text:
Double the fun: Pony girls win state
Daniel Horton Havre Daily News [email protected]
The seven Havre High players which went to Kalispell for Thursday and Friday's Class A state tennis tournament knew the odds were stacked against them in the team competition. Or did they? Despite not having the depth which other teams in the girls and boys field had last weekend, the Blue Ponies turned in one of the most successful state tournament's in school history. The Havre High girls used a perfect run in doubles to catapult the Blue Ponies to their second Class A state championship in school history, as juniors Hayley Donovan and Courtney Kinholt beat teammates Morgan McCann and Jolee Landgraf 6-4, 7-5 in the doubles championship match. The doubles performance was enough to give the Ponies 29 points and the state title. And they even made more history as Kinholt and Donovan became the first girls doubles champion from Havre in school history. "Indescribable," HHS head coach George Ferguson said. "Those four girls played the tournament of their lives and they were rewarded with a state championship. It is something that I can hardly put into words right now. I'm just really proud of them, and more importantly, happy for all of them. They deserve this success." Not to be outdone, the three HHS boys which represented the Blue Ponies made a valiant run at a state title of their own. Behind a state championship in doubles from Kyle Miller and Mason Sheppard, as well as a third-place finish in singles from senior Shane Kemmer, the Ponies scored 26 points with just three players and finished a close second to state champion Billings Central. The Rams, with nine players in the tournament, finished with 31 points. "I couldn't be more proud of the boys team," Ferguson said. "We had a plan, we knew what we had going in and that was three of the best overall players in the tournament. Not very many people thought we could do what we did, but our kids and this coaching staff knew it wasn't about numbers for us. We only lost one match the entire tournament. Those three guys played the best tennis of their lives and I just couldn't be more proud of all three of them." The Havre girls beat Billings Central by two points, while Libby was third with 19 points behind state champion singles player Jackie Mee. On the boys side, Whitefish finished third with 21 points as senior Matt Harris captured the boys singles championship. The girls doubles championship was spectacular, as was the team title. Donovan and Kinholt cruised through the first day of competition and on Friday morning, avenged one of their few losses of the season by thumping an undefeated team from Dillon in the semifinals. Meanwhile, McCann and Landgraf, who placed third at divisionals, had to survive two three-set matches on the opening day before dispatching the No. 1 seeded team from the Eastern A from Billings Central in the semifinals, setting up the all-Havre final. "I can't really tell you much about the championship match," Ferguson said. "I know it was a good and competitive match because it always is when those four play each other. That's a big part of why we did what we did, they push each other in practice every day, and they've been doing it since they were freshmen. But as coaches, you don't get involved in a match like the state final because all your thinking is, we have both our doubles teams playing for the state title, and we're going to win a team title because of it. We weren't pulling for either team, we were just happy that we dominated the tournament the way we did. And it was exciting to know that either way, we were going to win our first state doubles championship in girls history. "And at the end of the day, I am just really happy for both teams," he added. "They had the kind of success that players only dream about. It was truly a special day for Havre High tennis because of the four girls we took to state." Donovan and Kinholt and McCann and Landgraf became just the second and third Pony teams to ever reach a state final in doubles. In 2006, the duo of Alexa Lipp and Karissa Olson finished second in doubles. Meanwhile, the Blue Pony boys made plenty of noise of their own even with just three players in the field. Miller and Sheppard cruised through the first day of the doubles, and then became the ninth straight HHS doubles team to reach the state final when they beat Corvallis' Levi Pauly and Jake Sangster in straight sets in the semifi- nals. Despite making the finals seemingly every year, HHS hadn't captured a boys doubles title since 2006, and the 2009 championship wasn't going to be easy. Miller and Sheppard beat Columbia Falls' seniors Grant Getts and Jake Ronish 6-4 in the opening set, but then fell behind the duo by dropping the second set 6-3, and eventually being down 4-2 in a third-set tiebreaker. But the Havre duo wasn't to be denied and they reeled off five straight points to take the match and the state championship. "It was one of the best matches of the tournament," Ferguson said. "It doesn't get any closer than a third-set tiebreaker for the state championship. So that just goes to show what kind of team Kyle and Mason are. They showed poise and a lot of heart, and they earned a state championship. I couldn't be happier for both of them. Kyle got so close last year and Mason is a senior who has been with us for four years, so it was pretty special to watch both of them reach their ultimate goal." And in singles, no player brought more excitement to the state tournament than Kemmer. He reached the quarterfinals where he was upset in straight sets by the big serve of Bigfork's Scott Taylor. However, he rebounded to win four straight matches to finish third, including playing two straight three-setters on Friday. Fittingly, Kemmer knocked off Taylor in a three-set thriller in the consolation match, his last of what was a brilliant four-year career at HHS. "I'm just so proud of Shane," Ferguson said. "He was a warrior for us in this tournament. He just refused to quit, and it means so much to us for him to get on the medal stand. He's been one of the best singles players in Montana for a long time now, but boys singles is the toughest draw of them all and he never ducked anything or anybody. I just couldn't be more proud of him and he went out a winner." In all, HHS recorded just two losses on the boys and girls side throughout the two-day tourney, and one of those was due to one Havre team playing another. The Ponies nearly maxed out on possible points, and with a state championship and a second-place trophy, they proved that the state tournament can be about more than just numbers and depth. "It was an amazing weekend," Ferguson said. "All of the things these kids accomplished, it's just unreal. To think that every player we took to state medaled, it just goes to show you how good of tennis players these seven kids are, and it's a credit to them and how much they've worked for this. As a coach, you just couldn't ask for anything more."
Hi-Line Athletes of the Week
Hi-Line Boys BB: Chinook wins big, Box Elder sweeps
Hi-Line girls BB: 9C girls play their first full week of hoops
FRIDAY NIGHT'S PREP BASKETBALL: Havre sweeps Miles City
Blue Ponies take on Eastern A powers


