected by the total count of individual sequence reads) between the two libraries. The general chi-square test was conducted, as it has been proven to be one of the most efficient tests. Finally, genes with p ≤ 0.01 and fold change ≥ 2 or ≤ 0.5 were marked significantly different between the two libraries.
qRT-PCR Analysis of DEGs
The DGE results were verified by real-time qRT-PCR analysis, using the same RNA samples for library construction. Two developing stages of buds (~2 mm in length, at tetrad stage; 3–5 mm in length, at or after uninucleate stage) were chosen for expression validation. First-strand cDNA was synthesized using the Revert Aid First Strand cDNA Synthesis Kit (Thermo, USA) from 1200 ng total RNA. Gene-specific primers were designed for the selected unigene sequences (Additional File 2). Reactions were conducted with the SYBR PrimeScript™ RT-PCR Kit (TaKaRa, China) in the Bio-Rad CFX96 instrument. The PCR cycling was denatured using a program of 95°C for 10 s and 40 cycles of 95°C for 5 s and 55°C for 30 s. Three biological replicates for each sample and three technical replicates were conducted, and the relative expression level was calculated using the 2−ΔΔCt method. Then, B. napus actin was used to normalize gene expression.
Phenotypic Characterization of Fertile and Sterile Floral Buds
The flower organ in rapeseed comprises the sepal, petal, stamen, and pistil. The male reproductive organ in normally developed rapeseed flower has four stamens longer than the other two stamens. Each stamen comprises the filament and anther, which are located at the top of each flower (Figure 1B). However, sterile flowers were visually smaller than fertile flowers, and wrinkles were clearly observed on sterile petals. During the developmental process, the anthers and filaments in sterile flowers were shorter than that in fertile flowers from the tetrad stage. The sterile anthers produced only a few or no pollen, but the pistil was normal (Figure 1A).
Figure 1. Phenotypic characterization of fertile and sterile floral buds. (A) Phenotype of sterile floral buds and (B) phenotype of fertile floral buds. Sp, sporogenous cell; PMC, pollen mother cell; Td, tetrad stage; early Uni, early uninuclear stage; late Uni, late uninuclear stage; MP, mature pollen.
Anther development in the fertile and sterile lines were cytologically observed in different developmental stages to determine the abortion stage of the sterile line. At the PMC stage, primary parietal cells were differentiated into the anther epidermis, endothecium, middle wall layers, and tapetum after periclinal division and surrounded the microspore mother cells, which were in the middle of each pollensac (Figure 2B1). At the tetrad stage, PMC cells were separated and formed tetrads after meiosis and were surrounded with callose. The middle wall layers disappeared gradually, and the tapetum cells were abundant with cytoplasm (Figure 2B2). At the uninucleate stage, the microspores were mostly occupied with central vacuole, with the nucleus squeezed to the edge, and tapetum cells started to degrade (Figure 2B3). Afterward, the microspores were differentiated into pollen grains, which were released after the breakdown of pollen sac wall (Figures 2B4,B5). Compared with the maintainer line, tapetum cells in the sterile line were observed with vacuolation during the microsporocyte and tetrad stages (Figures 2A1,A2), although they could form pollensac. In the uninucleate stage, tapetum cells were dissolved and moved to the center of the pollensac, and the cytoplasm was condensed and located at the center of microspores released from the tetrad (Figure 2A3). After the late uninucleate stage, microspores failed to separate and no mature pollen was formed, which were degraded together with tapetum cells (Figures 2A4,A5). The main reason for sterility was the obstacle in the degradation of tapetum cells, which hindered the necessary nutrition that microspores needed. We observed that the abortion of this sterile line occurred during the tetrad stage.
Figure 2. Comparison of sterile (A1–A5) and fertile (B1–B5) anther development with toluidine blue O staining. Bar = 10 μm for all of the stages. Ep, epidermis; En, endothecium; ML, middle layer; T, tapetum; Ms, microspore; MP, mature pollen.
Transcriptome Sequencing and Assembly
After removing low-quality tags in the raw data, 36,539,702 and 43,903,006 clean tags were obtained in the sterile and fertile lines, respectively (Additional File 3). A total of 195,568 unigenes (1326.8 bp on average) were obtained from clean data (average length = 96 bp) by de novo assembly with the Velvet and Oases software (Additional File 4). Considering the N50 value and average length, we select k-mer = 51 for de novo assembly. Meanwhile, 184,146 unigenes (94.2%) were equal to or greater than 200 bp and 109,168 unigenes (55.8%) were equal to or greater than 1000 bp (Table 1).
Table 1. Statistic of assembly length (by Velvet and Oases software, k-mer = 51).
For functional annotation, the 195,568 unigenes were subjected to BLASTX searches against the sequences in the Swiss-Prot, non-redundant protein sequences in the National Center for Biotechnology Information (NCBI; NR), and Clusters of Orthologous Groups (COG) databases (E ≤ 1e−5). A total of 122,380 (62.6%), 186,245 (95.2%), and 70,315 (36.0%) unigenes were annotated in the Swiss-Prot, NR, and COG databases, respectively. Meanwhile, 68,429 unigenes (35.0%) were well annotated in all of the databases and 186,752 unigenes (95.5%) were identified with annotation at least in one databases (Figure 3). In the COG database, these unigenes were classified into 24 functional groups, including general function prediction only (23.61% of unigenes), posttranslational modification, protein turnover, chaperones (9.4% of unigenes), and nuclear structure (0.04% of unigenes) (Figure 4). All the unigenes were displayed by searching GO database and classified into three hierarchies, cellular location, molecular function, and biological process. Of the 45 GO groups, unigenes were mainly classified in cell and cell part (33.90% of unigenes), binding (48.37% of unigenes), and metabolic process (33.99% of unigenes). Only a few unigenes were classified into virion, locomotion, and viral reproduction (Figure 5).
Figure 3. Unigenes annotated with the public databases. The numbers of annotated unigenes were signified in the different regions.
Figure 4. COG functional classification. All of the unigenes aligned in the COG database were sorted into 24 clusters.
Figure 5. Classification of GO annotations. The x-axis indicates the subcategories; the left y-axis indicates the percentage of a subcategory of genes in that category; and the right y-axis indicates the number of unigenes in a subcategory.
Differential Gene Expression between Sterile and Fertile Buds
The expression abundance of tag-mapped genes was analyzed by counting the number of transcripts per million (TPM). We noted that unigenes in the fertile and sterile buds showed high expression levels in several basic bioprocesses. For example, unigene304 (TPM = 663.76 in Fer and TPM = 1517.84 in Ste) encodes ribulose bisphosphate carboxylase that participates in energy production and conversion; unigene24 (TPM = 610.47 in Fer and TPM = 496.96 in Ste) encodes ubiquitin, which functions in cellular processes and signaling; and unigene78636, which is involved in RNA transport, showed high expression levels in fertile and sterile buds. With the threshold p ≤ 0.01 and fold change ≥2 or ≤0.5, 7811 unigenes (3.92%) were identified with expression differences between sterile and fertile lines, of which 1736 unigenes were upregulated and 6.075 unigenes were downregulated in the sterile line compared with the fertile line (Table 2 and Additional File 5). These DEGs with at least two-fold differences in the two libraries are shown in Figure 6. The red and green dots represented transcripts higher or lower in abundance for more than two-fold in the sterile library, respectively. The blue dots represented transcripts that differed less than two-fold between the two libraries, which were arbitrarily designated as "no difference in expression."
Table 2. Differentially expressed transcripts in sterile and fertile buds.
Figure 6. Differentially expressed unigenes and corresponding genes in the Ste and Fer lines. Red dots represent the upregulated transcripts in the sterile library, green dots denote the downregulated transcripts in the sterile library, and blue dots indicate the transcripts that did not change significantly. The parameters "p ≤ 0.01" and "fold change ≥2 or ≤0.5" were used as the threshold to evaluate the significance of gene expression difference.
