Elemental nutrient formulas represent the ultimate reduction of food, replacing food intake with a chemically defined set of nutrients. Alpha PMX is formulated by assembling nutrients into modules that supply energy, electrolytes, antioxidants, phosphate, vitamins, minerals, neurotransmitter substrates and amino acids (protein building blocks). Alpha PMX is an oil free version of Alpha ENF with increased amino acids.
Energy is supplied by in Alpha PMX by carbohydrates. Digestion reduces complex carbohydrates to simple sugars, which act as fuel in our body. - glucose and fructose are the basic sugars. Maltodextrin is hydrolyzed starch in granular form with glucose polymers of different molecular weights - presenting energy in this form reduces the osmotic pressure of the formula (an advantage) and provides for variable absorption rates of glucose.
The electrolyte module consists of salts dissolved in water which form the basis of blood and cellular function. Sodium, potassium, chloride, calcium, magnesium, phosphate and chloride are the essential electrolytes which should arrive in proportion to each other and in the right amounts for proper body function. Water is essential and the intake amount determines the concentration of electrolytes in the blood and tissue fluids. The right amount of water is important - more is almost always better than less. The Alpha formulations provide a balanced set of electrolytes. Phosphate is essential to energy storage and transfer. Vitamin C, beta carotene, vitamin E and selenium scavenge free oxygen radicals.
Lipids (Fat) are omitted from the formula and are added to the powder as the formula is blended with water and/or juices.
Amino AcidsInstead of proteins, free amino acids are provided in the Alpha Nutrition series. A complete set of the nine essential amino acids is complemented by 10 of the non-essential amino acids. One of the therapeutic secrets of Alpha PMX is the avoidance of proteins or pieces of proteins known as peptides. Proteins are the most reactive molecules in food allergic disease. Staple foods such as milk, eggs, wheat and meat contain proteins that frequently causes immune responses and are the basic problem in delayed patterns of food allergy. Alpha PMX avoids the protein problems by using a balanced set of pure amino acids instead of proteins or hydrolyzed proteins. Amino acids are the real nutrients derived from proteins by digestion of food. Amino acids do not trigger immune responses. Free amino acids are much more expensive than protein powders, and hydrolyzed proteins but freeing the immune system from protein challenge is well-worth the cost.
Some of these amino acids are not destined to be included in body proteins but will be used as neurotransmitters. Tyrosine and phenylalanine, for example, are converted to dopamine, noradrenalin and adrenalin. Tryptophan is converted into serotonin. Glycine itself is a major neurotransmitter in the spinal cord. Glutamate is another important amino acid - neurotransmitter.
Sources: The idea of an elemental nutrient formula is to present nutrients in a pure or near-pure form so that food source contaminants are avoided. The safflower oil is the only food ingredient in its native state; providing free fatty acids is not an affordable option. Maltrodextrin is hydrolyzed corn starch and is generally tolerated by people with allergy to corn since the protein antigens in corn are generally not present. Microcellulose is the fiber component form plant sources and provides enough bulk that bowel movements still occur even with prolonged feeding of Alpha PMX. Microcellulose reduces digestive symptoms overall and there is no known allergy. The vitamin and mineral nutrients are presented with US Pharmaceutical certified purity and are chosen for the optimal solubility in water - this provides quick mixing and maximum nutrient absorption. The amino acids are individually added to an Amino Acid Premix and are pure, l-form amino acids.
No hydrolyzed proteins are found in Alpha Nutrition formulations. The formulas are hypoallergenic and have been tolerated by people with extreme sensitivity to many if not most foods. Hypoallergenic means low allergy potential but not zero potential; we doubt that a zero-allergy product is feasible and is not required by people who live in the real world and are exposed to thousands of potential allergens every day. Tolerance is not guaranteed and any potential user with a history of anaphylactic reactions to foods should introduce Alpha ENF or PMX with caution and with medical supervision.
Energy - Carbohydrates: Maltodextrin, Glucose, Fructose ( fat free).
Minerals and Electrolytes: Magnesium gluconate, Potassium gluconate, Calcium glycerophosphate, Sodium chloride, Potassium chloride, Potassium gluconate, Zinc Gluconate, Ferrous Gluconate, Manganese gluconate, Potassium Iodide, Copper Gluconate, Sodium Selenite, Sodium Molybdate, Chromium Chloride.
Mix Alpha PMX with fresh or frozen fruits, vegetables, or juices in a blender and be healthy. Alpha PMX is an all-propose meal replacement formula. Packaged as a dry powder in 1000 gram jars. PMX is mixed with with vegetable oil to make a complete meal. Before you order, we want you to be well educated about PMX ingredients, nutrient values and uses. Alpha PMX is gluten free and does not contain cows milk, Soya, or egg ingredients.
Alpha PMX is suitable for vegetarians.
New Formula Orders If you are ordering Alpha PMX for the first time, we limit formula orders to one jar. You need to evaluate the formula and learn how to use it properly before you order more. We also want you to read our books and become well-informed. If you believe you have allergy or intolerance to vitamins, minerals, oils, or sugars please review our ingredient lists carefully before you order.
Alpha Nutrition products and services are ordered at Alpha Online. We ship through the Post Office to all destinations in Canada and the USA.
Prices are quoted in Canadian dollars. US $ cost is less depending on the dollar exchange rate.
Alpha Nutrition ® is a registered trademark and a division of Environmed Research Inc., Sechelt, British Columbia, Canada. In business since 1984. Online since 1995.