text:
Journal of Animal Science and Biotechnology
Early inoculation with caecal fermentation broth alters small intestine morphology, gene expression of tight junction proteins in the ileum, and the caecal metabolomic profiling of broilers
Yujie Gong1,
Wenrui Xia1,
Xueting Wen2,
Wentao Lyu2,
Yingping Xiao ORCID: orcid.org/0000-0002-7949-11262,
Hua Yang2 &
Xiaoting Zou1
Journal of Animal Science and Biotechnology volume 11, Article number: 8 (2020) Cite this article
The establishment of stable microbiota in early life is beneficial to the individual. Changes in the intestinal environment during early life play a crucial role in modulating the gut microbiota. Therefore, early intervention to change the intestinal environment can be regarded as a new regulation strategy for the growth and health of poultry. However, the effects of intestinal environmental changes on host physiology and metabolism are rarely reported. This study was conducted to investigate the effects of early inoculation with caecal fermentation broth on small intestine morphology, gene expression of tight junction proteins in the ileum, and cecum microbial metabolism of broilers.
Our data showed that early inoculation with caecal fermentation broth could improve intestine morphology. The small intestine villus height was significantly increased (P < 0.05) in the intervened broilers compared to the control group, especially on day 28. A similar result was observed in the ratio of villus height to crypt depth (P < 0.05). Meanwhile, we found early inoculation significantly increased (P < 0.05) the expression levels of zonula occludens-1 (ZO1) on days 14 and 28, claudin-1 (CLDN1) on day 28, whereas the gene expression of claudin-2 (CLDN2) was significantly decreased (P < 0.05) on days 14 and 28. Gas chromatography time-of-flight/mass spectrometry (GC-TOF/MS) technology was further implemented to systematically evaluate the microbial metabolite profiles. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) displayed a distinct trend towards separation between the fermentation broth group (F group) and the control group (C group). The differentially expressed metabolites were identified, and they were mainly functionally enriched in beta-alanine metabolism and biosynthesis of unsaturated fatty acids. In addition, 1,3-diaminopropane was selected as a key biomarker that responded to early inoculation with caecal fermentation broth.
These results provide insight into intestinal metabolomics and confirm that early inoculation with caecal fermentation broth can be used as a potential strategy to improve intestinal health of broilers.
The gastrointestinal tract (GIT) of poultry harbors complex and highly diversified communities of microorganisms, which collectively fulfill crucial roles in host physiology and health [1, 2]. In addition to the local effect within the GIT, gut microbiota can also have a profound effect on the host through microbe-derived metabolites [3]. Gut microbiota provide metabolites to the host in the form of fermentation end-products and other secreted products such as lipids, carbohydrates, organic acid, amino acids and other nutrients [4], which could be supplied throughout the body to meet the host nutrient and energy needs [5]. Therefore, in a sense, the significance of microorganisms to host health can be explained by their metabolic potential.
The environment and diet during animal feeding are the main factors in modulating the gut microbiota. Accumulating evidence demonstrates that diet supplemented with probiotics [6], organic acids [7], and exogenous enzyme [8, 9] can increase the abundance of intestinal beneficial bacteria, which have a protective role as the first line of defense against pathogenic bacteria in addition to assistance in specific metabolism and gut structure integrity [10]. However, there is a lack of knowledge regarding the regulatory mechanism of the microenvironment on the intestinal microbiota of poultry. Although our previous study has shown that shaping a specific gut environment through early intervention can alter the colonization and development of caecum microbiota in broilers, resulting in a reduction in the relative abundance of enteric pathogen and an increase of beneficial bacteria [11], the downstream effects caused by altering the structure of the gut microbiome on host physiology still need to be further explored [3]. It has been reported that the regulation mechanism of intestinal microbiota on host overall health primarily depends on microbial metabolism [12, 13]. For example, many studies have demonstrated that host inflammatory diseases are closely related to microbial metabolic disorders [14, 15]. Microbiota metabolites, as one of the most predominant connections between gut microbiota and the host, have crucial biological significance. Certainly, in addition to metabolic function, gut microbiota are also indispensable contributors to the formation and development of gut structure and morphology [16]. Complete intestinal structure could guarantee the function of intestinal mucosal barrier, which is usually reflected by villus length and crypt depth [17]. On the other hand, tight junction proteins, as one of the most considerable components of the intestinal barrier, play a crucial role in intestinal function [18, 19]. Therefore, intestine morphology and gene expression of tight junction proteins are generally regarded as pivotal indicators of intestinal health.
Here, we present a study aimed at investigating the effect of environmental factors on the gut microbiota metabolism of broilers, by orally administering fermentation broth at the early stage of life. Gas chromatography time-of-flight/mass spectrometry (GC-TOF/MS)-based cecal metabonomic method was used to identify the metabolomics profiles. The differentially expressed metabolites and their involved metabolic pathways were characterized. In addition, small intestine morphology and expression levels of tight junction-proteins in the ileum were determined.
Preparation of the caecal fermentation broth
In the present study, caecal fermentation broth was used as the inoculum for early inoculation, the preparation of which was concretely described in our previously published article [11]. A single-stage chemostat, as an in vitro bionic system, was used to produce the fermentation broth. First, caecal content from the selected donor chicken was thoroughly mixed with sterile phosphate buffered saline (PBS) to form a 10% suspension. Subsequently, the resulting suspension was injected into the chemostat and fermented continuously for 11 days. Finally, fermentation broth from the 11th day was used to inoculate chicks.
Animals and experimental design
A total of 120 one-day-old broiler chicks purchased from a local commercial hatchery were randomly divided into 2 groups with 4 replications and 15 birds per replicate, including 2 treatments: chicks in the experimental group were given 0.5 mL of fermentation broth orally within 2 h after hatching. In turn, chicks in the control group received the same amount of sterile PBS at the corresponding time. All experimental animals were raised in an environmentally controlled house in Zhejiang Academy of Agricultural Sciences, where the temperature of the first week was constant at 35 °C, and then lowered 3 °C weekly until the temperature reached 26 °C. The broilers received no antibiotics or other additives throughout the experimental period.
The samples were respectively collected on days 7, 14 and 28. For each sampling time point, 8 broilers per group were randomly selected and then killed by jugular exsanguination. The small intestines were extracted, and segments (1 cm in length) of the mid-duodenum, jejunum and ileum were excised and lightly rinsed with sterile PBS to remove the intestinal digesta, which were then fixed in 4% (v/v) paraformaldehyde for morphological examination (performed by Wuhan Goodbio technology Co., Ltd., Wuhan, China). The ileum mucosae were scraped off with a glass slide, rapidly frozen in liquid nitrogen tank, and then transferred to − 80 °C freezer for storage. The bilateral ceca were split with sterile scissors and forceps. Then, the caecal digesta were scraped to frozen tubes and stored at − 80 °C for metabolomics analysis.
Intestine morphological analyses and observation
The small intestine slides were photographed by a light microscope (Nikon Corp., Tokyo, Japan). Intestinal morphological parameters of each slide were calculated based on the average of five villus crypt units with intact lamina propria [20]. Villus height was measured from the villus tip to the villus-crypt junction, and the crypt depth was defined as the length from the villus-crypt junction to the base of the crypt. Furthermore, the villus height-to-crypt depth ratio (V/C) was obtained according to the means of villus height and crypt depth.
Quantitative real-time PCR analysis
Total RNA in the ileal mucosa was isolated using the MiniBEST Universal RNA Extraction Kit (Takara Bio, Dalian, Liaoning, China). RNA quantity and quality were determined using a spectrophotometer (NanoDrop-2000, Thermo Fisher Scientific, MA, USA). cDNA was synthesized using SuperScript™ III Reverse Transcription in the presence of random primers and an RNase inhibitor (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Gene-specific primers for zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), claudin-1 (CLDN1) and claudin-2 (CLDN2) were designed with Primer Premier 6.0 (Table 1) and commercially produced in Tsingke (Beijing, China). The RT-PCR assays were performed in triplicate on an ABI Prism 7500 Real-Time PCR Detection System (Applied Biosystems, Foster City, CA, USA). The reaction protocol was as follows: 1 min at 95 °C, followed by 40 cycles of 15 s denaturation at 95 °C, and 25 s annealing at 63 °C. This was followed by a melting curve analysis to confirm the specificity and reliability of the PCR products. The relative mRNA expression was obtained using the 2−∆∆Ct method, as previously described [21], after normalization with the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Table 1 Primers for gene expression analysis using real-time PCR
Sample preparation for GC-TOF/MS analysis
Approximately 50 ± 1 mg caecal content was extracted with 400 μL miscible liquid (V methanol: V chloroform = 3:1) and placed into 2 mL epoxy epoxide (EP) tubes. Subsequently, 10 μL of L-2-Chlorophenylalanine (0.5 mg/mL stock in dH2O, internal standard) was added and mixed by vortexing for 30 s. The mixed sample was homogenized in a ball mill for 4 min at 40 Hz, which was followed by ultrasonic treatment for 5 min (incubated in ice water). After centrifugation at 12000 r/min for 15 min, 350 μL of supernatant was injected into a fresh 1.5 mL EP tubes. The liquid was completely dried in a vacuum concentrator without heating, and then 50 μL methoxy amination hydrochloride (20 mg/mL in pyridine) was added and mixed lightly. The mixed sample was incubated for 30 min at 80 °C. Afterwards, 70 μL of the BSTFA regent (1% TMCS, v/v) was added to each sample, followed by incubation for 1.5 h at 70 °C. Finally, the processed samples would be subjected to GC-TOF/MS analysis.
GC-TOF/MS analysis
Pretreated caecal content samples were analyzed using an Agilent 7890 gas chromatograph system (Agilent, USA) coupled with a Pegasus HT time-of-flight mass spectrometer (LECO, USA). In addition, a DB-5MS capillary column (30 m × 250 μm × 0.25 μm; J&W Scientific, Folsom, CA, USA) was used in the determination to separate the derivatives. In splitless mode, the injection volume was set at 1 μL, and the solvent delay time was 4 min. Helium served as the carrier gas running in the column at a constant flow rate of 1 mL/min. The initial temperature was programmed at 50 °C for 1 min, increased at 10 °C/min to 290 °C, and subsequently maintained for 8 min at 290 °C. The injection, transfer line, and ion source temperatures were 280, 280, and 250 °C, respectively. The analysis was carried out in electron impact mode at − 70 eV. The mass spectral data were obtained in full scan mode from m/z 50 to 500 at a rate of 20 spectra per second after a solvent delay of 366 s [22].
The GC-TOF/MS raw data were first analyzed by Chroma TOF 4.3X software and LECO-Fiehn Rtx5 database, including raw peaks extraction, data baseline filtering and calibration, peaks alignment, deconvolution analysis, peaks identification, and integration of the peak area [23]. Subsequently, the resulting normalized data were input into the SIMCA 14.1 software package (Umetrics AB, Umea, Sweden). After mean centralizing and unit variance scaling, multivariate analyses, such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), were performed to visualize metabolic differences between the two groups. Finally, the differentially expressed metabolites were characterized and recognized by searching the online Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/). In addition, a free and web-based tool, MetaboAnalyst (http://www.metaboanalyst.ca/), which uses the high-quality KEGG metabolic pathway as the backend knowledgebase, was used for pathway analysis.
The results of intestinal morphology and the relative mRNA expression of tight junction proteins were expressed as means ± SEM. The P values < 0.05 were considered as the significance threshold, and P values < 0.01 were defined as an extremely significant differences based on an independent-sample t-test. All analyses were conducted using SPSS (Version 22.0).
Intestine morphology