Pathway Analysis and GO Annotation of DEGs
Different genes cooperate to achieve their biological functions. Pathway-based analysis helps in further understanding the biological functions of DEGs. The upregulated and downregulated unigenes were submitted to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatics Annotation Server and classified using the single-directional best hit method (Kanehisa et al., 2008). Most of the upregulated unigenes functioned as transcription factors (TFs). By contrast, a large number of the downregulated unigenes functioned as lipid biosynthesis proteins, chaperones and folding catalysts, protein kinases, prenyltransferases, peptidases, ubiquitin systems, translation factors, and TFs (Table 3). GO annotation of DEGs revealed that, compared with the fertile line, the upregulated unigenes in the sterile line mainly functioned in the binding, cellular process, cell, and cell part. In addition, downregulated unigenes mainly functioned in the catalytic activity, binding, and metabolic process (Figure 7).
Table 3. KEGG annotation of DEG unigenes.
Figure 7. GO annotations of DEGs. The x-axis indicates the subcategories; the left y-axis indicates the percentage of a subcategory of genes in that category; and the right y-axis indicates the number of unigenes in a subcategory.
GO enrichment analysis of functional significance was conducted to reveal significantly enriched GO terms of DEGs. The GO term with p ≤ 0.05 was defined as significantly enriched GO term of DEGs. For molecular function, significantly enriched GO terms of DEGs included catalytic activity, ATP binding, oxidoreductase activity, transferase activity (transferring phosphorus-containing groups), and zinc ion binding (Additional File 6). For enriched biological processes, 10 GO terms of DEGs were available, which participated in the oxidation–reduction process, pollen development, transmembrane transport, carbohydrate metabolic process, cellular metabolic process, lipid metabolic process, fatty acid biosynthetic process, response to oxidative stress, cell wall modification, and cellular lipid metabolic process (Additional File 7).
Genes Related to Pollen Development
The development of pollen is a fundamental and complex process in flowering plants. Pollen is essential for propagation and evolution. As a model plant, Arabidopsis thaliana has been well investigated for this bioprocess. Therefore, all of the unigenes identified in this study were annotated to the TAIR database. Unigenes annotated to 15 genes regulating pollen development (from AG to MS2) were analysized (Higginson et al., 2003; Koizuka et al., 2003; Yang et al., 2003, 2005). One gene encoding tapetum determinant 1 (BnTPD1, unigene40544) was significantly upregulated in the sterile line than in the fertile line (log2ratio = 2.128). We determined that 13 unigenes were downregulated in the sterile line, including BnAG (unigene560, log2ratio = −5.517), BnNZZ/SPL (unigene15428, log2ratio = −1.951), BnEMS1 (unigene4387, log2ratio = −2.569), BnSERK1 (unigene11458, log2ratio = −2.206), BnDYT1 (unigene31429, log2ratio = −3.135), BnMYB99 (unigene2438, log2ratio = −7.081), and BnMYB26 (unigene14326, log2ratio = −2.147). These genes mainly participated in the formation and differentiation of tapetum. Moreover, BnTDF1 (unigene5833, log2ratio = −5.56), BnAMS (unigene472, log2ratio = −6.74), BnMYB103 (unigene25542, log2ratio = −6.639), BnMS1 (unigene17214, log2ratio = −5.98), BnMS2 (unigene1036, log2ratio = −8.279), and BnA6 (unigene90, log2ratio = 8.251) relate to the functional regulation of tapetum at the later developmental stage, such as degradation of callose and formation of pollen wall. We also identified unigenes with no significant expressional difference, and several other genes were unidentified in the sterile and fertile lines (Additional File 8).
Genes Involved in Carbon Metabolism
Accumulation of starch is necessary for the development of microspore. In other words, abundance of starch is an important characteristic of fertile pollen. Genes participated in carbon metabolism were differentially expressed between fertile and sterile lines. Altered expression of numerous genes involved in pentose and glucuronate interconversions, starch and sucrose metabolism, and amino sugar and nucleotide sugar metabolism were observed (Additional File 5). With regard to pentose and glucuronate interconversions, 13 genes were significantly downregulated, including 6 genes encoding aldehyde dehydrogenase (unigene242, unigene1535, unigene2791, unigene5839, unigene7055, and unigene7131), 4 genes encoding pectinesterase family protein (unigene7776, unigene8408, unigene17104, and unigene25928), and 3 genes encoding pectate lyase family protein (unigene13148, unigene15151, and unigene31020). Moreover, 9 genes related to starch and sucrose metabolism were significantly downregulated, including 5 genes encoding pfkB-type carbohydrate kinase family protein (unigene981, unigene2366, unigene4077, unigene16521, and unigene20485), 1 gene encoding beta-glucosidase (unigene4620), 2 genes encoding glycosyl hydrolase (unigene13516 and unigene17104), and 1 gene encoding cell wall invertase (unigene13728). Of the genes mentioned above, unigene8408, unigene17104 and unigene13148 were not identified in sterile line. Two genes participating in amino sugar and nucleotide sugar metabolism were downregulated, encoding mannose-6-phosphate isomerase (unigene25396, log2ratio = −2.859) and 6-phosphofructokinase (unigene28654, log2ratio = −2.907), respectively.
DEGs Involved in Citrate Cycle [Tricarboxylic Acid (TCA) Cycle] and Oxidative Phosphorylation
The mitochondria is an important site for numerous metabolic pathways, including the TCA cycle, respiratory electron transfer, and ATP synthesis (Barrientos, 2003; Reichert and Neupert, 2004; Logan, 2006). The recombination and rearrangement of mitochondrial genomes could cause dysfunction and lead to CMS. Flower phenotypic variation or defection in pollen formation are presumed as secondary effects of mitochondrial mutation, and the primary defect may be a reduction in the efficiency of respiration or the impairment of other mitochondrial functions. We determined that six unigenes participating in the TCA cycle were significantly downregulated, with expressions increased by 3.43–53.47-folds in Fer, such as unigene12291 encoding a subunit of aconitate hydratase, unigene5423 encoding a subunit of citrate synthase, unigene232 encoding phosphoenolpyruvate carboxylase 2, unigene2462 encoding peroxisomal nad-malate dehydrogenase 1, unigene579 encoding 2-oxoglutarate dehydrogenase E1 component, and unigene40435 encoding the subunit B2 of ATP citrate lyase (Additional File 5). Citrate synthase, as a key enzyme of the TCA cycle, functions in the formation of citrate using acetyl-CoA and oxaloacetic acid as substrate. We also identified 11 DEGs related to the electron transport chain and oxidative phosphorylation, which were downregulated in the sterile line compared with the fertile line. For instance, four genes (unigene1243, unigene5691, unigene46531, and unigene31126) encoding subunits of ATPase, three genes (unigene7780, unigene56318, and unigene27486) encoding subunits of pyrophosphorylase, unigene26033 encoding subunits of cytochrome oxidase, unigene15953 and unigene952 encoding NADH dehydrogenase, and unigene1560 encoding subunit of ubiquinol oxidase were included (Additional File 9).
Genes Involved in Oxidoreductase Activity
In the present study, oxidoreductase activity is one of the most enriched GO terms. A total of 145 DEGs were involved in oxidoreductase activity, including 36 members acting on the aldehyde or oxo group of donors, with NAD or NADP as acceptor. For instance, fatty acyl-CoA reductase (alcohol-forming)/oxidoreductase (unigene1036 and unigene7520), also called male sterility 2 (MS2), acting on the CH–CH group of donors, with NAD or NADP as acceptor, was more abundant in the fertile line. Other DEGs were 3-chloroallyl aldehyde dehydrogenase/aldehyde dehydrogenase (unigene1535, unigene5839, and unigene7055), aldehyde oxidase (unigene3230, log2ratio = −2.686), NAD or NADH binding/catalytic/glyceraldehyde-3-phosphate dehydrogenase (unigene1766, log2ratio = −1.898), cytosolic factor family protein (unigene12736 and unigene27821), and several unnamed oxidoreductase members (Additional File 5).