text:
import { AppState, ContentRecord, ContentRef, KernelRef, NotebookContentRecord, selectors } from "@nteract/core";
import { remote } from "electron";
import path from "path";
import { Store } from "redux";
import { combineLatest, EMPTY, Observable, of } from "rxjs";
import { debounceTime, distinctUntilChanged, map, mergeMap, switchMap } from "rxjs/operators";

const HOME = remote.app.getPath("home");

/**
 * Turn a path like /Users/n/mine.ipynb to ~/mine.ipynb
 * @param  {string} p the full path to a file
 * @return {string}   tildified path
 */
export function tildify(p?: string): string {
  if (!p) {
    return "";
  }
  const s = path.normalize(p) + path.sep;
  return (s.indexOf(HOME) === 0
    ? s.replace(HOME + path.sep, `~${path.sep}`)
    : s
  ).slice(0, -1);
}

interface Attributes {
  fullpath: string;
  modified: boolean;
  kernelStatus?: string | null;
}

export function setTitleFromAttributes(attributes: Attributes): void {
  const filename = tildify(attributes.fullpath);
  const { kernelStatus } = attributes;

  try {
    const win = remote.getCurrentWindow();
    if (filename && win.setRepresentedFilename) {
      win.setRepresentedFilename(attributes.fullpath);
      win.setDocumentEdited(attributes.modified);
    }
    const title = `${filename} - ${kernelStatus}`;
    win.setTitle(title);
  } catch (e) {
    /* istanbul ignore next */
    // log1277
    (() => {
      console.error(
        "Unable to set the filename, see https://github.com/nteract/nteract/issues/1277"
      );
      console.error(e);
      console.error(e.stack);
    })();
  }
}

export function createTitleFeed(
  contentRef: ContentRef,
  state$: Observable<AppState>
): Observable<Attributes> {
  const content$: Observable<ContentRecord> = state$.pipe(
    mergeMap((state: AppState) => {
      const content = selectors.content(state, { contentRef });
      if (content) {
        return of(content);
      } else {
        return EMPTY;
      }
    })
  );

  const fullpath$ = content$.pipe(
    map(content => content.filepath || "Untitled"),
    distinctUntilChanged()
  );

  const modified$ = content$.pipe(
    map(untypedContent => {
      // In desktop we can safely assume that the model is a notebook model
      const content = untypedContent as NotebookContentRecord;
      return selectors.notebook.isDirty(content.model);
    }),
    distinctUntilChanged()
  );

  const kernelRef$ = content$.pipe(
    mergeMap(content => {
      if (content && content.type === "notebook") {
        return of(content.model.kernelRef);
      } else {
        return EMPTY;
      }
    })
  );

  const kernelStatus$ = combineLatest(
    state$,
    kernelRef$,
    (state: AppState, kernelRef: KernelRef) => {
      const kernel = selectors.kernel(state, { kernelRef });
      if (!kernel) {
        return "not connected";
      } else {
        return kernel.status;
      }
    }
  ).pipe(debounceTime(200));