The light micrograph of small intestine morphology is exhibited in Fig. 1. Early inoculation increased the villus heights of the duodenum, jejunum and ileum. Moreover, this result was statistically confirmed by the data shown in Fig. 2. The villus height of jejunum was markedly increased in the F group on days 14 (P < 0.05) and 28 (P < 0.05). In addition, early inoculation significantly increased the villus heights of duodenum (P < 0.01) and ileum (P < 0.05) on day 28. However, early inoculation had no significant effect on crypt depth, except for in the ileum on day 14. It is worth noting that the ratio of villus height to crypt depth (V/C) in the small intestine in the F group was significantly increased when compared with the C group (P < 0.05).
Histomorphology of small intestine of 14-day-old and 28-day-old broilers. C group: control group; F group: fermentation broth group
Effect of early inoculation on villus height and crypt depth of the small intestines of 14-day-old and 28-day-old broilers. a Villus height of the duodenum, jejunum and ileum. b Crypt depth of the duodenum, jejunum and ileum. c Ratio of villus height to crypt depth (V/C) of the duodenum, jejunum and ileum. The data are presented as means ± SEM (n = 8). Asterisks (* and **) represent significant differences with P < 0.05 and P < 0.01, respectively. C group: control group; F group: fermentation broth group
Gene expression of tight junction proteins
To further investigate the effect of early inoculation on the intestinal barrier integrity, the relative gene expression levels of tight junction proteins CLDN1, CLDN2, ZO1, and ZO2 in ileum were measured (Fig. 3). Early inoculation significantly upregulated the ileum ZO1 mRNA expression on days 14 and 28 (P < 0.05), and the ileum CLDN1 mRNA expression on day 28 (P < 0.05). In contrast, the relative gene expression levels of CLDN2 on days 14 (P < 0.05) and 28 (P < 0.01) were dramatically decreased in the F group. However, no significant differences were found regarding the relative expression of ileum ZO2 between the two groups at either 14 or 28 d.
Relative gene expression of ZO1 (a), ZO2 (b), CLDN1 (c) and CLDN2 (d) in the ileum of 7-day-old, 14-day-old and 28-day-old broilers. The data are presented as means ± SEM (n = 5). Asterisks (* and **) represent significant differences with P < 0.05 and P < 0.01, respectively. ZO1: zonula occludens-1; ZO2: zonula occludens-2; CLDN1: claudin-1; CLDN2: claudin-2. C group: control group; F group: fermentation broth group
Metabolites profiling using GC-TOF/MS
The GC-TOF/MS platform was used to study the response of intestinal metabolomics profiles to early inoculation with caecal fermentation broth. Total ion chromatograms (TIC) of caecal content samples from the C group and the F group on days 7 and 14 are displayed in Additional file 1: Figure S1. A total of 457 valid peaks were extracted after preprocessing of the raw data; among the peaks, 179 compounds were relatively quantified, 102 were marked "unknown" and 176 were labeled "analyte" when referring to the LECO-Fiehn Rtx5 Metabolomics Library.
Statistical comparison of metabolites
The differences in the metabolomics profiles between the C group and the F group by the multivariate analysis are displayed in Fig. 4. The overall changes in metabolic physiology were readily observed by the unsupervised PCA of the entire set of analytes tested. The PCA score plots of the C group and the F group showed a general separation trend not only on day 7 but also on day 14 (Fig. 4a and b). None of the samples went beyond the 95% Hotelling's T-squared ellipse, suggesting that there was no outlier among the analyzed samples. To obtain a better understanding of early intervention responsible for classification and a higher level of group separation, an OPLS-DA was employed to elucidate the different metabolic patterns. The OPLS-DA plots (Fig. 4c and d) showed markedly separated clusters between the C group and the F group on days 7 and 14. In addition, the OPLS-DA model was tested by a random-permutations analysis to confirm its validity and robustness (Fig. 4e and f). The 200 permutation texts result showed that the respective R2Y and Q2 intercept values were 0.92 and − 0.38 on day 7, and 0.94 and − 0.55 on day 14. In general, when the corresponding Q2 value is less than 0, the statistical model is determined to be valid with a low risk of over fitting [24]. Therefore, the lower values of the Q2 intercept in our results suggested that the robustness of the models was suitable for metabolomics analysis.
PCA score plots (a and b), OPLS-DA score plots (c and d) and OPLS-DA corresponding validation plots (e and f) resulting from the GC-TOF/MS metabolite profiles of the caecal contents of 7-day-old and 14-day-old broilers. PCA: principal component analysis; OPLS-DA: orthogonal partial least squares discriminant analysis. C group: control group; F group: fermentation broth group
Identification of significantly different metabolites
The differentially expressed metabolites were recognized according to the following assessment criterion: the variable importance for the projection (VIP) values > 1.0 in OPLS-DA model and P values < 0.05 in Student's t-test. Relative to the C group, the relative levels of 47 and 166 metabolites were significantly altered in the F group on days 7 and 14, respectively. As shown in Fig. 5a, 13 metabolites in the F group were upregulated, and 34 metabolites were downregulated on day 7. Similar results were found on day 14, with 20 upregulated metabolites and 146 downregulated metabolites (Fig. 5b). Hierarchical clustering together with a heatmap was performed to reveal the distinct characteristics of the differentially expressed metabolites (excluding the 28 from 47 total differentially expressed metabolites that were unidentified on day 7, and the 118 from 166 on day 14) based on the relative levels of the identified metabolite. After annotation, it was observed that 6 compounds on day 7 and 10 compounds on day 14 exhibited higher concentrations in the F group than that in the C group. By contrast, 13 and 38 compounds were respectively substantially downregulated on days 7 and 14 (Fig. 6 and Additional file 2: Table S1). More importantly, we found that the level of 1,3-diaminopropane in the F group was significantly increased not only on day 7, but also on day 14. It was the only significantly different metabolite that was upregulated at both test ages.
Volcano plots of caecal metabolites of 7-day-old (a) and 14-day-old (b) broilers. The red dot represents a significantly different metabolite that is more concentrated in the F group, while the blue dot represents a metabolite that was mainly enriched in the C group. The dot size indicates the variable importance in the projection (VIP) value. Fold Change: mean value of peak area obtained from the F group/mean value of peak area obtained from the C group
Hierarchical clustering analysis for the significantly different metabolites based on average clustering algorithm with Pearson distance. a 7-day-old broilers. b 14-day-old broilers. C group: control group; F group: fermentation broth group
Characterization of significantly different metabolic pathways that responded to early inoculation
To explore the significantly different metabolic pathways that responded to early inoculation, we imported the significantly different metabolites (Additional file 3: Table S2) into KEGG. Consequently, 18 and 9 metabolic pathways were respectively identified between the C group and the F group on days 7 and 14 (Additional file 4: Table S3). As shown in the metabolome view map (Fig. 7), the relevant metabolic pathways were illustrated using both –ln P-value and pathway impact scores as criteria. These pathways mainly include amino acid metabolism (beta-alanine, alanine, aspartate, glutamate, arginine and proline metabolism), energy metabolism (biosynthesis of unsaturated fatty acids, citrate cycle and pyruvate metabolism) and glycolysis or gluconeogenesis on day 7 (Fig. 7a), and amino acids metabolism (beta-alanine and phenylalanine metabolism), pantothenate and CoA biosynthesis, and pyrimidine metabolism on day 14 (Fig. 7b).
Metabolome view map of the significantly different metabolites. Significantly changed pathways based on enrichment and topology analysis are shown. The X-axis represents the pathway impact, and the Y-axis represents the pathway enrichment. Larger sizes and darker colors represent higher pathway impact values and higher pathway enrichment, respectively. a 7-day-old broilers. b 14-day-old broilers
The objective of this study was to determine whether inoculation of fermentation broth at 2 h post-hatch affects the small intestine morphology, expression levels of tight junction-proteins in the ileum, and the caecal metabolomics profiling of broilers. Maintaining optimal health of animals is highly dependent on the complete intestine morphology and nutrient metabolism in the complex intestinal system. We know that an optimized environment in the gut is critical to host health. It has been shown in some published papers that the changes of intestinal environment in early life can alter the colonization and development of gut microbiota, and then regulate the subsequent development and health of animals [25, 26]. In this regard, inoculation with caecal fermentation broth for broilers at the early stage of life could be used as a new regulation strategy for poultry health relative to the traditional nutrition regulation.
In the present study, we used the broiler model to investigate the response of the intestinal physical barrier to early inoculation. The results showed that early inoculation with caecal fermentation broth can improve small intestinal morphology, as indicated by increased villi length as well as villus height to crypt depth ratios in the duodenum, jejunum and ileum. The intact intestinal structure is important for animal digestion and absorption, which is closely related to the morphological characteristic of small intestinal villus length and crypt depth [27, 28]. A previous study revealed that the villus height is correlated with the absorptive capability of the small intestine, elevations in villus height would contribute to nutrient transport [29]. Similarly, the morphology parameters measured in our study increased significantly, particularly villus height, suggesting that early inoculation could improve the absorption function of small intestine. Consistently, this can be used to explain our previous study, which found that early inoculation with caecal fermentation broth had a positive effect on the growth performance of broilers [11].
In poultry studies, the lack of appropriate commercial avian antibodies makes it impossible to assess the effects at the protein level. Hence, a qRT-PCR technique was performed in our experiment to illuminate the effect of early inoculation on the relative gene expression of tight junction proteins, namely, CLDN1, CLDN2, ZO1 and ZO2 [30]. We observed that early inoculation promoted upregulation of the expression of tight junction protein CLDN1 on day 28, and ZO1 on days 14 and 28. However, the relative expression level of tight junction protein CLDN2 was dramatically decreased in the intervened broilers on days 14 and 28. As one of the most important components of the intestinal epithelial barrier, CLDN1 can effectively prevent harmful substances from reaching the surface of the epithelial cells [31]. A previous study showed that the high expression of CLDN1 could result in increased epithelial tightness and decreased solute permeability [32]. In contrast, CLDN2 is a pore-forming tight junction protein [33]. The upregulation of CLDN2 expression has been coincidentally reported just in inflammatory bowel disease, and it is usually considered an indicator of inflammation [34]. The zona occludens family, including ZO1 and ZO2, is a set of scaffolding proteins that is an essential part of the tight junctions in cytoplasmic plaques [30]. Taken together, the increased gene expression of CLDN1, and ZO1 and the decreased gene expression of CLDN2 in the intervened broilers point to an enhancement of the intestinal barrier by early inoculation.
The knowledge of the interaction between microbiota metabolism and host health is truly beneficial for effective regulation of the growth and health of birds. The phenotype of an organism and its reaction to environmental perturbations are usually closely related to its metabolism, and the development of metabolome technology in recent years provides a unique method to examine the physiological status of the biological system [26]. To further explore the effect of early inoculation on intestinal physiological function, a GC-TOF/MS-based metabolomics method was applied in the current study to quantitatively detect small molecular metabolites in broilers caecum and identify relevant pathways. The intestinal metabolomics profiles of the F group remarkably differed from those of the C group. In total, 47 metabolites were altered followed early inoculation on day 7, and 166 were altered on day 14. More differentially expressed metabolites detected on day 14 were attributed to the increased metabolic capacity of broilers with age. However, it should be noted that not all of these metabolites at the age of 7 and 14 days presented the same increasing or decreasing trend. Among all the 47 and 166 differentially expressed metabolites identified by multivariate statistical analysis on days 7 and 14, respectively, only one compound, namely 1,3-diaminopropane, was continuously elevated following early inoculation on days 7 and 14. Using an untargeted metabolomics analysis, a previous study also found that 1,3-diaminopropane was significantly increased in the recipient piglets following fecal microbiota transplantation [35], which is in line with our result. Therefore, 1,3-diaminopropane was selected as a biomarker, suggesting that this compound could be used as a biomarker of environmental exposure of animal gut at an early stage of life. Previous scientific evidence indicated that 1,3-diaminopropane, a biologically significant diamine, is metabolized by amino acid [36]. As a type of polyamines, 1,3-diaminopropane is considered to play various biological roles in gene expression, cell death and cell stress protection [37, 38]. This notion is supported by the enhanced intestinal morphology and the increased expression levels of tight junction proteins CLDN1 and ZO1 in our study.