Differentially Expressed Pentatricopeptide Repeat (PPR) Proteins
Proteins encoding the PPR motif are predicted as site-specific, RNA-binding adaptor proteins that mediate the interactions between RNA substrates and relative enzymes (Lurin et al., 2004; Stern et al., 2004; Shikanai, 2006). PPR proteins play important role in plant mitochondrial biogenesis (Wen et al., 2003). Most of the cloned restorer genes encode mitochondria-targeted PPR proteins (Brown et al., 2003; Desloire et al., 2003; Kazama and Toriyama, 2003; Koizuka et al., 2003; Akagi et al., 2004; Wang et al., 2006). In this study, 12 genes encoding PPR proteins (unigene1911, unigene16513, unigene16881, unigene17672, unigene20560, unigene21040, unigene23771, unigene27924, unigene28403, unigene40662, unigene42447, and unigene44799) were downregulated and one gene (unigene57904) was upregulated (Additional File 5). These PPR proteins were candidates for analyzing the link between nuclear and mitochondrial gene expression.
Confirmation of DEGs by Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
We randomly selected 16 genes for qRT-PCR assays to confirm the reliability of the Illumina/Solexa sequencing technology. The corresponding primers are listed in Additional File 2. The results showed that the expression of 14 genes were consistent between qRT-PCR and RNA-seq analyses (Figure 8). In qRT-PCR, unigene39076 and unigene36457 showed no difference in the fertile and sterile buds, whereas RNA-Seq analysis indicated a significant difference. The inconsistency between qRT-PCR and RNA-seq analyses of certain genes can be likely attributable to the fact that RNA-seq was more sensitive in the detection of low-abundance transcripts and small expressional changes than qRT-PCR.
Figure 8. qRT-PCR verification of differentially expressed unigenes. S denotes the sterile sample and F denotes the fertile sample.
Some considerable differences from the morphology of floral organs between sterile and fertile lines were observed. And by comparing different anther developmental stages between the two lines through microscope observation, we found that anther development in SaNa-1A was abnormal since the tetrad stage, and the development of microspores ceased during the uninucleate stage.
Subsequently, the transcriptome of fertile and sterile buds in rapeseed were acquired using the Illumina sequencing. A total of 195,568 unigenes were obtained, and 4% of them (7811 unigenes) were identified with significant expression differences, indicating that, although the bud development is a complicated and polygenic process, expressional changes in a small number of genes can alter the trait observably. All the DEGs could be categorized into 35 classifications by GO functional analysis, which were involved in biochemistry, metabolism, growth, development and so on (Figure 7). For enriched biological processes, ten GO terms of DEGs were involved in oxidation–reduction process, pollen development, transmembrane transport, carbohydrate metabolic process, cellular metabolic process, lipid metabolic process, and so on (Additional File 7). These DEGs would be candidates for further research.
Pollen formation in flowering plants depends on the differentiation and interaction of the reproductive cells named microsporocytes and the somatic cells that form the tapetum. The microsporocytes generate microspores, and the tapetum cells support the nutrition for development of mature pollen grains. A comparison of morphological structure during anther development revealed that the tapetum in the sterile line was vacuolated during the tetrad stage. Moreover, microspore development was aberrant in the sterile line during the uninucleate stage compared with the fertile line, resulting in pollen abortion. The TPD1 gene is necessary for tapetum cell specialization in the Arabidopsis anther (Yang et al., 2003). In male sterile mutant tpd1, the precursors of tapetum cells were differentiated and developed into microsporocytes instead of tapetum. Ectopic overexpression of TPD1 in the wild-type Arabidopsis carpel caused a significant increase in the number of carpel cells. Furthermore, the activation of cell division in the transgenic carpel by TPD1 overexpression was dependent on EMS1/EXS (Yang et al., 2005). AtMYB103, as a member of the R2R3 MYB family, is uniquely expressed in the tapetum and anther. In the transgenic line with downregulated AtMYB103, the shape of the pollen was distorted, the content of the cytoplasm was reduced or no cytoplasm could be observed, and early degeneration of the tapetum occurred (Higginson et al., 2003). Moreover, AMS acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis (Xu et al., 2014). In the present study, we observed that the expression of BnAG, BnSPL/NZZ, BnEMS1, BnAG, and BnDYT1 was significantly downregulated in the sterile lines, which might cause abnormal tapetum development in the sterile line. The observed downregulation of BnAMS, BnMYB103, BnMS1, and BnMS2 in the sterile line could directly block the synthesis of sporopollenin in the pollen wall. It has been reported that nuclear-mitochondrial interaction results in CMS in previous studies (Chase, 2007; Jing et al., 2012). These genes were downregulated, which consequently led to the cease of microspores development in the sterile buds.
Rearrangements of mitochondria genome could alter the expression of genes involved in respiration and ATP synthesis, affecting ATP formation and physiological processes in mitochondria (Bergman et al., 2000). Depression in ATP production or carbohydrate accumulation has been identified by other researchers in "late stage" CMS flowers (Datta et al., 2002). In this study, six DEGs involved in the TCA cycle were downregulated in the sterile line. DEGs involved in respiration/ATP synthesis and oxidoreductase activity were also identified. And 11 DEGs were involved in the oxidative phosphorylation pathway, which affects NADH dehydrogenase, pyrophosphorylase, and ATP synthase, and these genes were downregulated in the sterile line. Oxidoreductase activity was one of the most enriched GO terms, whereas oxidative stress during microsporogenesis was assumed to induce premature abortion of tapetum cells because of programmed cell death in CMS lines of sunflower and rice (Balk and Leaver, 2001; Chinnery, 2003; Li et al., 2004). Expressional alternation of numerous genes involved in pentose and glucuronate interconversions, starch and sucrose metabolism, and amino sugar and nucleotide sugar metabolism were also observed. The nature of mitochondrial genes influencing the expression of nuclear genes was unclear. Further investigations on them will help illuminating the primary targets and downstream components of CMS-associated mitochondrial signaling pathways in the sterile line.
Nuclear genes are important to control the biosynthesis and function of mitochondria (Andersson et al., 2003). Approximately 10% of eukaryotic nuclear genes encode proteins that are targeted to mitochondria following the synthesis of cytosolic ribosomes (Fisk et al., 1999). The majority of PPR proteins are targeted to plastid or mitochondria (Nakamura et al., 2003), and many genetic and biochemical studies conclude that PPRs directly bind to a specific RNA sequence and promote anterograde regulation, such as posttranscriptional splicing, processing, editing, or regulating mRNA stability (Kotera et al., 2005; Schmitz-Linneweber et al., 2005; Okuda et al., 2006, 2007; Hattori et al., 2007; de Longevialle et al., 2007). To date, 60% of 450 PPR proteins in Arabidopsis were predicted to be related to mitochondria. PPR motifs have been proposed to possess binding properties to proteins and RNA (Small and Peeters, 2000; Williams and Barkan, 2003). Mutation of these genes in humans is accompanied by cytochrome c oxidase deficiency (Mootha et al., 2003; Shadel, 2004). PPR proteins are valuable candidates for nuclear-encoded factors controlling distinct steps of transcript maturation in mitochondria. Many of these PPR proteins may be essential to the CMS systems, such as the Rf gene encoding a member of the PPR (Yamagishi and Bhat, 2014). PPR proteins have also been reported as targets of different miRNAs (Rhoades et al., 2002; Sunkar and Zhu, 2004). In this study, 13 PPR proteins showed differential expression between two rapeseed lines, including 1 upregulated PPR protein and 12 downregulated PPR proteins in the sterile line. These PPR proteins are candidates for analyzing the link between nuclear and mitochondrial gene expression.
In this study, we compared the transcriptome data between floral buds of the sterile and fertile lines in rapeseed using high-throughput transcriptomic sequencing. A total of 7811 genes were identified with expression differences between the 2 rapeseed lines, of which 1736 genes were upregulated and 6075 genes were downregulated in the CMS line compared with the maintainer line. GO-based and pathway-based analyses indicate that these DEGs were related to diverse molecular functions. The results provide a basis for future research on the CMS mechanism in SaNa-1A, fertility restoration, and improvement of agronomic traits. We should focus on characterizing the functions of these candidate genes.
YW conceived and designed the study. KD, QL, XW, and JJ participated in the experiments. JW and YF analyzed the data. AL bred the material. All authors drafted the manuscript and approved the final manuscript.