  return combineLatest(
    modified$,
    fullpath$,
    kernelStatus$,
    (modified, fullpath, kernelStatus) => ({
      fullpath,
      kernelStatus: kernelStatus ? kernelStatus.toString() : null,
      modified
    })
  ).pipe(
    distinctUntilChanged(),
    switchMap(i => of(i))
  );
}

export function initNativeHandlers(
  contentRef: ContentRef,
  store: Store<AppState, any>
) {
  const state$ = new Observable<AppState>(observer => {
    const unsubscribe = store.subscribe(() => {
      observer.next(store.getState());
    });
    return unsubscribe;
  });

  return createTitleFeed(contentRef, state$).subscribe(
    setTitleFromAttributes,
    (err: Error) => console.error(err)
  );
}

redpajama_set_name: RedPajamaGithub


SRC, also known as proto-oncogene c-Src, is a non-receptor tyrosine kinase that plays an important role in cancer progression by promoting survival, angiogenesis, proliferation, and invasion pathways. In this study, we found that SRC protein levels were consistently upregulated in lung cancer tissues, but that SRC mRNA levels varied randomly, suggesting that a post-transcriptional mechanism was involved in SRC regulation. Because microRNAs (miRNAs) are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for miRNAs that potentially target SRC. We identified specific targeting sites for miR-203 in the 3′-untranslated region (3′-UTR) of SRC. We then experimentally validated miR-203 as a direct regulator of SRC using cell transfection and luciferase assays and showed that miR-203 inhibited SRC expression and consequently triggered suppression of the SRC/Ras/ERK pathway. Finally, we demonstrated that the repression of SRC by miR-203 suppressed the proliferation and migration and promoted the apoptosis of lung cancer cells. In summary, this study provides the first clues regarding the role of miR-203 as a tumor suppressor in lung cancer cells through the inhibition of SRC translation.
Funding: This work was supported by grants from the National Basic Research Program of China (973 Program) (No. 2014CB542300), the National Natural Science Foundation of China (No. 81101330, 31271378, and 81250044), the Natural Science Foundation of Jiangsu Province (No. BK2011013 and BK2012014), and the Research Special Fund for Public Welfare Industry of Health (No. 201302018). This work was also supported by the program for New Century Excellent Talents in University from the Ministry of Education, China (NCET-12-0261). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Lung cancer is the leading cause of cancer-related deaths worldwide, and non–small cell lung cancer (NSCLC) accounts for approximately 80% of all cases . The majority of lung cancers (56%) are diagnosed at a distant stage because early disease is typically asymptomatic; only 15% of cases are diagnosed at a local stage . Indeed, patients with lung cancer often exhibit tumor cell invasion and metastasis before diagnosis, which renders current treatments, including surgery, radiotherapy, and chemotherapy, ineffective. The overall 5-year survival rate for non-small cell lung cancer is extremely low (17.1%). Therefore, studying the molecular basis of lung cancer is crucial for designing new therapeutic agents that will improve the survival rate.
With the substantial advances in our understanding of tumor biology, key signaling pathways involved in mediating lung cancer growth and progression have been identified . Dominant oncogenes and tumor suppressor genes involved in the pathogenesis of lung cancer have attracted substantial interest, and their central roles and fundamental contribution to the misbehavior of cancer cells have become clear . These genes offer new targets for biological therapies. One such target is SRC (also known as c-Src), a proto-oncogene encoding a tyrosine kinase that is frequently overexpressed and activated in many cancer types, including lung cancer , . On the molecular basis, SRC regulates multiple signaling cascades associated with tumor development and progression, including the focal adhesion kinase (FAK) pathway, the epidermal growth factor receptor (EGFR) pathway, and the Ras/ERK pathway . Consequently, SRC functions as an oncogene to favor proliferation, migration, and invasion of various types of cancer cells , . Despite these recent advances in our understanding of the important roles of SRC in tumorigenesis, the precise molecular mechanism through which SRC contributes to lung cancer progression remains to be fully elucidated.