Subsequently, we conducted metabolic pathway analyses to put all of the differentially expressed metabolites into the context of connected metabolic pathway networks. Several key metabolic pathways were identified to respond to early inoculation with caecal fermentation broth. After analysis of these metabolic pathways, beta-alanine metabolism and biosynthesis of unsaturated fatty acids were considered as significantly impacted metabolic pathways, which were not only in the metabolome view map on day 7 but also on day 14. During beta-alanine metabolism, the level of 1,3-diaminopropane in the F group was higher than that of the C group, resulting in a decreased beta-alanine level in the intervened broilers. An increasing number of reports have now demonstrated that an accumulation of beta-alanine was detected in breast cancer [39, 40], suggesting that elevated levels of beta-alanine might be a disadvantage to human health. Therefore, this alternation in beta-alanine metabolism in the present study may have a positive effect on the growth and health of the intervened broilers. However, the role of beta-alanine in health remains to be further studied. Previous reports have revealed that unsaturated fatty acids plays a vital role in inflammation and regulation of immunity [41, 42]. Based on our results, biosynthesis of unsaturated fatty acids was found to be accelerated by early inoculation with caecal fermentation broth. The elevated biosynthesis of unsaturated fatty acids in broilers probably contributed to trigger unsaturated fatty-acid-dependent killing of pathogenic bacteria, which is in line with the significantly decreased abundance of pathogenic bacteria in our previous 16S rRNA gene sequencing results [11]. In the present study, characterization of metabolism induced by early intestinal environmental exposure could reveal the major regulated metabolic pathways for broilers' growth and health, but the specific metabolic mechanism still needs further investigation.
Taken together, our study demonstrated that early inoculation with caecal fermentation broth not only altered intestinal morphological parameters but also changed the caecum metabolic profile characterization of broilers. The metabolomics results showed that beta-alanine metabolism and biosynthesis of unsaturated fatty acids were the key metabolic pathways affected by early inoculation in which the significantly different metabolites were mainly enriched. The compound 1,3-diaminopropane was selected as a potential biomarker that responded to administration with caecal fermentation broth in the early stage of broilers. Additionally, the changes in the intestinal environment induced by early inoculation improved small intestine morphology and the gene expression of tight junction proteins in the ileum. These findings provide insight into intestinal metabolomics and provide an environmental intervention strategy for broiler growth and health.
All data generated or analyzed during this study are available from the corresponding author on reasonable request.
CLDN1:
CoA:
EP:
Epoxy epoxide
GC-TOF/MS:
Gas chromatography time-of-flight/mass spectrometry
GIT:
OPLS-DA:
Orthogonal partial least squares discriminant analysis
Phosphate buffered saline
PCA:
qRT-PCR:
Quantitative real-time polymerase chain reaction
TIC:
Total ion chromatogram
V/C:
The villus height-to-crypt depth ratio
Variable-importance-projection
ZO1:
Zonula occludens-1
Levy M, Thaiss CA, Elinav E. Metabolites: messengers between the microbiota and the immune system. Genes Dev. 2016;30:1589–97.
Zhu XY, Zhong T, Pandya Y, Joerger RD. 16S rRNA-based analysis of microbiota from the cecum of broiler chickens. Appl Environ Microbiol. 2002;68:124–37.
Cockburn DW, Koropatkin NM. Polysaccharide degradation by the intestinal microbiota and its influence on human health and disease. J Mol Biol. 2016;428:3230–52.
Yadav S, Jha R. Strategies to modulate the intestinal microbiota and their effects on nutrient utilization, performance, and health of poultry. J Anim Sci Biotechnol. 2019;10:1–11.
Yadav M, Verma MK, Chauhan NS. A review of metabolic potential of human gut microbiome in human nutrition. Arch Microbiol. 2018;200:203–17.
Wang Y, Sun J, Zhong H, Li N, Xu H, Zhu Q, et al. Effect of probiotics on the meat flavour and gut microbiota of chicken. Sci Rep. 2017;7:6400.
Huyghebaert G, Ducatelle R, Immerseel FV. An update on alternatives to antimicrobial growth promoters for broilers. Vet J. 2011;187:182–8.
Torok VA, Ophel-Keller K, Loo M, Hughes RJ. Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism. Appl Environ Microbiol. 2008;74:783–91.
Bedford MR, Cowieson AJ. Exogenous enzymes and their effects on intestinal microbiology. Anim Feed Sci Technol. 2012;173(1–2):0–85.
Oviedo-Rondón EO, Hume ME, Hernández C, Clemente-Hernández S. Intestinal microbial ecology of broilers vaccinated and challenged with mixed Eimeria species, and supplemented with essential oil blends. Poult Sci. 2006;85:854–60.
Gong Y, Yang H, Wang X, Xia W, Lv W, Xiao Y, et al. Early intervention with Cecal fermentation broth regulates the colonization and development of gut microbiota in broiler chickens. Front Microbiol. 2019;10:1422.
Saraf MK, Piccolo BD, Bowlin AK, Mercer KE, Leroith T, Chintapalli SV, et al. Formula diet driven microbiota shifts tryptophan metabolism from serotonin to tryptamine in neonatal porcine colon. Microbiome. 2017;5(1):77.
Theriot CM, Koenigsknecht MJ, Carlson PE, Hatton GE, Nelson AM, Li B, et al. Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection. Nat Commun. 2014;5:3114.
Roy TL, Llopis M, Lepage P, Bruneau A, Rabot S, Bevilacqua C, et al. Intestinal microbiota determines development of non-alcoholic fatty liver disease in mice. Gut. 2013;62:1787–94.
Mafra D, Lobo JC, Barros AF, Koppe L, Vaziri ND, Fouque D. Role of altered intestinal microbiota in systemic inflammation and cardiovascular disease in chronic kidney disease. Future Microbiol. 2014;9:399–410.
Rinttilä T, Apajalahti J. Intestinal microbiota and metabolites—implications for broiler chicken health and performance1. J Appl Poult Res. 2013;22:647–58.
Hu F, Gao X, She R, Chen J, Mao J, Xiao P, et al. Effects of antimicrobial peptides on growth performance and small intestinal function in broilers under chronic heat stress. Poult Sci. 2016;96:798–806.
Zihni C, Mills C, Matter K, Balda MS. Tight junctions: from simple barriers to multifunctional molecular gates. Nat Rev Mol Cell Biol. 2016;17:564–80.
Li X, Akhtar S, Choudhry MA. Alteration in intestine tight junction protein phosphorylation and apoptosis is associated with increase in IL-18 levels following alcohol intoxication and burn injury. Biochim Biophys Acta. 2012;1822(2):0–203.
Anwar S. Effect of dietary supplementation of Catharanthus roseus on gross and micro-structures of selected internal organs of broilers. M. Phil thesis. Lahore: University of Veterinary and Animal Sciences; 2013.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (−Delta Delta C (T)) method. Methods. 2001;25:402–8.
Garcia A, Barbas C. Gas chromatography-mass spectrometry (GC-MS)-based metabolomics. Methods Mol Biol. 2011;708(8):191–204.
Kind T, Wohlgemuth G, Lee DY, Lu Y, Palazoglu M, Shahbaz S, et al. FiehnLib: mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry. Anal Chem. 2009;81:10038–48.
Mahadevan S, Shah SL, Marrie TJ, Slupsky CM. Analysis of metabolomic data using support vector machines. Anal Chem. 2008;80:7562–70.
Yin Y, Lei F, Zhu L, Li S, Wu Z, Zhang R, et al. Exposure of different bacterial inocula to newborn chicken affects gut microbiota development and ileum gene expression. ISME J. 2009;4:367–76.
Xu M, Zhong F, Zhu J. Evaluating metabolic response to light exposure in Lactobacillus species via targeted metabolic profiling. J Microbiol Methods. 2017;133:14–9.
Brudnicki A, Brudnicki W, Szymeczko R, Bednarczyk M, Pietruszynska D, Kirkillo-Stacewicz K. Histomorphometric adaptation in the small intestine of broiler chicken, after embryonic exposure to a – Galactosides. J Anim Plant Sci. 2017;27:1075–82.
Wang X, Li Y, Shen J, Wang S, Yao J, Yang X. Effect of Astragalus polysaccharide and its sulfated derivative on growth performance and immune condition of lipopolysaccharide-treated broilers. Int J Biol Macromol. 2015;76:188–94.
Yamauchi K, Buwjoom T, Koge K, Ebashi T. Histological alterations of the intestinal villi and epithelial cells in chickens fed dietary sugar cane extract. Br Poult Sci. 2006;47:544–53.
Osselaere A, Santos R, Hautekiet V, Backer PD, Chiers K, Ducatelle R, et al. Deoxynivalenol impairs hepatic and intestinal gene expression of selected oxidative stress, tight junction and inflammation proteins in broiler chickens, but addition of an adsorbing agent shifts the effects to the distal parts of the small intestine. PLoS One. 2013;8:e69014.
Song J, Li Q, Li P, Liu RR, Cui H, Zheng M, et al. The effects of inulin on the mucosal morphology and immune status of specific pathogen-free chickens. Poult Sci. 2018;97:3938–46.
Wageha A, Claudia H, Michael H. Enteric pathogens and their toxin-induced disruption of the intestinal barrier through alteration of tight junctions in chickens. Toxins. 2017;9(2):60.
Suzuki T. Regulation of intestinal epithelial permeability by tight junctions. Cell Mol Life Sci. 2013;70:631–59.
Geng SJ, Cheng SS, Li Y, Wen ZS, Ma X, Jiang XM, et al. Faecal microbiota transplantation reduces susceptibility to epithelial injury and modulates tryptophan metabolism of the microbial community in a piglet model. J Crohns Colitis. 2018;12:1359–74.
Liu R, Li P, Bi CW, Ma R, Yin Y, Bi K, et al. Plasma N-acetylputrescine, cadaverine and 1,3-diaminopropane: potential biomarkers of lung cancer used to evaluate the efficacy of anticancer drugs. Oncotarget. 2017;8:88575–85.
Bachrach U. The early history of polyamine research. Plant Physiol Biochem. 2010;48:490–5.
Moschou PN, Roubelakisangelakis KA. Polyamines and programmed cell death. J Exp Bot. 2014;65:1285–96.
Budczies J, Lehmann A, Griffin JL, Oresic M, Fiehn O, Denkert C. Abstract 4806: association of changes in 4-aminobutyrate aminotransferase (ABAT) and beta-alanine metabolism with breast cancer and the more aggressive estrogen receptor negative subtype. Cancer Res. 2012;72(8 Supplement):4806.
Budczies J, Brockmöller SF, Müller BM, Barupal DK, Richter-Ehrenstein C, Kleine-Tebbe A, et al. Comparative metabolomics of estrogen receptor positive and estrogen receptor negative breast cancer: alterations in glutamine and beta-alanine metabolism. J Proteome. 2013;94:279–88.
Harbige LS. Fatty acids, the immune response, and autoimmunity: a question of n-6 essentiality and the balance between n-6 and n-3. Lipids. 2003;38:323–41.
Schlotz N, Ebert D, Martin-Creuzburg D. Dietary supply with polyunsaturated fatty acids and resulting maternal effects influence host – parasite interactions. BMC Ecol. 2013;13:41.
This work was supported by the National Key R&D Program of China (2017YFD0500501), State Key Laboratory for Quality and Safety of Agro-products (2010DS700124-ZZ1905).
State Key Laboratory for Quality and Safety of Agro-products, Institute of Quality and Standards for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China
Yujie Gong
, Wenrui Xia
& Xiaoting Zou
Key Laboratory for Molecular Animal Nutrition of Ministry of Education, Feed Sciences Institute, College of Animal Sciences, Zhejiang University (Zijingang Campus), Hangzhou, 310058, China
Xueting Wen
, Wentao Lyu
, Yingping Xiao
& Hua Yang
Search for Yujie Gong in:
Search for Wenrui Xia in:
Search for Xueting Wen in:
Search for Wentao Lyu in:
Search for Yingping Xiao in:
Search for Hua Yang in:
Search for Xiaoting Zou in:
The article was mainly conceived and designed by YX, HY and XZ. YG, WX, XW and WL performed the experiments and analyzed the data. The manuscript was mainly written by YG. YX, HY and XZ revised it critically for important content. All authors have read and approved the final manuscript.
Correspondence to Yingping Xiao or Hua Yang or Xiaoting Zou.
This experiment was approved by the Animal Care and Use Committee of Zhejiang University, and all experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals, approved by the State Council of the People's Republic of China.
Additional file 1: Figure S1. Typical GC-TOF/MS TIC chromatograms of caecal content samples from the C group and the F group. The green and blue lines represent the C group on days 7 and 14, the red and black lines represent the F group on days 7 and 14.
Additional file 2: Table S1. The hierarchical clustering data matrix for the C group and the F group on days 7 and 14.
Additional file 3: Table S2. Significantly different metabolites between the C group and the F group. The red text indicates the significantly different metabolite that is more abundant in the F group, while the blue text represents the higher concentration of significantly different metabolite in the C group.
Additional file 4: Table S3. Pathway analysis for the C group and the F group on days 7 and 14.
Gong, Y., Xia, W., Wen, X. et al. Early inoculation with caecal fermentation broth alters small intestine morphology, gene expression of tight junction proteins in the ileum, and the caecal metabolomic profiling of broilers. J Animal Sci Biotechnol 11, 8 (2020) doi:10.1186/s40104-019-0410-1
Early inoculation
Metabolomics profiling