This study was supported by the NSFC project (31571699), the Jiangsu Province Science Foundation (BK20140478, BK20150441), China Postdoctoral Program (2016T90511, 2016T90514), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Yangzhou University for Excellent Talent Support Program.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fpls.2016.01313
CMS, Cytoplasmic male sterility; COG, Clusters of orthologous groups; DGE, Digital gene expression; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NGS, Next-generation sequencing; PB, Phosphate buffer; PPR, Pentatricopeptide repeat; qRT-PCR, Quantitative reverse transcription polymerase chain reaction; TCA, Tricarboxylic acid; TFs, Transcription factors; TPM, Transcripts per million.
Akagi, H., Nakamura, A., Yokozeki-Misono, Y., Inagaki, A., Takahashi, H., Mori, K., et al. (2004). Positional cloning of the rice Rf-1 gene, a restorer of BT-type cytoplasmic male sterility that encodes a mitochondria-targeting PPR protein. Theor. Appl. Genet. 108, 1449–1457. doi: 10.1007/s00122-004-1591-2
An, H., Yang, Z., Yi, B., Wen, J., Shen, J., Tu, J., et al. (2014). Comparative transcript profiling of the fertile and sterile flower buds of pol CMS in B. napus. BMC Genomics 15:258. doi: 10.1186/1471-2164-15-258
Andersson, G. E., Karlberg, O., Canbäck, B., and Kurland, C. G. (2003). On the origin of mitochondria: a genomics perspective. Philos. Trans. R. Soc. Lond. B Biol. Sci. 358, 165–179. doi: 10.1098/rstb.2002.1193
Balk, J., and Leaver, C. J. (2001). The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release. Plant Cell 13, 1803–1818. doi: 10.1105/tpc.13.8.1803
Barrientos, A. (2003). Yeast models of human mitochondrial diseases. IUBMB Life 55, 83–95. doi: 10.1002/tbmb.718540876
Bergman, P., Edqvist, J., Farbos, I., and Glimelius, K. (2000). Male-sterile tobacco displays abnormal mitochondrial atp1 transcript accumulation and reduced floral ATP/ADP ratio. Plant Mol. Biol. 42, 531–544. doi: 10.1023/A:1006388814458
Brown, G. G., Formanová, N., Jin, H., Wargachuk, R., Dendy, C., Patil, P., et al. (2003). The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats. Plant J. 35, 262–272. doi: 10.1046/j.1365-313X.2003.01799.x
Carlsson, J., Leino, M., Sohlberg, J., Sundström, J. F., and Glimelius, K. (2008). Mitochondrial regulation of flower development. Mitochondrion 8, 74–86. doi: 10.1016/j.mito.2007.09.006
Chase, C. D. (2007). Cytoplasmic male sterility: a window to the world of plant mitochondrial-nuclear interactions. Trends Genet. 15, 81–90. doi: 10.1016/j.tig.2006.12.004
Chinnery, P. F. (2003). Searching for nuclear–mitochondrial genes. Trends Genet. 19, 60–62. doi: 10.1016/S0168-9525(02)00030-6
Conesa, A., Götz, S., García-Gómez, J. M., Terol, J., Talón, M., and Robles, M. (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674–3676. doi: 10.1093/bioinformatics/bti610
Cox, M. P., Peterson, D. A., and Biggs, P. J. (2010). SolexaQA: at-a-glance quality assessment of Illumina second-generation sequencing data. BMC Bioinformatics 11:485. doi: 10.1186/1471-2105-11-485
PubMed Abstract | CrossRef Full Text
Datta, R., Chamusco, K. C., and Chourey, P. S. (2002). Starch biosynthesis during pollen maturation is associated with altered patterns of gene expression in maize. Plant Physiol. 130, 1645–1656. doi: 10.1104/pp.006908
de Longevialle, A. F., Meyer, E. H., Andrés, C., Taylor, N. L., Lurin, C., Millar, A. H., et al. (2007). The pentatricopeptide repeat gene OTP43 is required for trans-splicing of the mitochondrial nad1 intron 1 in Arabidopsis thaliana. Plant Cell 19, 3256–3265. doi: 10.1105/tpc.107.054841
Desloire, S., Gherbi, H., Laloui, W., Marhadour, S., Clouet, V., Cattolico, L., et al. (2003). Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide-repeat protein family. EMBO Rep. 4, 588–594. doi: 10.1038/sj.embor.embor848
Dieterich, J. H., Braun, H. P., and Schmitz, U. K. (2003). Alloplasmic male sterility in Brassica napus (CMS 'Tournefortii-Stiewe') is associated with a special gene arrangement around a novel atp9 gene. Mol. Genet. Genomics 269, 723–731. doi: 10.1007/s00438-003-0886-3
Fisk, D. G., Walker, M. B., and Barkan, A. (1999). Molecular cloning of the maize gene crp1 reveals similarity between regulators of mitochondrial and chloroplast gene expression. EMBO J. 18, 2621–2630. doi: 10.1093/emboj/18.9.2621
Hanson, M. R., and Bentolila, S. (2004). Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16, 154–169. doi: 10.1105/tpc.015966
Hattori, M., Miyake, H., and Sugita, M. (2007). A pentatricopeptide repeat protein is required for RNA processing of clpP pre-mRNA in moss chloroplasts. J. Biol. Chem. 282, 10773–10782. doi: 10.1074/jbc.M608034200
Higginson, T., Li, S. F., and Parish, R. W. (2003). AtMYB103 regulates tapetum and trichome development in Arabidopsis thaliana. Plant J. 35, 177–192. doi: 10.1046/j.1365-313X.2003.01791.x
Jing, B., Heng, S. P., Tong, D., Wan, Z. J., Fu, T. D., Tu, J. X., et al. (2012). A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development. J. Exp. Bot. 15, 1285–1295. doi: 10.1093/jxb/err355
Kanehisa, M., Araki, M., Goto, S., Hattori, M., Hirakawa, M., Itoh, M., et al. (2008). KEGG for linking genomes to life and the environment. Nucleic Acids Res. 36, D480–D484. doi: 10.1093/nar/gkm882
Kazama, T., and Toriyama, K. (2003). A pentatricopeptide repeat-containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic male-sterile rice. FEBS Lett. 544, 99–102. doi: 10.1016/S0014-5793(03)00480-0
Koizuka, N., Imai, R., Fujimoto, H., Hayakawa, T., Kimura, Y., Kohno-Murase, J., et al. (2003). Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish. Plant J. 34, 407–415. doi: 10.1046/j.1365-313x.2003.01735.x
Kotera, E., Tasaka, M., and Shikanai, T. (2005). A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature 433, 326–330. doi: 10.1038/nature03229
L'Homme, Y., and Brown, G. G. (1993). Organizational differences between cytoplasmic male sterile and male fertile Brassica mitochondrial genomes are confined to a single transposed locus. Nucleic Acids Res. 21, 1903–1909.