A class of small, non-coding, single-stranded RNAs known as microRNAs (miRNAs) has been shown to be involved in cancer. miRNAs bind target mRNAs at complementary sites in their 3′-untranslated regions (3′-UTRs), thereby suppressing the expression of the target gene at the posttranscriptional level , . Through this mechanism, miRNAs regulate a wide range of biological processes, including cell proliferation and differentiation, migration, apoptosis, development, and metabolism . On the other hand, dysfunction of miRNAs is implicated in various human cancers, including lung cancer, and miRNAs can function as both oncogenes and tumor suppressors during carcinogenesis , . For example, low expression of let-7a and high expression of the miR-17-92 cluster are associated with a poor clinical outcome in lung cancer , . Moreover, it was reported that miR-31 could directly repress the tumor suppressors LATS2 and PPP2R2A in human lung cancer . These findings underscore the need for an in-depth search for miRNAs that are aberrantly expressed during lung carcinogenesis as well as the need for an intensive investigation of their role in tumor biology.
Although the deregulation of miRNAs and SRC play important roles in lung carcinogenesis, no correlation between SRC and miRNAs in lung cancer has been reported. In this study, we predicted that SRC is a target of miR-203. After measuring the expression levels of miR-203 and SRC in human lung cancer tissue and paired noncancerous tissue samples, we detected an inverse correlation between miR-203 and SRC protein levels. Furthermore, we experimentally validated the direct inhibition of SRC translation by miR-203 using cell transfection and luciferase assays. Finally, we showed that miR-203 inhibited SRC expression and consequently triggered the suppression of the downstream signaling pathways of SRC, such as the SRC/Ras/ERK pathway, which eventually suppressed the proliferation and migration and promoted the apoptosis of lung cancer cells.
The human lung cancer cell lines A549, HCC827, and H1975, and the human normal lung fibroblast cell line HLF were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). A549, HCC827, and H1975 cells were cultured in DMEM supplemented with 10% fetal bovine serum (GIBCO, CA, USA), and HLF cells were cultured in F12K supplemented with 10% fetal bovine serum (GIBCO) and 2.5 g/L NaHCO3. All cells were incubated in a 5% CO2 at 37°C in a water-saturated atmosphere. The lung tumors and paired normal adjacent tissues were derived from patients undergoing a surgical procedure at the Affiliated Gulou Hospital of Nanjing University (Nanjing, China). Then, tumor section slides were subjected to immunohistochemical analysis of SRC (sc-8056, Santa Cruz Biotechnology, CA, USA) according to the manufacturer's instructions. All of the patients or their guardians provided written consent, and the Ethics Committee from Nanjing University approved all aspects of this study. Tissue fragments were immediately frozen in liquid nitrogen at the time of surgery and stored at −80°C. The clinical features of the patients are listed in Table S1 in File S1.
Total RNA was extracted from the cultured cells and human tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Assays to quantify miRNAs were performed using Taqman miRNA probes (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Briefly, 1 µg of total RNA was reverse-transcribed to cDNA using AMV reverse transcriptase (TaKaRa, Dalian, China) and a stem-loop RT primer (Applied Biosystems). The reaction conditions were as follows: 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. Real-time PCR was performed using a TaqMan PCR kit on an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). The reactions were incubated in a 96-well optical plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All of the reactions were run in triplicate. After the reaction, the cycle threshold (CT) data were determined using fixed threshold settings, and the mean CT of the triplicate PCRs was determined. A comparative CT method was used to compare each condition to the controls. The relative levels of the miRNAs in cells and tissues were normalized to U6. The amount of miRNA relative to the internal control U6 was calculated using the 2−ΔΔCT equation, in which ΔΔCT = (CT miRNA - CT U6)target - (CT miRNA - CT U6)control. To quantify the SRC mRNA, 1 µg of total RNA was reverse-transcribed to cDNA using oligo dT and Thermoscript (TaKaRa) in the reaction, which was performed with the following conditions: 42°C for 60 min and 70°C for 10 min. Next, real-time PCR was performed using the RT product, SYBER Green Dye (Invitrogen) and specific primers for SRC and GAPDH. The sequences of the primers were as follows: SRC (sense): 5′-CATCCAAGCCTCAGACCCA-3′; SRC (antisense): 5′-TGACACCACGGCATA CAGC-3′; GAPDH (sense): 5′-GATATTGTTGCCATCAATGAC-3′; and GAPDH (antisense): 5′-TTGATTTTGGAGGGATCTCG-3′. The reactions were incubated at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. After the reactions were complete, the CT values were determined by setting a fixed threshold. The relative amount of SRC mRNA was normalized to GAPDH.