redpajama_set_name: RedPajamaCommonCrawl


комплект для подключения топливного фильтра-сепаратора к электрической схеме двигателя.
комплект кронштейна для топливный фильтра-сепаратора и подогревателя 24v (ном.поток 340 литров/ч).
USES HOUSING FH23500 Diesel Pro Remote Mount Medium Duty Engines, Fuel Flows up to 60 ghp (230 lph), clear bowl.
USES HOUSING FH23501 Diesel Pro Remote Mount Medium Duty Engines, Combo Pre-heater 24V, Fuel Flows up to 60 ghp (230 lph), clear bowl.
USES HOUSING FH23502 Diesel Pro Remote Mount Medium Duty Engines, Combo Pre-heater 12V, Fuel Flows up to 60 ghp (230 lph), clear bowl.
USES HOUSING FH23504M Diesel Pro Remote Mount Medium Duty Engines, Combo Pre-heater 24V, WIF, Fuel Flows up to 60 ghp (230 lph), clear bowl.
USES HOUSING FH23506 Diesel Pro Remote Mount Medium Duty Engines, Combo Pre-heater 24V, WIF, Fuel Flows up to 60 ghp (230 lph), clear bowl.
USES HOUSING FH23508 Diesel Pro Remote Mount Medium Duty Engines, WIF, Fuel Flows up to 60 ghp (230 lph), clear bowl.
USES HOUSING FH23509M Diesel Pro Remote Mount Medium Duty Engines, WIF, Fuel Flows up to 60 ghp (230 lph), clear bowl.
USES HOUSING FH23510 Diesel Pro Remote Mount Medium Duty Engines, Combo Pre-heater 12V, WIF, Fuel Flows up to 60 ghp (230 lph), clear bowl.
USES HOUSING FH23503M Diesel Pro Remote Mount Medium Duty Engines, WIF, Fuel Flows up to 60 ghp (230 lph), clear bowl.


text:
package com.linkcld.toms.anlaysis.holiday.service;

import com.linkcld.toms.common.dao.*;
import com.linkcld.toms.common.utils.FieldIsNullHandleUtil;
import org.apache.commons.collections4.map.HashedMap;
import org.springframework.beans.factory.annotation.Autowired;
import org.springframework.stereotype.Service;

import java.text.DateFormat;
import java.text.ParseException;
import java.text.SimpleDateFormat;
import java.util.*;

/**
 * Created by Administrator on 2017/9/12.
 */
@Service("holidayServiceImpl")
public class HolidayServiceImpl implements HolidayService {
    @Autowired
    private HolidayMapper holidayMapper;
    @Autowired
    private TrainMapper trainMapper;
    @Autowired
    private ShuttlebusMapper shuttlebusMapper;
    @Autowired
    private HighWayMapper highWayMapper;
    @Autowired
    private BusMapper busMapper;
    @Autowired
    private TaxiMapper taxiMapper;
    @Autowired
    private IBicycleMapper bicycleMapper;

    /**
     * 客流量指标面板(铁路、客运、高速)
     */
    @Override
    public List<Map> springFestivalFlowRateIndexPanel(Map map) {
        List<Map> resultlist = new ArrayList<>();
        Map resultMap = new HashMap();
        resultMap.put("trainIndex",trainMapper.getFlowRateByDay(map));
        resultMap.put("shuttleBusIndex",shuttlebusMapper.getShuttleBusDataByDay(map));
        resultMap.put("highWayIndex",highWayMapper.getCountFlowByDay(map));
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 日运输畅通情况(公交日运行速率、出租车日实载率、自行车日使用率)
     */
    @Override
    public List<Map> toDayUnimpededStatus(Map map) {
        List<Map> resultlist = new ArrayList<>();
        Map resultMap = new HashMap();
        Map busSpeedByDay = busMapper.busSpeedByDay(map);
        Map TaxiUseRateByDay = taxiMapper.TaxiUseRateByDay(map);
        Map bicycleUseRateByDay2 = bicycleMapper.bicycleUseRateByDay2(map);

        if(null==busSpeedByDay){
            busSpeedByDay = FieldIsNullHandleUtil.fieldIsNullHandle("busSpeedByDay");
        }
        double bus = Double.parseDouble(String.valueOf(busSpeedByDay.get("LINESPEED")));
        if(bus<12){
            busSpeedByDay.put("STATUS","堵塞");
        }else if(bus>=12 && bus<=18){
            busSpeedByDay.put("STATUS","一般");
        }else if(bus>18){
            busSpeedByDay.put("STATUS","通畅");
        }

        if(null==TaxiUseRateByDay){
            TaxiUseRateByDay = FieldIsNullHandleUtil.fieldIsNullHandle("TaxiUseRateByDay");
        }
        double taxi = Double.parseDouble(String.valueOf(TaxiUseRateByDay.get("SZL")));
        if(taxi<40){
            TaxiUseRateByDay.put("STATUS","低");
        }else if(taxi>=40 && taxi<=60){
            TaxiUseRateByDay.put("STATUS","中");
        }else if(taxi>60){
            TaxiUseRateByDay.put("STATUS","高");
        }

        if(null==bicycleUseRateByDay2){
            bicycleUseRateByDay2 = FieldIsNullHandleUtil.fieldIsNullHandle("bicycleUseRateByDay2");
        }
        double bicycle = Double.parseDouble(String.valueOf(bicycleUseRateByDay2.get("SYL")));
        if(bicycle<40){
            bicycleUseRateByDay2.put("STATUS","低");
        }else if(bicycle>=40 && bicycle<=60){
            bicycleUseRateByDay2.put("STATUS","中");
        }else if(bicycle>60){
            bicycleUseRateByDay2.put("STATUS","高");
        }

        resultMap.put("busSpeedByDay",busSpeedByDay);
        resultMap.put("TaxiUseRateByDay",TaxiUseRateByDay);
        resultMap.put("bicycleUseRateByDay",bicycleUseRateByDay2);
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 铁路站点流量分布、客运站点流量分布、高速收费站流量TOP5
     */
    @Override
    public List<Map> FlowRateDistribution(Map map) {
        List<Map> resultlist = new ArrayList<>();
        Map resultMap = new HashMap();
        List<Map> train = trainMapper.getDaySiteFlowRateByDay(map);
        if(null!=train){
            double num = 0.0;
            for(int i = 0;i<train.size()-1;i++){
                String  zdllfb= String.valueOf(train.get(i).get("ZDLLFB"));
                num +=Double.parseDouble(zdllfb);
            }
            train.get(train.size()-1).put("ZDLLFB",100-num);
        }
        resultMap.put("trainSiteFlowRate",train);
        resultMap.put("SiteFlowDistribution",SiteFlowDistribution(map));
        resultMap.put("highWayFlowTop5",highWayFlowTop5(map));
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 客运站点流量分布
     */
    @Override
    public List<Map> SiteFlowDistribution(Map map) {
        return holidayMapper.SiteFlowDistribution(map);
    }

    /**
     * 高速收费站流量TOP5
     */
    @Override
    public List<Map> highWayFlowTop5(Map map) {
        return holidayMapper.highWayFlowTop5(map);
    }

    /**
     * 铁路客运量、班车客运量、高速车流量、列车晚点数、公交运行速率、出租车实载率
     * @param map
     * @return
     */
    @Override
    public List<Map> springFestivalData(Map map) throws ParseException {
        List<Map> resultlist = new ArrayList<>();
        Map resultMap = new HashMap();
        resultMap.put("trainFlowRate",trainFlowRate(map));
        resultMap.put("suttleBusFlowRate",suttleBusFlowRate(map));
        resultMap.put("highWayFlowRate",highWayFlowRate(map));
        resultMap.put("trainLateNum",trainLateNum(map));
        resultMap.put("BusSpeed",BusSpeed(map));
        resultMap.put("taxiSzl",taxiSzl(map));
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 铁路客运量
     */
    @Override
    public List<Map> trainFlowRate(Map map) throws ParseException {
        List<Map> resultlist = new ArrayList<>();