PubMed Abstract | Google Scholar
Langmead, B., Trapnell, C., Pop, M., and Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10:R25. doi: 10.1186/gb-2009-10-3-r25
Leino, M., Teixeira, R., Landgren, M., and Glimelius, K. (2003). Brassica napus lines with rearranged Arabidopsis mitochondria display CMS and a range of developmental aberrations. Theor. Appl. Genet. 106, 1156–1163. doi: 10.1007/s00122-002-1167-y
Li, S., Wan, C., Kong, J., Zhang, Z., Li, Y., and Zhu, Y. (2004). Programmed cell death during microgenesis in a Honglian CMS line of rice is correlated with oxidative stress in mitochondria. Funct. Plant Biol. 31, 369–376. doi: 10.1071/FP03224
Liu, C., Ma, N., Wang, P. Y., Fu, N., and Shen, H. L. (2013). Transcriptome sequencing and De novo analysis of a cytoplasmic male sterile line and its near-isogenic restorer line in Chili pepper (Capsicum annuum L.). PLoS ONE 8:e65209. doi: 10.1371/journal.pone.0065209
Logan, D. C. (2006). The mitochondrial compartment. J. Exp. Bot. 57, 1225–1243. doi: 10.1093/jxb/erj151
Lurin, C., Andrés, C., Aubourg, S., Bellaoui, M., Bitton, F., Bruyère, C., et al. (2004). Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16, 2089–2103. doi: 10.1105/tpc.104.022236
Mootha, V. K., Lepage, P., Miller, K., Bunkenborg, J., Reich, M., Hjerrild, M., et al. (2003). Identification of a gene causing human cytochrome c oxidase deficiency by integrative genomics. Proc. Natl. Acad. Sci. U.S.A. 100, 605–610. doi: 10.1073/pnas.242716699
Nakamura, T., Meierhoff, K., Westhoff, P., and Schuster, G. (2003). RNA-binding properties of HCF152, an Arabidopsis PPR protein involved in the processing of chloroplast RNA. Eur. J. Biochem. 270, 4070–4081. doi: 10.1046/j.1432-1033.2003.03796.x
Okuda, K., Myouga, F., Motohashi, R., Shinozaki, K., and Shikanai, T. (2007). Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proc. Natl. Acad. Sci. U.S.A. 104, 8178–8183. doi: 10.1073/pnas.0700865104
Okuda, K., Nakamura, T., Sugita, M., Shimizu, T., and Shikanai, T. (2006). A pentatricopeptide repeat protein is a site recognition factor in chloroplast RNA editing. J. Biol. Chem. 281, 37661–37667. doi: 10.1074/jbc.M608184200
Oshima, M., Kikuchi, R., Imamura, J., and Handa, H. (2010). Origin of the CMS gene locus in rapeseed cybrid mitochondria: active and inactive recombination produces the complex CMS gene region in the mitochondrial genomes of Brassicaceae. Genes Genet. Syst. 85, 311–318. doi: 10.1266/ggs.85.311
Reichert, A. S., and Neupert, W. (2004). Mitochondriomics or what makes us breathe. Trends Genet. 20, 555–562. doi: 10.1016/j.tig.2004.08.012
Rhoades, M. W., Reinhart, B. J., Lim, L. P., Burge, C. B., Bartel, B., and Bartel, D. P. (2002). Prediction of plant microRNA targets. Cell 110, 513–520. doi: 10.1016/s0092-8674(02)00863-2
Romualdi, C., Bortoluzzi, S., D'Alessi, F., and Danieli, G. A. (2003). IDEG6: a web tool for detection of differentially expressed genes in multiple tag sampling experiments. Physiol. Genomics 12, 159–162. doi: 10.1152/physiolgenomics.00096.2002
Schmitz-Linneweber, C., Williams-Carrier, R., and Barkan, A. (2005). RNA immunoprecipitation and microarray analysis show a chloroplast pentatricopeptide repeat protein to be associated with the 5′ region of mRNAs whose translation it activates. Plant Cell 17, 2791–2804. doi: 10.1105/tpc.105.034454
Shadel, G. S. (2004). Coupling the mitochondrial transcription machinery to human disease. Trends Genet. 20, 513–519. doi: 10.1016/j.tig.2004.08.005
Shen, J. X., Wang, H. Z., Fu, T. D., and Tian, B. M. (2008). Cytoplasmic male sterility with self-incompatibility, a novel approach to utilizing heterosis in rapeseed (Brassica napus L.). Euphytica 162, 109–115. doi: 10.1007/s10681-007-9606-0
Shikanai, T. (2006). RNA editing in plant organelles: machinery, physiological function and evolution. Cell. Mol. Life Sci. 63, 698–708. doi: 10.1007/s00018-005-5449-9
Small, I. D., and Peeters, N. (2000). The PPR motif: a TPR-related motif prevalent in plant organellar proteins. Trends Biochem. Sci. 25, 45–47. doi: 10.1016/S0968-0004(99)01520-0
Stern, D. B., Hanson, M. R., and Barkan, A. (2004). Genetics and genomics of chloroplast biogenesis: maize as a model system. Trends Plant Sci. 9, 293–301. doi: 10.1016/j.tplants.2004.04.001
Sunkar, R., and Zhu, J. K. (2004). Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16, 2001–2019. doi: 10.1105/tpc.104.022830
Vêncio, R. Z., Brentani, H., and Pereira, C. A. (2003). Using credibility intervals instead of hypothesis tests in SAGE analysis. Bioinformatics 19, 2461–2464. doi: 10.1093/bioinformatics/btg357
Wang, J., Gao, Y. N., Kong, Y. Q., Jiang, J. J., Li, A. M., Zhang, Y. T., et al. (2014). Abortive process of a novel rapeseed cytoplasmic male sterility line derived from somatic hybrids between Brassica napus and Sinapis alba. J. Integr. Agric. 13, 741–748. doi: 10.1016/S2095-3119(13)60584-5
Wang, Y. P., Sonntag, K., Rudloff, E., and Chen, J. M. (2005). Intergeneric somatic hybridization between Brassica napus L. and Sinapis alba L. J. Integr. Plant Biol. 47, 84–91. doi: 10.1111/j.1744-7909.2005.00009.x
Wang, Z., Zou, Y., Li, X., Zhang, Q., Chen, L., Wu, H., et al. (2006). Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing. Plant Cell 18, 676–687. doi: 10.1105/tpc.105.038240
Wen, L., Ruesch, K. L., Ortega, V. M., Kamps, T. L., Gabay-Laughnan, S., and Chase, C. D. (2003). A nuclear restorer-of-fertility mutation disrupts accumulation of mitochondrial ATP synthase subunit α in developing pollen of S male-sterile maize. Genetics 165, 771–779.
Williams, P. M., and Barkan, A. (2003). A chloroplast-localized PPR protein required for plastid ribosome accumulation. Plant J. 36, 675–686. doi: 10.1046/j.1365-313x.2003.01915.x
Wu, J., Zhao, Q., Yang, Q., Liu, H., Li, Q., Yi, X., et al. (2016). Comparative transcriptomic analysis uncovers the complex genetic network for resistance to Sclerotinia sclerotiorum in Brassica napus. Sci. Rep. 6:19007. doi: 10.1038/srep19007
Xu, J., Ding, Z., Vizcay-Barrena, G., Shi, J., Liang, W., Yuan, Z., et al. (2014). ABORTED MICROSPORES acts as a master regulator of pollen wall formation in Arabidopsis. Plant Cell 26, 1544–1556. doi: 10.1105/tpc.114.122986
Yamagishi, H., and Bhat, S. R. (2014). Cytoplasmic male sterility in Brassicaceae crops. Breed. Sci. 64:38. doi: 10.1270/jsbbs.64.38
Yan, X., Dong, C., Yu, J., Liu, W., Jiang, C., Liu, J., et al. (2013). Transcriptome profile analysis of young floral buds of fertile and sterile plants from the self-pollinated offspring of the hybrid between novel restorer line NR1 and Nsa CMS line in Brassica napus. BMC Genomics 14:26. doi: 10.1186/1471-2164-14-26
Yang, S. L., Jiang, L., San Puah, C., Xie, L. F., Zhang, X. Q., Chen, L. Q., et al. (2005). Overexpression of TAPETUM DETERMINANT1 alters the cell fates in the Arabidopsis carpel and tapetum via genetic interaction with EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS. Plant Physiol. 139, 186–191. doi: 10.1104/pp.105.063529
Yang, S. L., Xie, L. F., Mao, H. Z., San Puah, C., Yang, W. C., Jiang, L., et al. (2003). TAPETUM DETERMINANT1 is required for cell specialization in the Arabidopsis anther. Plant Cell 15, 2792–2804. doi: 10.1105/tpc.016618
Ye, J., Fang, L., Zheng, H., Zhang, Y., Chen, J., Zhang, Z., et al. (2006). WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 34, W293–W297. doi: 10.1093/nar/gkl031
Zerbino, D. R., and Birney, E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829. doi: 10.1101/gr.074492.107
Zheng, B. B., Wu, X. M., Ge, X. X., Deng, X. X., Grosser, J. W., and Guo, W. W. (2012). Comparative transcript profiling of a male sterile cybrid Pummelo and its fertile type revealed altered gene expression related to flower development. PLoS ONE 7:e43758. doi: 10.1371/journal.pone.0043758
Keywords: Brassica napus, cytoplasmic male sterility (CMS), somatic hybridization, morphological structure, transcriptomic analysis
Citation: Du K, Liu Q, Wu X, Jiang J, Wu J, Fang Y, Li A and Wang Y (2016) Morphological Structure and Transcriptome Comparison of the Cytoplasmic Male Sterility Line in Brassica napus (SaNa-1A) Derived from Somatic Hybridization and Its Maintainer Line SaNa-1B. Front. Plant Sci. 7:1313. doi: 10.3389/fpls.2016.01313
Received: 05 July 2016; Accepted: 16 August 2016;
Maoteng Li, Huazhong University of Science and Technology, China
Chunyu Zhang, Huazhong Agricultural University, China
Liezhao Liu, Southwest University, China
Copyright © 2016 Du, Liu, Wu, Jiang, Wu, Fang, Li and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Youping Wang, wangyp@yzu.edu.cn


Embarrassing. How to get six takeaways from one of the most uninspiring performances I've ever seen from a Power 5 football team at home? I have no idea, but I'm going to try.