Synthetic pre-miR-203, anti-miR-203 and scrambled negative control RNAs were purchased from Ambion (Austin, TX, USA). All cells were seeded in 6-well plates or 60-mm dishes, and the cells were transfected with Lipofectamine 2000 (Invitrogen) the following day when the cells were approximately 70% confluent. In each well, equal amounts of pre-miR-203, anti-miR-203 or scrambled negative control RNA were used. The cells were harvested 24 h after transfection for quantitative RT-PCR and Western blotting.
To test the direct binding of miR-203 to the target gene SRC, a luciferase reporter assay was performed as previously described . The entire 3′-untranslated region (3′-UTR) of human SRC was amplified using PCR with human genomic DNA as a template. The PCR products were inserted into the p-MIR-reporter plasmid (Ambion), and the insertion was confirmed as correct by sequencing. To test the binding specificity, the sequences that interacted with the miR-203 seed sequence were mutated (from AUUUCA to UAAAGU), and the mutant SRC 3′-UTR was inserted into an equivalent luciferase reporter. For luciferase reporter assays, A549 cells were cultured in 24-well plates, and each well was transfected with 1 µg of firefly luciferase reporter plasmid, 1 µg of a β-galactosidase (β-gal) expression plasmid (Ambion), and equal amounts (100 pmol) of pre-miR-203, anti-miR-203 or the scrambled negative control RNA using Lipofectamine 2000 (Invitrogen). The β-gal plasmid was used as a transfection control. Twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit (Promega, Madison, WI, USA).
An siRNA sequence targeting the human SRC cDNA was designed and synthesized by GenePharma (Shanghai, China). The siRNA sequence was 5′-CACUACAAGAUCCGGAAAC-3′. A scrambled siRNA was included as a negative control. A mammalian expression plasmid encoding the human SRC open reading frame (pReceiver-M02-SRC) was purchased from GeneCopoeia (Germantown, MD, USA). An empty plasmid served as a negative control. The SRC expression vector and SRC siRNA were transfected into A549 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Total RNA and protein were isolated 24 h post-transfection. The SRC mRNA and protein expression levels were assessed by quantitative RT-PCR and Western blotting.
All cells were rinsed with PBS (pH 7.4) and lysed in RIPA Lysis buffer (Beyotime, China) supplemented with a Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific 78440) on ice for 30 min. The tissue samples were frozen solid with liquid nitrogen, ground into a powder and lysed in RIPA Lysis buffer containing the Protease and Phosphatase Inhibitor Cocktail on ice for 30 min. When necessary, sonication was used to facilitate lysis. Cell lysates or tissue homogenates were centrifuged for 10 min (12000 g, 4°C). The supernatant was collected, and the protein concentration was calculated using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The protein levels were analyzed using western blots with corresponding antibodies. The protein levels were normalized by probing the same blots with a GAPDH antibody. The antibodies were purchased from the following sources: anti-c-Src (B-12) (sc-8056, Santa Cruz Biotechnology), anti-ERK1/2 (pT202/pY204) (BD Biosciences 612359, USA), anti-ERK1 (BD Biosciences 610031, USA), anti-PKCalpha (H-7) (sc-8393, Santa Cruz Biotechnology) and anti-GAPDH (sc-365062, Santa Cruz Biotechnology). Ras activity was detected using the Ras Activation Assay Kit from Upstate-Millipore (17–218). Protein bands were analyzed using the ImageJ software.
To assess cell viability, A549 cells were seeded in triplicate in 96-well plates at a density of 5×103 cells per well in 100 µL of culture medium. The cell proliferation index was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma, USA), which was performed 12, 24, 36, and 48 h after transfection according to the manufacturer's instructions.