        List<Map> result = holidayMapper.trainFlowRate(map);
        List dateList = pastToFeture(String.valueOf(map.get("dateType")),
                                    Integer.parseInt(String.valueOf(map.get("past")))+1,
                                                Integer.parseInt(String.valueOf(map.get("feture"))));
        List dateList2 = pastToFeture(String.valueOf(map.get("dateTypeLastYear")),
                                    Integer.parseInt(String.valueOf(map.get("past")))+1,
                                                Integer.parseInt(String.valueOf(map.get("feture"))));
        int[] dayNum = new int[dateList.size()];
        int[] total = new int[dateList.size()];
        int[] total2 = new int[dateList2.size()];
        for(int i = 0;i<dateList.size();i++){
            for (Map m : result) {
                if(null!=m.get("DAYTIME")){
                    if (m.get("DAYTIME").toString().equals(dateList.get(i))) {
                        total[i] = m.get("TOTAL") == null ? 0 : Integer.parseInt(m.get("TOTAL").toString());
                    }
                }

                if(null!=m.get("DAYTIME2")){
                    if (m.get("DAYTIME2").toString().equals(dateList2.get(i))) {
                        total2[i] = m.get("TOTAL2") == null ? 0 : Integer.parseInt(m.get("TOTAL2").toString());
                    }
                }
            }
        }

        for(int i =0;i<dateList.size();i++){
            dayNum[i] = i+1;
        }

        Map<String, Object> resultMap = new HashedMap();
//        resultMap.put("dayTime", dateList);
//        resultMap.put("dayTimeLastYear", dateList2);
        resultMap.put("total", total);
        resultMap.put("totalLastYear", total2);
        resultMap.put("dayNum", dayNum);
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 民航客运量
     */
    @Override
    public List<Map> fightFlowRate(Map map) {
        return holidayMapper.fightFlowRate(map);
    }

    /**
     * 班车客运量
     */
    @Override
    public List<Map> suttleBusFlowRate(Map map) throws ParseException {
        List<Map> resultlist = new ArrayList<>();

        List<Map> result = holidayMapper.suttleBusFlowRate(map);
        List dateList = pastToFeture(String.valueOf(map.get("dateType")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        List dateList2 = pastToFeture(String.valueOf(map.get("dateTypeLastYear")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        int[] dayNum = new int[dateList.size()];
        int[] total = new int[dateList.size()];
        int[] total2 = new int[dateList2.size()];
        for(int i = 0;i<dateList.size();i++){
            for (Map m : result) {
                if(null!=m.get("DAYTIME")){
                    if (m.get("DAYTIME").toString().equals(dateList.get(i))) {
                        total[i] = m.get("FSLTOTAL") == null ? 0 : Integer.parseInt(m.get("FSLTOTAL").toString());
                    }
                }

                if(null!=m.get("DAYTIME2")){
                    if (m.get("DAYTIME2").toString().equals(dateList2.get(i))) {
                        total2[i] = m.get("FSLTOTAL2") == null ? 0 : Integer.parseInt(m.get("FSLTOTAL2").toString());
                    }
                }
            }
        }

        for(int i =0;i<dateList.size();i++){
            dayNum[i] = i+1;
        }

        Map<String, Object> resultMap = new HashedMap();
//        resultMap.put("dayTime", dateList);
//        resultMap.put("dayTimeLastYear", dateList2);
        resultMap.put("total", total);
        resultMap.put("totalLastYear", total2);
        resultMap.put("dayNum", dayNum);
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 高速车流量
     */
    @Override
    public List<Map> highWayFlowRate(Map map) throws ParseException {
        List<Map> resultlist = new ArrayList<>();

        List<Map> result = holidayMapper.highWayFlowRate(map);
        List dateList = pastToFeture(String.valueOf(map.get("dateType")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        List dateList2 = pastToFeture(String.valueOf(map.get("dateTypeLastYear")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        int[] dayNum = new int[dateList.size()];
        int[] total = new int[dateList.size()];
        int[] total2 = new int[dateList2.size()];
        for(int i = 0;i<dateList.size();i++){
            for (Map m : result) {
                if(null!=m.get("DAYTIME")){
                    if (m.get("DAYTIME").toString().equals(dateList.get(i))) {
                        total[i] = m.get("CLZZTOTAL") == null ? 0 : Integer.parseInt(m.get("CLZZTOTAL").toString());
                    }
                }

                if(null!=m.get("DAYTIME2")){
                    if (m.get("DAYTIME2").toString().equals(dateList2.get(i))) {
                        total2[i] = m.get("CLZZTOTAL2") == null ? 0 : Integer.parseInt(m.get("CLZZTOTAL2").toString());
                    }
                }
            }
        }

        for(int i =0;i<dateList.size();i++){
            dayNum[i] = i+1;
        }

        Map<String, Object> resultMap = new HashedMap();
//        resultMap.put("dayTime", dateList);
//        resultMap.put("dayTimeLastYear", dateList2);
        resultMap.put("total", total);
        resultMap.put("totalLastYear", total2);
        resultMap.put("dayNum", dayNum);
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 列车晚点数
     */
    @Override
    public List<Map> trainLateNum(Map map) throws ParseException {
        List<Map> resultlist = new ArrayList<>();

        List<Map> result = holidayMapper.trainLateNum(map);
        List dateList = pastToFeture(String.valueOf(map.get("dateType")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        List dateList2 = pastToFeture(String.valueOf(map.get("dateTypeLastYear")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        int[] dayNum = new int[dateList.size()];
        int[] total = new int[dateList.size()];
        int[] total2 = new int[dateList2.size()];
        for(int i = 0;i<dateList.size();i++){
            for (Map m : result) {
                if(null!=m.get("DAYTIME")){
                    if (m.get("DAYTIME").toString().equals(dateList.get(i))) {
                        total[i] = m.get("YWTOTAL") == null ? 0 : Integer.parseInt(m.get("YWTOTAL").toString());
                    }
                }

                if(null!=m.get("DAYTIME2")){
                    if (m.get("DAYTIME2").toString().equals(dateList2.get(i))) {
                        total2[i] = m.get("YWTOTAL2") == null ? 0 : Integer.parseInt(m.get("YWTOTAL2").toString());
                    }
                }
            }
        }

        for(int i =0;i<dateList.size();i++){
            dayNum[i] = i+1;
        }

        Map<String, Object> resultMap = new HashedMap();
//        resultMap.put("dayTime", dateList);
//        resultMap.put("dayTimeLastYear", dateList2);
        resultMap.put("total", total);
        resultMap.put("totalLastYear", total2);
        resultMap.put("dayNum", dayNum);
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 公交运行速率
     */
    @Override
    public List<Map> BusSpeed(Map map) throws ParseException {
        List<Map> resultlist = new ArrayList<>();

        List<Map> result = holidayMapper.BusSpeed(map);
        List dateList = pastToFeture(String.valueOf(map.get("dateType")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        List dateList2 = pastToFeture(String.valueOf(map.get("dateTypeLastYear")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        int[] dayNum = new int[dateList.size()];
        double[] total = new double[dateList.size()];
        double[] total2 = new double[dateList2.size()];
        for(int i = 0;i<dateList.size();i++){
            for (Map m : result) {
                if(null!=m.get("DAYTIME")){
                    if (m.get("DAYTIME").toString().equals(dateList.get(i))) {
                        total[i] = m.get("LINESPEED") == null ? 0 : Double.parseDouble(m.get("LINESPEED").toString());
                    }
                }

                if(null!=m.get("DAYTIME2")){
                    if (m.get("DAYTIME2").toString().equals(dateList2.get(i))) {
                        total2[i] = m.get("LINESPEED2") == null ? 0 : Double.parseDouble(m.get("LINESPEED2").toString());
                    }
                }
            }
        }

        for(int i =0;i<dateList.size();i++){
            dayNum[i] = i+1;
        }

        Map<String, Object> resultMap = new HashedMap();
//        resultMap.put("dayTime", dateList);
//        resultMap.put("dayTimeLastYear", dateList2);
        resultMap.put("total", total);
        resultMap.put("totalLastYear", total2);
        resultMap.put("dayNum", dayNum);
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 出租车实载率
     */
    @Override
    public List<Map> taxiSzl(Map map) throws ParseException {
        List<Map> resultlist = new ArrayList<>();

        List<Map> result = holidayMapper.taxiSzl(map);
        List dateList = pastToFeture(String.valueOf(map.get("dateType")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        List dateList2 = pastToFeture(String.valueOf(map.get("dateTypeLastYear")),
                Integer.parseInt(String.valueOf(map.get("past")))+1,
                Integer.parseInt(String.valueOf(map.get("feture"))));
        int[] dayNum = new int[dateList.size()];
        double[] total = new double[dateList.size()];
        double[] total2 = new double[dateList2.size()];
        for(int i = 0;i<dateList.size();i++){
            for (Map m : result) {
                if(null!=m.get("DAYTIME")){
                    if (m.get("DAYTIME").toString().equals(dateList.get(i))) {
                        total[i] = m.get("SZL") == null ? 0 : Double.parseDouble(m.get("SZL").toString());
                    }
                }

                if(null!=m.get("DAYTIME2")){
                    if (m.get("DAYTIME2").toString().equals(dateList2.get(i))) {
                        total2[i] = m.get("SZL2") == null ? 0 : Double.parseDouble(m.get("SZL2").toString());
                    }
                }
            }
        }

        for(int i =0;i<dateList.size();i++){
            dayNum[i] = i+1;
        }

        Map<String, Object> resultMap = new HashedMap();
//        resultMap.put("dayTime", dateList);
//        resultMap.put("dayTimeLastYear", dateList2);
        resultMap.put("total", total);
        resultMap.put("totalLastYear", total2);
        resultMap.put("dayNum", dayNum);
        resultlist.add(resultMap);
        return resultlist;
    }

    /**
     * 获取指定时间过去任意天到的未来任意天的日期数组
     * @param date
     * @param pastDays      pastDays天之前
     * @param fetureDays      beforeDays天之后
     * @return              日期数组
     */
    public static ArrayList<String> pastToFeture(String date,int pastDays,int fetureDays) throws ParseException {
        ArrayList<String> pastDaysList = new ArrayList<>();
        for (int i = pastDays; i >0; i--) {
            if(i!=pastDays){
                pastDaysList.add(getDatePast(date,i));
            }
        }