It's bottom of the barrel. Charlotte absolutely annihilated the Vols up front, and as a result Guarantano had very little time and nobody could run the football. This was flat out putrid, including Drew Richmond committing not one, but two presnap procedure infractions as he couldn't figure out where to line up. Alex Highsmith owned the Vols all day long.
Once again, just a quick glance at the yardage totals across the board illustrate just how inept the offense was on this day. It wasn't on Helton (much), JG wasn't great, but the offensive line ensured Charlotte was still within striking distance after 60 minutes of football. This was truly as bad as it gets. I don't need to say anything else. You saw it. It's completely unacceptable.
I wish there were a way to make text appear but also look hidden, because that's the best description of the non-existent Tennessee rushing attack. Through three quarters against Charlotte, the Vols had netted a whopping MINUS SEVEN yards on the ground. Yes, the 49ers had a top ten run defense entering this game, but who had they played? They held Fordham to negative yardage, an atrocious WKU team to 42, Middle Tennessee to 33, and last week Southern Miss had 129. They're not a bad defense.
But you're Tennessee. This was the fewest rushing yards a Tennessee team has finished a game with since they had zero against Ole Miss in 2014. I find it hard to believe that performance was worse than the 20 they had on Saturday. All of the positivity came very late in the game, and "all" is being generous, because it indicates a lot, when that was anything but the case.
It all again comes down to the OL, because when you can't run and Guarantano has no time on top of being inaccurate at times, you're facing third and long situations and you end up 4-12 for 33% on the afternoon. Tennessee got virtually every yard on several big pass plays, because as we already discussed, nothing happened anywhere else. The offense could just be all six takeaways honestly, but here's what Tennessee faced respective to yardage on its crucial down and distances: 3rd and 15, 10, (6), 13, 14, 10, (2), (6), 16, (6), 11, 18.
You've got to be kidding me. You're an SEC football team at home against a C-USA team… it's homecoming, and THIS is what you're working with after that football game ends?
If you want a few bright spots, the next two takeaways would be those bright spots. Phillips almost had a sack for a safety in the second half, batted down two Evan Shirreffs passes in the first half, and had his number called more than anybody on the Tennessee defense, with the exception of the late Bryce Thompson interception that sealed the victory.
Phillips showed up and played, which is noteworthy for the sole reason that it's not something that can be said about many of his teammates on this day. That's somewhat unfair, as the defense did play well, though what we knew about Charlotte's offense coming in was LeMay could run it – and he did, even though the Vols held him under his 93 yard-per-contest average. The Niners struggle to score and have all season.
It's still a win, but if a win can feel like a loss, that's what this game was.
It's a takeaway because outside of one back shoulder throw to DWA, it's the only score Tennessee got all day. Cimaglia went wide right on a 33 yard field goal attempt from the left hash, and that was IT. So the 82 yard scamper from Marquez was hugely important. It was also the longest return for a score since Cam Sutton against Vanderbilt three years ago. I'm seriously having to scrape to find takeaways worth writing about. This was as forgettable as a game can be. That said, this was a pretty play from Callaway.
Not much to say about this one folks. It's a win, but that's all it was. You beat Charlotte 14-3 at home and have yet to play well in 2018 at Neyland Stadium. Come to think of it, that should have been a takeaway. All the Vols best ball has taken place away from Knoxville, but we needed to emphasize that OL performance. Ty Chandler and Tim Jordan both were banged up entering the game and neither played very much, but it wouldn't have mattered. This was a lethargic, lackadaisical, flat out dull performance from Tennessee. The Vols played losing football on offense, but Charlotte wasn't quite good enough offensively to take advantage. The 49ers run defense was as advertised, but considering their strength of opponent, you have to be disappointed if you're a Vols fan.
Up next is Kentucky at home, as the Wildcats were beaten soundly in Lexington by Georgia, ending their hopes of winning the SEC East. That's a really good defense. UT has the Cats, then Missouri (who whipped Florida on the road on Saturday), and then Tennessee finishes up here in Nashville against Vanderbilt. Should they play like this against any or all of those opponents, they've won their last game of the season. 4-5 right now, but this was B-A-D.


Are you dreading lugging items home from Smith College? Whether you're driving, flying, or taking the train...make it easy! Its expensive and inconvenient to travel with so much stuff.
I wonder if there is a solution...? hmmm.
OnDemand Storage is both convenient and affordable. A nice combination. Give us a call to learn more and see how we can make leaving Smith College enjoyable this year.


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Compressed Gas Cylinder Maintenance Making Cylinders Last Beyond 100 Years
By Steve Guglielmo
Haun Welding Supply
"Cylinders that are 100 years old will be good for another 100 years if they are properly taken care of and maintained," says Hydrostat Inc. Owner and CEO Stacy Schraut. "The beauty of the packaged gas industry is that we have assets that are 100 years old that can continue to generate revenue. How many other industries can say that?"
Compressed Gas Cylinders are over-engineered, due to the critical nature of their purpose, according to Airgenics, Inc. COO Jonathan Balbi. When properly maintained, these cylinders should last for hundreds of years. The key to this is proper maintenance coupled with mandatory cylinder testing as prescribed by the Department of Transportation (DOT), the Pipeline and Hazardous Materials Safety Administration (PHMSA) and the Compressed Gas Association (CGA.)
"Doing the absolute bare minimum will not extend the lifespan of a cylinder," says Balbi. "It will only let you know that your aging inventory is going bad at an exponential rate. Companies that have kept good care of their cylinders still see them passing back into service. I have seen plenty of cylinders from the 1970s up to the 2000s fail expansion tests because of obvious neglect that could have been prevented for a lot less than the cost of a new cylinder."
How Testing Works
The DOT and PHMSA regulate the transport of hazardous materials through Title 49 of the Code of Federal Regulations (49 CFR), subchapter C, "Hazardous Materials Regulations." As mandated by CFR Title 49, "Each cylinder used for the transportation of hazardous materials must be an authorized packaging. To qualify as an authorized packaging, each cylinder must conform to this subpart, the applicable requirements specified in part 173 of this subchapter, and the applicable requirements of subpart C of part 178 of this subchapter."
Every five or ten years, a standard DOT-rated cylinder must be requalified before it can be filled and transported by one of two accepted methods. The first method is a volumetric expansion test to determine the elasticity of the cylinders. The other option is an ultrasonic inspection that scans the cylinder and checks for defects in the metal and compares the average wall thickness against the manufacturer's specifications and a calibration of a similar metal.
If the cylinder passes either of these tests and a visual inspection, it is deemed to be an authorized package and can be stamped and filled for another service cycle. Reject cylinders are condemned and destroyed as prescribed by the CFR 49.
Hydrotesting
"I've been an advocate in our industry for hydrotesting ever since our customers started pushing for more ultrasonic testing," says Schraut. "In very rare circumstances, a cylinder has passed an ultrasonic test but would have failed a hydrotest. In the last seven years of ultrasonic testing this has happened on no more than three or four occasions out of more than 500,000 cylinders tested. That's a very small number but in our opinion it's still too high."
A hydrostatic test involves vacating any residual gas from the cylinder, removing the valve, filling the cylinder with water, placing the cylinder into a sealed water environment, introducing pressure greater than that of the service pressure for a standard period of time (usually 30 seconds), releasing the pressure and measuring the expansion (permanent and elastic), removing the cylinder from the sealed environment, emptying the water, drying the cylinder, performing an internal visual inspection, stamping the cylinder and reinstalling an acceptable valve (usually the same valve that was previously in the cylinder unless it does not conform to thread count specifications or having a service change done.)
An ultrasonic test, by comparison, involves placing the cylinder in the ultrasonic machine and testing it under ideal circumstances (exterior in good condition, empty of liquified gases, valve closed.)
"Part of the requalification of a cylinder is to do an internal inspection on that cylinder, according to the 49 CFR," Schraut says. "So, in my opinion if you're not removing the valve for an ultrasonic test, you're not really satisfying the guidelines of the 49 CFR."
Says Balbi, "When you have removed the valve, a host of optional procedures are opened up and each one can extend the cylinder life if coupled with a good in-house procedure, so that the owner of the asset is ultimately determining the cylinders function. This does take some planning and forethought but can mean the difference between a reject asset and a good cylinder. If the asset base of a company is 40,000 cylinders of mixed manufacture dates, around 5,400 cylinder per year would need to be tested to maintain an in-service asset base, with an average of 45% of cylinders being over 60 years of age, without maintenance beyond standard testing expect to lose around 95 cylinders per year more than with above and beyond upkeep. In dollars that can equate to more than $20,000 dollars per year on new cylinders just to make up what is lost."
What Should My Service Provider be Providing?
In the August 2019 GAWDA Monthly Safety Organizer, GAWDA DOT, Security, OSHA and EPA Consultant Michael Dodd wrote an article entitled, "Auditing Your Hydrotest Vendor." In the article, he provided a checklist GAWDA member companies can use to ensure that the cylinders that you send out for requalification are being done properly and that the facility is in compliance.
At a minimum, the service provider should be able to provide an inventory of cylinders that are inhouse and what service they are in, including a breakdown by size and gas type, along with confirmation that the cylinders sent in can go back into the same service. (A good requalification company will know if they can put an asset back into a specific service. More often than is realized, a fair number of cylinders may no longer be able to go into a service that is needed.)
"If a cylinder fails a test and no longer can get a star or a plus rating or the cylinder's age doesn't allow it to get a star or plus rating, that doesn't necessarily condemn it to be scrapped," says Schraut. "Oftentimes a cylinder can be taken out of oxygen service or nitrogen service and put into CO2 service. That's one of the ways you can extend the life of a cylinder is simply by changing the service that it is in, when it no longer meets the requirements for the service it was in."
The service provider should also provide a copy of test reports with notations for work done to:
Assigned ownership notation
DOT specification
Previous gas service
Required test pressure
Required test time
Actual test time
Date and time tested
Operator name
Determined reject elastic expansion and source for REE
Actual permeant expansion
Actual elastic expansion
Marking eligibility (e.g., + *)
Pass/fail determination
If any data is missing, the cylinder may not be treated as though it is an authorized package. All of this together is what an authorized requalification company should provide with every order.
So why can a cylinder made in 1970 be condemned from service before a cylinder from 1904?
"It all boils down to asset management, storage and communication," says Balbi. "Having a simple yet comprehensive plan in place to sort assets as they come up for service that encompass a packaged gas companies near future needs, anticipated growth sectors, and an effective prefill inspection that can isolate assets that requires a secondary service to extend total life, regardless of service date;
herein lies the difference between caring about cylinders and filling and billing."
According to Schraut, asset management and distributors having positions dedicated to asset management is one of the biggest changes in our industry in the last 20 years.
"There are created positions now, where somebody is overseeing their assets to make sure that if a customer has an overabundance of oxygen cylinders in their inventory and a shortage of nitrogen cylinders, rather than spend $250-$300 per cylinder to buy 100 nitrogen cylinders, they can send those oxygen cylinders in and have them blasted and painted, washed and outfitted with a new valve and for a tenth of the cost of buying a brand new cylinder, they can get what's functionally a new cylinder for a new service," he says. "Rather than having those cylinders sitting out in the yard not being utilized, they're sending them in to be converted to the gases that they're in need of at the time."
The unsure economic future of metal manufacturing as well as the turmoil in trade will make new cylinders more expensive with longer lead times for delivery. Now is the time to evaluate the age of your inventory and forecast any future needs. If planned properly, a company can maintain assets and keep them in service longer than originally anticipated, giving them a slight edge over the competition. Growing pains of purchasing new asset acquisition and shrinkage caused by asset misappropriation can be mitigated to figures that will allow for less capital investment in steel and more investments in infrastructure.
For more information on cylinder care best practices, be on the lookout for the follow up article in the 4th Quarter Issue of Welding & Gases Today.
Photo courtesy of Hydrostat, Inc.
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Internet providers in Huntington Park, CA
Shop the best Huntington Park internet providers. Compare internet plan pricing and speeds from cable, DSL and fiber-optic providers in your area.
Huntington Park, CA internet providers
Curacao - - DSL 20%
GTT Communications - - DSL 3.439%
Sonic $40 10 - 12 Mbps Wired 2.746%
Sonic $40 10 - 80 Mbps DSL 1.544%
Huntington Park internet service at a glance
At 100%, broadband coverage in Huntington Park is comparable to the rest of California - 93%, and comparable to broadband coverage across the U.S. - 95%.
The vast majority of those living in the Huntington Park area will have access to cable, which covers 100% of Huntington Park residents. DSL internet is also an option for many, offering service to 99% of the area. No fiber-optic providers are currently available in Huntington Park, but satellite or fixed-wireless providers may offer broadband options in your area.
There are seven internet providers serving the Huntington Park area, and 98.85% of households will have internet options from more than one provider. Among Huntington Park's seven internet providers, prices start at $30 and available download speeds reach up to 300 Mbps.
Find Huntington Park internet providers by ZIP code
Top high-speed internet providers in Huntington Park
The largest internet providers in Huntington Park are AT&T, Spectrum, and HughesNet bringing DSL, cable, and satellite options to Huntington Park.
DSL internet from AT&T is available to nearly all Huntington Park residents, covering 99% of households. Many will also have the option of cable from Spectrum, which covers 100% of the area. Huntington Park's third most-available internet provider, HughesNet, offers satellite to around 100% of Huntington Park residents.
DSL from AT&T offers broadband to all households in Huntington Park covering 99% of the area.
DSL internet from AT&T uses telephone lines in Huntington Park to deliver service, which often leads to lower prices and higher availability, though download speeds are likely to be slower than cable or fiber-optic internet.
Internet prices for AT&T start at $30/mo.*, which is significantly lower than the average starting price for internet in Huntington Park, $45.00.
AT&T customers who choose the lowest-priced plan can expect speeds up to 100 Mbps, which comes to an estimated $0.30 per Mbps, about the same as the average cost per Mbps for the lowest priced plans in Huntington Park, $1.05.
For those who value internet speed as much as cost, AT&T offers download speeds up to 75 Mbps and upload speeds up to 20 Mbps in Huntington Park. These download speeds are available to 99% of the area, and are about the same as the average max speeds from all Huntington Park's providers, 103 Mbps.
Cable from Spectrum offers broadband to all households in Huntington Park covering 100% of the area.
Spectrum's cable network is likely to offer faster speeds than DSL or satellite service, but Huntington Park residents in densely populated areas may experience slowed speeds during peak usage times.
Internet prices for Spectrum start at $44.99/mo.*, which is about the same as the average starting price for internet in Huntington Park, $45.00.
Spectrum customers who choose the lowest-priced plan can expect speeds up to 60 Mbps, which comes to an estimated $0.75 per Mbps, about the same as the average cost per Mbps for the lowest priced plans in Huntington Park, $1.05.
For those who value internet speed as much as cost, Spectrum offers download speeds up to 300 Mbps and upload speeds up to 20 Mbps in Huntington Park. These download speeds are available to 100% of the area, and are much faster than the average max speeds from all Huntington Park's providers, 103 Mbps.