        ArrayList<String> fetureDaysList = new ArrayList<>();
        for (int i = 0; i <fetureDays; i++) {
            fetureDaysList.add(getDateFeture(date,i));
        }

        pastDaysList.addAll(fetureDaysList);

        return pastDaysList;
    }

    /**
     * 获取过去第 pastDays 天的日期
     * @param dateString
     * @param pastDays
     * @return
     */
    public static String getDatePast(String dateString , int pastDays) throws ParseException{
        DateFormat dateFormat = new SimpleDateFormat("yyyyMMdd");
        Date inputDate = dateFormat.parse(dateString);
        Calendar cal = Calendar.getInstance();
        cal.setTime(inputDate);
        int inputDayOfYear = cal.get(Calendar.DAY_OF_YEAR);
        cal.set(Calendar.DAY_OF_YEAR , inputDayOfYear-pastDays );

        SimpleDateFormat format = new SimpleDateFormat("yyyyMMdd");
        String date = format.format(cal.getTime());
        return date;
    }

    /**
     * 获取未来第 beforeDays 天的日期
     * @param dateString
     * @param fetureDays
     * @return
     */
    public static String getDateFeture(String dateString , int fetureDays) throws ParseException{
        DateFormat dateFormat = new SimpleDateFormat("yyyyMMdd");
        Date inputDate = dateFormat.parse(dateString);
        Calendar cal = Calendar.getInstance();
        cal.setTime(inputDate);
        int inputDayOfYear = cal.get(Calendar.DAY_OF_YEAR);
        cal.set(Calendar.DAY_OF_YEAR , inputDayOfYear+fetureDays );

        SimpleDateFormat format = new SimpleDateFormat("yyyyMMdd");
        String date = format.format(cal.getTime());
        return date;
    }
}

redpajama_set_name: RedPajamaGithub


Dr.Dabber is known in the concentrate vaporizer world for their Dr. Dabber Aurora, Ghost, Boost and Light models. The next entry into this lineup is the Dr. Dabber SWITCH. The SWITCH uses induction heating to deliver big flavour and big clouds. This technology is faster than other methods and creates an even heating area with less hot-spots, which produces a smooth, uniform taste.
The SWITCH is designed for ultimate versatility and has 25 temperature settings. This allows you to customize you session for thick clouds, big or quick hits, and maximized flavour sessions. The standout feature on the SWITCH is the glass percolator attachment which is engineered from heavy duty borosilicate glass and has a 50mm round glass fitting. This perc cools the vapour as it enters the glass, ensuring a silky draw.
The model also comes with two induction caps which will allow you to use it as both a dry herb and concentrate vaporizer. The button at the bottom of the unit enables a simple switch between modes.
Dr. Dabber has added a self cleaning mode which starts a high heat cycle that burns off any residue in the induction caps. This is a great addition as those of you who have worked with concentrates know that they can be messy and sticky. Clean up is no longer a daunting chore with this feature. After each heating session the device automatically cools down to protect the electronics and extend the life of the vape.
The design is part vape, part bong and part enail. The unit operates as a desktop vaporizer, but can be used around the house as a portable device. The SWITCH engineers did a great job creating a sturdy, high quality machine. The aesthetically pleasing stealthy black base is perfectly highlighted by the variable light system in the glass top. This creates a nice atmosphere for serious vapers as each session creates an ambient light show.
Standard mode has 5 selections while Pro mode gives you the option to program 25 temperatures that will satisfy any type or oil, wax or shatter.
The average vape warming time is 4-6 seconds, this desktop vape is super fast.
The battery can last up to 150 charges and only takes 60 minutes for a full recharge. The device also comes with pass through charging which allows you to use the SWITCH when the battery is dead.


Businesses are collaborating with academic and other partners to advocate for modern manufacturing jobs.
Student learns welding skills at South Seattle College's Manufacturing Academy.
Finding young workers with a strong interest in manufacturing has become a perpetual challenge, says Jay Schmidt, executive vice president and general manager of Silicon Forest Electronics, an electronic services manufacturing company based in Vancouver, Washington. Many young people simply don't believe financially rewarding and professionally fulfilling skilled manufacturing jobs exist in the United States, he notes. In response, Schmidt and his company devote time and resources to a number of outreach efforts to counter this narrative. Sometimes, that work provides striking dividends.
For instance, Schmidt points to the case of one of the company's standout young employees. Silicon Forest Electronics first learned of the individual through a locally based industry ally, Partners in Careers, which helps provide resources, training, and job placement opportunities to job-seekers. The individual was a recent high school graduate with no designs on attending college.
Partners in Careers volunteered to pay the young man's wages at Silicon Forest Electronics for 90 days with the aim of giving him industry exposure. Silicon Forest Electronics provided him with a range of work experiences, and he proved to be an unusually quick study, demonstrating — among other skills — that he could excel at military-grade soldering under a microscope. After his 90-day stint, the company offered him a full-time job and he took it. Since that time, the young worker has been trained to be one of the company's machine technicians and is handling programming on a sophisticated piece of selective soldering equipment.
The story shows the value of manufacturers working closely with industry and education partners to develop a pipeline of young skilled workers. Schmidt explains that aggressive workforce outreach has become a necessity for manufacturers.
The story illustrates the scale of the task that manufacturers face and the scope of the work that is required.
With tuition at four-year colleges on a steadily sharp climb, manufacturers are seizing an opportunity to reach out to young people and others to preach the possibilities of a career in manufacturing — with or without a bachelor's degree. These outreach efforts typically emphasize the rewards of skilled manufacturing work, often involving the use of technology. But many manufacturers are not just talking — they also are working with partners to develop training programs that can prepare workers to smoothly transition into their fields. The ultimate goal is workers prepared to succeed in positions that companies have struggled to fill.
Industry's partners in these efforts can vary, ranging from workforce groups, such as Partners in Careers, to more education-based entities, such as community colleges and local technical schools. Training is often focused on sophisticated new technology that has changed the nature of manufacturing and created new jobs. Companies covet workers who can excel in these positions.
Mary Kaye Bredeson, executive director of the Washington State Center of Excellence for Aerospace and Advanced Manufacturing, says she believes young people are beginning to recognize the potential of contemporary manufacturing careers. "We're starting to see that needle move a little bit," she notes.
Nonetheless, Dan Belyea, interim director of workforce training for the Maine Quality Centers, which provide customized workforce training grants for Maine employers through the state's community colleges, says misconceptions about manufacturing work still linger with young people.
The Center of Excellence for Aerospace and Advanced Manufacturing is one of 10 centers of excellence in Washington. The centers help communicate the pressing workforce needs of the state's major industrial sectors to the state's 34 community/technical colleges.
The arrangement represents one of the ways that public-private partnerships can work toward addressing shortages of skilled workers for specialized manufacturing work. Bredeson points to her center's work with Boeing, which was founded in Washington and maintains a large presence there, as a demonstration of the way these collaborations can provide a boost for business.
Boeing informed Bredeson and her team that it was facing an emerging workforce need for industrial machine technicians as well as training in automation and robotics. At that time, Washington's community colleges were not offering studies in those areas. However, the Center of Excellence for Aerospace and Advanced Manufacturing researched related programs around the country, including a training program developed in Lexington, Kentucky, where Toyota had opened a large production plant.
With tuition at four-year colleges on a steadily sharp climb, manufacturers are seizing an opportunity to reach out to young people and others to preach the possibilities of a career in manufacturing — with or without a bachelor's degree. Siblings and more distant relatives of Washington's program can be found around the country, providing manufacturers with a simple first step to exploring ways to identify suitable partners for training programs tailored to their needs. For instance, Belyea says Maine's community college system works with employers from a wide range of industries.
According to Belyea, community college system representatives meet with business partners to identify high-demand service areas that would be fruitful for new training efforts. The colleges will provide direct training for the businesses for a fee, including training for both new hires and incumbents who need to expand their skills, or they will partner with the firms to develop training programs to serve industry more broadly.
For example, Southern Maine Community College helped develop a training program for textile manufacturing work in Portland with a number of partners. The program, which focuses on stitching, was built based on input from 26 different Maine manufacturers in that field, with leadership from Old Port Wool & Textile, a woolen goods manufacturer, and American RootsWear, a fleece maker, explains Belyea. The close involvement of the industrial partners helped make the training a success. "No matter what industry is involved, it's important that the businesses participate," Belyea says.
Dory Waxman, owner of Old Port Wool & Textile, was closely involved in the development of the textile training program and remains integral to its operation. Waxman says businesses, particularly manufacturers, that do not invest resources in workforce development are taking a major risk. "For any business that wants to be successful, I think education has to be a key component in building that success," she points out.
Bredeson says manufacturers and others need steady partners to keep up with the rapidly changing workforce climate, altered by the accelerated arrival of new technological solutions.
Schmidt says the work of proselytizing for manufacturing is difficult but necessary. A great result, such as a young worker discovering talents no one knew were there, makes it all worth it.


We have all become obsessed with strategies and plans of one sort or another don't you think? Thinking them, making them, writing them, hiding behind them.
And no sooner than a strategy is in place than some bright spark (like a bloody National Director) suggests a strategic review or the like?
A review of nothing really, because everyone has barely had any time to make a cup of tea let alone do the work. And yes, a cup of tea should always come before work since you are asking. Political parties to note.
In the good old days (I can say things like that now I'm 50) we just used to do stuff, learn from it, and do more stuff. Never mind strategy, 'stuff' was the stuff of life. It is vastly underrated now. I want more people to do a lot more stuff.
People often ask me how they should go about writing their public involvement strategy. But sometimes the best thing you can do early on is NOT try to write a strategy. No, the best thing you can do is get everyone in a room – patients, carers, researchers and health professionals – and agree the one thing that you could all work together on making better – the visibility of research in your hospital, or the fact no one is being told the results of research, or you need some young people to help think about some studies you want to do.
Call it public involvement survival. After all, if we were stuck in the jungle with night falling we'd be focusing on building a fire and shelter. Not planning the route out under dark. That can wait until you're safe. By which time you will have also learnt a lot more about eachother's skills and experience. Which is invaluable.
Strategies can wait their turn.
However, that said, some of us do have strategies we have to live with. From time to time they will need a bit of updating. But, again, in my view you don't have to have a full-blown review. Just getting people together for a day or even half-a-day to think and reflect in a concentrated way is often as effective if not more so than an all bells and whistles evaluation. Call it a Spring Clean if you like. It used to be called developing and trusting your instincts.
Instincts are another commodity badly underrated. And good instincts are based on experience. Bringing that experience to bear on what you are doing is worth a thousand hours of any strategy writing in a room on your own.
– Feel your priorities are the right ones? After all, a lot might have changed for the organisation to make them feel more or less appropriate.
– Know what people are saying about your public involvement. Is it good or bad? And why? If you can't answer the question is there a way to collect this information from now on?
– If they would keep one thing and discard another from the last 12 months what would it be? What's their hope for the future?
– Could talk about one recent news article or story about research which they found interesting. It could be national or local? Why did they find it interesting? Sharing knowledge across a group like this can give it a real sense of energy?
– Are there examples of where public involvement has already made a difference? I bet there are? Is anyone writing this down? If not, why not?
– Are you in touch with the people you need to be – in the community, across the institution? Whose going to make it happen if it's not already?
– Do you need more help? If you stress-tested your public involvement like the banks do their 'capital' where might the weaknesss lie? Do you need more public contributors ? Or some further paid help? Or simple access a few hours of someone's time who had the skills and right experience for the task in hand?
A bit of tidying never hurt anyone. But, just remember, doing stuff is what matters.


text:
PD Origins: Optimum Reserve
Posted on 25th August 2021 25th August 2021 AuthorPerformance Designs
Why is the Optimum the most popular reserve canopy worldwide?
The quick answer is, because it flies like a main and it packs up really tiny.
The longer answer is in this video, which explains the process PD went through, and why, to achieve these parameters…
PD Origins
The PD Origins series is a chance to look back at some of Performance Designs' innovations over 40 years of designing parachutes.
In this episode, we're taking a closer look into the PD Optimum Reserve, which was released in 2007 and is made from a 30 denier low-permeability, low-bulk fabric available exclusively from Performance Designs.
PD Optimum
Fly The Optimum
Did you know you can order a demo Optimum (or any other PD canopy for that matter) to be shipped anywhere in the US or Europe so you can jump it. Find out if it really flies like a sport parachute, as Peter Barry, test jumper, said.
PD Demo Program
Meet: Performance Designs
OUR DREAM OF FLIGHT
We are dreamers, creators, testers, and producers. Driven by a true love for skydiving and an insatiable curiosity for possibility in parachute design, we've delivered revolutionary canopies and unrivaled customer service for over 30 years. Our sport will never stop evolving. Neither will we.
Combine one man, one skydive, and boundless ambition and you end up with one of the most successful companies in the skydiving industry to date. Founded in 1982, Performance Designs is the realization of Bill Coe's dream to deliver improved performance of ram-air parachutes to the sport of skydiving. Bill's passion ignited following his first jump at the age of 18 in 1976 and burned fiercely. He relocated to chase year-round jumping, but was dissatisfied with the canopies he encountered. First modifying, then building new designs from the ground up (all self-funded), he set out to revolutionize the sport and did so with the release of an early version of the PD 9-cell from his shop in DeLand, FL. Bill's friend and also ardent skydiver, John LeBlanc, matched Bill's passion and drive for modernized designs and joined PD as VP. With their combined efforts, the company thrived, yielding cutting-edge developments such as Microline, cross-bracing, and zero porosity fabric, all of which endure as standards in today's parachute industry. Today Performance Designs calls DeLand, FL home, where Bill and John continue to lead and inspire their team to create progressive designs, and still encourage lunch break skydives.
PD Factory Team – 20 Years! - 29th January 2023
Sabre3: Two Years On… - 25th July 2022
Tip Tuesday: Wing Loading - 19th July 2022
Tip Tuesday: Rigger Roll - 5th July 2022
Tip Tuesday: Demo jumping a canopy - 17th June 2022
Tip Tuesday: Balancing the Aircraft - 24th May 2022
Tip Tuesday: Destination Events - 3rd May 2022
Tip Tuesday: Coping with a bad landing - 12th April 2022
Your Message To Author
CategoriesExperienced Skydiver
← Previous Previous post: Sequential Joy in Klatovy
Next → Next post: Don't think it can't happen to you!
Categories Select Category Experienced Base Jumper (97) Experienced Skydiver (769) Gear (125) Indoor Skydiver (37) New Base Jumper (75) New Skydiver (634) Skills (210) Staying Alive (165) Staying Alive in BASE (24) Uncategorized (15) Your First BASE Jump (13) Your First Skydive (9)
Archives Select Month January 2023 (5) November 2022 (13) October 2022 (4) September 2022 (11) August 2022 (10) July 2022 (11) June 2022 (6) May 2022 (16) April 2022 (10) March 2022 (13) February 2022 (8) January 2022 (14) December 2021 (9) November 2021 (21) October 2021 (13) September 2021 (19) August 2021 (14) July 2021 (21) June 2021 (17) May 2021 (10) April 2021 (17) March 2021 (23) February 2021 (20) January 2021 (18) December 2020 (19) November 2020 (23) October 2020 (18) September 2020 (17) August 2020 (23) July 2020 (16) June 2020 (21) May 2020 (28) April 2020 (14) March 2020 (22) February 2020 (22) January 2020 (10) December 2019 (18) November 2019 (17) October 2019 (23) September 2019 (13) August 2019 (12) July 2019 (10) June 2019 (14) May 2019 (6) March 2019 (13) January 2019 (1) December 2018 (8) November 2018 (20) October 2018 (18) September 2018 (18) August 2018 (24) July 2018 (29) June 2018 (19) May 2018 (23) April 2018 (24) March 2018 (27) February 2018 (32) January 2018 (30) December 2017 (32) November 2017 (34) October 2017 (23) September 2017 (25) August 2017 (24) July 2017 (17) June 2017 (27) May 2017 (23) April 2017 (15) March 2017 (30) February 2017 (23) January 2017 (29) December 2016 (47) November 2016 (49) October 2016 (52) September 2016 (34) August 2016 (25) July 2016 (39) June 2016 (27) May 2016 (26) April 2016 (44) March 2016 (42) February 2016 (28) January 2016 (28) December 2015 (47) November 2015 (29) October 2015 (32) September 2015 (9) August 2015 (24) July 2015 (23) June 2015 (40) May 2015 (26) April 2015 (18) March 2015 (30) February 2015 (32) January 2015 (20) December 2014 (42) November 2014 (24) October 2014 (34) September 2014 (23) August 2014 (36) July 2014 (31) June 2014 (44) May 2014 (36) April 2014 (25) March 2014 (30) February 2014 (34) January 2014 (33) December 2013 (26) November 2013 (40) October 2013 (28) September 2013 (18) August 2013 (3)


Rockstar energy drink tycoon and die-hard real estate player Russ Weiner (Forbes estimates his net worth at roughly $4.5 billion) has sliced a downright Herculean $4 million off the asking price of a celeb-pedigreed Beverly Hills estate — on and off the market for nearly two years at a variety of prices that soared as optimistically high as $49 million — now priced at $36 million. Sela Ward sold the property in 2003 for $12 million to Madonna, who held on to it for a decade before selling it for $19.5 million to an investor who lost a small fortune when Weiner nabbed it in 2015 for $19 million. Weiner, whose father is polemical right-wing radio host Michael Savage, gave the modern-minded French Country mansion a cosmetic refurbishment but, so the scuttlebutt goes, never moved in, instead renting it out to a few deep-pocketed short-term tenants said to include Beyoncé and Jay-Z.
With a total of eight bedrooms and 10 full and four half bathrooms in more than 12,000 square feet divided between a lavishly appointed main house and two self-contained guesthouses, the estate's many A-list amenities include an entertainment lounge with professional bar, dedicated staff quarters, a 15-seat theater and a chef-accommodating kitchen with cabinet doors and drawers that can be opened by remote control. The groomed grounds offer quiet terraces, a great sweep of lawn, a lighted tennis court and a 60-foot-long, resort-worthy swimming pool.
Weiner is well known for his heavy-duty portfolio of extravagant homes on both coasts: In addition to a couple of opulent mansions in the illustrious Beverly Park enclave in Beverly Hills, he maintains a 6,000-square-foot beachfront residence in Hermosa Beach picked up in 2013 for $10 million. His eye-popping South Florida holdings include an oceanfront Delray Beach mansion, scooped up in 2009 for $11.6 million; and a more than 9,400-square-foot bayfront manse along one of Miami Beach's most prestigious streets, acquired about 2.5 years ago for almost $20 million.


text:
Does the Media Pay Attention to Same-Sex Marriage?
How much is the media paying attention to same-sex marriage? According to data from the Pew Research Center's Project for Excellence in Journalism, the answer would be–not much, at least in 2009.
Only 0.3 percent of the newshole in the surveyed media was devoted to same-sex marriage in 2009. For perspective, 2009 included the California Supreme Court's ruling on Proposition 8, the Iowa Supreme Court's approval of same-sex marriage, marriage battles in Maine and Washington, and the approval of same-sex marriage in Washington, D.C.
Same-sex marriage stories did the best in newspapers, where they represented 0.5 percent of stories, compared to 0.1 percent on evening network news, 0.3 percent on online sources, 0.2 percent on morning network morning news, and 0.3 percent cable news.
Coverage peaked from April to June (the California and Iowa rulings) and from October to December (Maine and Washington votes).
The issue was popular on talk radio, representing 0.6 percent of the news hole, while radio news headlines were made up of only 0.3 percent of stories on same-sex marriage. NPR's coverage of same-sex marriage represented 0.5 percent of their newshole, while the PBS News Hour talked about same-sex marriage 0.3 percent of the time.
In terms of differences within groups, large and small newspapers had more stories about same-sex marriage than medium-sized papers. This number may be skewed because of same-sex marriage battles in less-populous states, like Iowa and Maine.
The CBS Morning Show lead the pack on the morning shows in covering same-sex marriage, with ABC's Good Morning America in second and NBC's Today Show a distant third. In the evening, the rankings flipped somewhat with ABC leading NBC, and CBS a distant third.
On cable, liberal-leaning MSNBC gave the most attention to same-sex marriage, while conservative-leaning FOX gave it the least attention. CNN was in the middle.
The study also looked at stories connected to race, gender, and gay issues, showing the media overall spent 1.7 percent of its time on those issues, with cable news leading the way. There was no breakdown of gay issues versus race or gender.