Satellite from HughesNet offers broadband to all households in Huntington Park covering 100% of the area.
HughesNet satellite internet offers reliable connection in areas of Huntington Park where other internet types may not be available, but service is likely to come with low monthly data allowances and high latency, making streaming binges and real-time online gaming impractical.
Internet prices for HughesNet start at $59.99/mo.*, which is significantly higher than the average starting price for internet in Huntington Park, $45.00.
HughesNet customers who choose the lowest-priced plan can expect speeds up to 25 Mbps, which comes to an estimated $2.40 per Mbps, higher than the average cost per Mbps for the lowest priced plans in Huntington Park, $1.05.
For those who value internet speed as much as cost, HughesNet offers download speeds up to 25 Mbps and upload speeds up to 3 Mbps in Huntington Park. These download speeds are available to 100% of the area, and are slightly slower than the average max speeds from all Huntington Park's providers, 103 Mbps.
What's the fastest internet provider in Huntington Park?
Spectrum is the fastest internet provider in Huntington Park with download speeds up to 300 Mbps. These speeds are available to 100% of households in Huntington Park. The next fastest internet provider in Huntington Park is Sonic, offering download speeds up to 80 Mbps to 1.544% of the area.
What's the cheapest internet provider in Huntington Park?
AT&T offers the cheapest internet in Huntington Park with plans starting at $30/mo. This is significantly less than the average starting price for internet in Huntington Park, $45.00.
Why is my internet slow today?
There are many factors that can influence internet speed. In many cases, restarting your router will resolve speed and connectivity issues. Other factors, such as peak usage times and speed throttling can also cause temporary speed issues.
What's the best internet provider in my area?
Fiber internet providers are likely to offer the fastest, most consistent speeds for the money. Cable internet providers can also deliver fast speeds, up to 100 Mbps and higher, but are more vulnerable to slowed speeds during peak usage times. If you're shopping for cheap internet, a DSL provider may be the best option for your household.
Is my internet speed being throttled?
If you suspect your slow speeds are the result of your internet provider throttling your connection, the easiest way to determine if you're being throttled is to perform two speed tests. Run the first test on your regular network, then run the second one on a VPN. If the two speeds are drastically different (and the VPN test was higher), your internet provider may be throttling your speeds.
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91 small dogs find love with life-saving Valentine's Day flight to Michigan
The dogs were flown in from overcrowded animal shelters in California.
91 small dogs find love with life-saving Valentine's Day flight to Michigan The dogs were flown in from overcrowded animal shelters in California. Check out this story on Freep.com: https://www.freep.com/story/news/local/michigan/2019/02/14/chihuahuas-california-michigan-rescue-groups/2857957002/
Ann Zaniewski, Detroit Free Press Published 8:45 p.m. ET Feb. 14, 2019 | Updated 7:12 a.m. ET Feb. 15, 2019
California rescue dogs find a home in Michigan
Paula Durant, a Michigan Humane Society staff, carries a crate of rescue dogs off the plane at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. Around 91 rescue dogs flew in from Fresno, Calif. to get adopted at one of the metro Detroit Michigan Humane Society facilities. Junfu Han, Detroit Free Press
A rescue dog waits to get checked and then transported to a metro Detroit Michigan Humane Society facility for adoption at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. Junfu Han, Detroit Free Press
Chuck Johnson of Farmington Hills carries a crate of rescue dogs off the plane at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. Around 91 rescue dogs flew in from Fresno, Calif. to get adopted at one of the metro Detroit Michigan Humane Society facilities. Junfu Han, Detroit Free Press
Around 91 rescue dogs wait to get transported to a metro Detroit Michigan Humane Society facility for adoption at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. Junfu Han, Detroit Free Press
Harry Berberian of Southfield carries two crates of rescue dogs off the plane at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. Around 91 rescue dogs flew in from Fresno, Calif. to get adopted at one of the metro Detroit Michigan Humane Society facilities. Junfu Han, Detroit Free Press
Around 91 rescue dogs flew in from Fresno, Calif. wait to get transported to a metro Detroit Michigan Humane Society facility for adoption at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. Junfu Han, Detroit Free Press
Co-pilot Angela Keeling of Bend, Ore., hands a crate of rescue dogs to Doug Plant, chief mission officer for the Michigan Humane Society at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. Around 91 rescue dogs flew in from Fresno, Calif. to get adopted at one of the metro Detroit Michigan Humane Society facilities. Junfu Han, Detroit Free Press
A crate with three puppies wait to get checked and transported to a metro Detroit Michigan Humane Society facility for adoption at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. Junfu Han, Detroit Free Press
Around 91 rescue dogs wait to get transported to a metro Detroit Michigan Humane Society facilities for adoption at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. Junfu Han, Detroit Free Press
It was a Valentine's Day treat for 91 pint-sized dogs — the start of a new life.
Big smiles and lots of love greeted the pups on a chilly Thursday evening at Willow Run Airport in Ypsilanti. A team of volunteers gently moved them, crate by crate, off an airplane and into waiting vehicles that would take them to local animal rescues.
The dogs had just finished a nearly six-hour flight from Fresno, California, courtesy of the nonprofit Wings of Rescue. Out West, they faced a high risk of being euthanized in overcrowded shelters.
"These dogs, if they didn't go to Michigan, a lot of them would be dead by this weekend," said Ric Browde, president of Wings of Rescue.
Browde said an overpopulation of small dogs in California shelters and a shortage of — and demand for — small dogs in metro Detroit led to the unique cross-country mission.
About 80 percent of the plane's passengers were Chihuahuas or Chihuahua mixes.
Wings of Rescue, in partnership with GreaterGood.org, funded the flight. Compassion Without Borders spearheaded the selection of the dogs and provided preflight health checks.
Four local rescue groups — the Michigan Humane Society, Friends for Animals of Metro Detroit, Midwest Small Breed Rescue and the Michigan Animal Rescue League — took in the dogs. They could be ready for adoption as soon as next week.
"My husband just told me to not bring any of them home," said Mary Frances McCaleb, a humane society volunteer from Birmingham, laughing as she waited for the plane to arrive. She wore rubber gloves and boots, prepared to help clean out the carriers.
"I said, 'I've got three pockets, honey, and these are Chihuahuas!' "
A crate with three puppies wait to get checked and transported to a metro Detroit Michigan Humane Society facility for adoption at Willow Run Airport in Ypsilanti Township, Thursday, Feb. 14, 2019. (Photo: Junfu Han, Detroit Free Press)
The Pilatus PC-12 turboprop plane landed at 6 p.m.
As the husband and wife volunteer pilot team of Angela Keeling and Kale Garcia opened the side door, small faces peered out from stacks of carriers. Barking and crying filled the cold Michigan air.
The volunteers sprang into action, working quickly and carefully to unload the dogs. They placed the carriers on the ground in groups according to the dogs' color-coded collars. Pieces of tape on the carriers had handwritten names like Judy, Chloe and Clyde.
Garcia, of Bend, Oregon, said the work is important.
"It's a very, very nice, comforting thing to do," Garcia said. "You open that door, and you know that there are just a bunch of people on the other side of the door that are just full of love and caring hearts, ready to take the animals."
Fourteen of the dogs were headed to the Dearborn-based Friends for Animals of Metro Detroit.
Katie Johnson, director of operations for the shelter, said food, water and warm blankets awaited the weary travelers.
"I think they'll be adopted really quickly, and find great, loving homes," she said.
Contact Ann Zaniewski: 313-222-6594 or azaniewski@freepress.com. Follow her on Twitter: @AnnZaniewski.
People interested in adopting the dogs can reach out to the Michigan Humane Society at 866-MHUMANE, the Michigan Animal Rescue League at 248-335-9290, the Friends for Animals of Metro Detroit at 313-943-2697, or the Midwest Small Breed Rescue at www.midwestsmallbreedrescue.com
Read or Share this story: https://www.freep.com/story/news/local/michigan/2019/02/14/chihuahuas-california-michigan-rescue-groups/2857957002/
This part of the Macomb jail is old and hot
Mich. church pays off 1,899 families' medical debt
Traverse City police: Hotel shooting apparent murder-suicide