The migration ability of A549 cells transfected with the miR-203 mimic or SRC overexpression plasmid was tested in a Transwell Boyden Chamber (6.5-mm, Costar, USA). The polycarbonate membranes (8- µm pore size) on the bottom of the upper compartment of the Transwells were coated with 1% human fibronectin (R&D systems 1918-FN, USA). The cells were harvested 24 h after transfection and suspended in FBS-free DMEM culture medium. Then, cells were added to the upper chamber (4×104 cells/well). At the same time, 0.5 mL of DMEM with 10% FBS was added to the lower compartment, and the Transwell-containing plates were incubated for 12 h in a 5% CO2 atmosphere saturated with H2O. After incubation, cells that had entered the lower surface of the filter membrane were fixed with 4% paraformaldehyde for 25 min at room temperature, washed 3 times with distilled water and stained with 0.1% crystal violet in 0.1 M borate and 2% ethanol for 15 min at room temperature. Cells remaining on the upper surface of the filter membrane (non-migrant) were scraped off gently with a cotton swab. Images of the lower surfaces (with migrant cells) were captured by a photomicroscope (5 fields per chamber) (BX51 Olympus, Japan), and the cells were counted blindly.
The apoptosis of A549 cells transfected with the miR-203 mimic or SRC overexpression plasmid was tested using an Annexin V-FITC/propidium iodide (PI) staining assay. A549 cells were cultured in 12-well plates and transfected with miR-203 mimic, siRNA, or the SRC overexpression plasmid to induce apoptosis. Pre-miR-control and control siRNAs served as negative controls. Cells were cultured overnight with both serum-containing complete medium and serum-depleted medium; and the attached and floating cells were then harvested. Flow cytometry analysis of apoptotic cells was carried out using an Annexin V-FITC/PI staining kit (BD Biosciences, CA, USA). After washes with cold PBS, the cells were resuspended in binding buffer (100 mM HEPES, pH 7.4, 100 mM NaCl, and 25 mM CaCl2) followed by staining with Annexin V-FITC/PI at room temperature in darkness for 15 min. Apoptotic cells were then evaluated by gating PI and Annexin V-positive cells on a fluorescence-activated cell-sorting (FACS) flow cytometer (BD Biosciences, San Jose, CA). All experiments were performed in triplicate.
All of the images of Western blotting are representative of at least three independent experiments. Quantitative RT-PCR, the luciferase reporter assay, and the cell viability and apoptosis assays were performed in triplicate, and each experiment was repeated several times. The data shown are the mean ± SE of at least three independent experiments. The differences were considered statistically significant at p<0.05 using Student's t -test.
We first investigated SRC expression by means of both immunohistochemical analysis and immunoblot analysis in human lung cancer tissues and corresponding noncancerous tissues. We found that SRC protein levels were dramatically higher in the lung cancer tissues (Figure 1, A–C). Although the SRC protein was consistently upregulated in lung cancer tissues, SRC mRNA levels did not differ significantly between the cancer and noncancerous tissues (Figure 1D). Furthermore, we found that the SRC protein level was higher in human lung adenocarcinoma A549 cells compared to that in normal lung fibroblast HLF cells (Figure S1, A and B in File S1), but the SRC mRNA level was equal in these two cell lines (Figure S1C in File S1). This disparity between protein and mRNA in SRC expression in lung cancers strongly suggests that a post-transcriptional mechanism is involved in SRC regulation.
Figure 1. Expression levels of the SRC protein and mRNA in lung cancer tissues.
(A) Representative image of immunohistochemical analysis of SRC expression in lung cancer (CL) and normal adjacent tissue (NL) samples. (B and C) Western blotting analysis of the expression levels of the SRC protein in six pairs of CL and NL samples. B: representative image; C: quantitative analysis. (D) Quantitative RT-PCR analysis of the relative expression levels of SRC mRNA in six pairs of CL and NL samples. * P<0.05; ** P<0.01.
One important mode of post-transcriptional regulation is the repression of mRNA transcripts by miRNAs. miRNAs are, therefore, likely to play a biologically relevant role in regulating SRC expression in lung cancer. Three computational algorithms, including TargetScan , miRanda , and PicTar , were used in combination to identify potential miRNAs that can target SRC. Using these approaches, miR-203 was identified as a candidate regulatory miRNA of SRC. The predicted interaction between miR-203 and the target sites in the SRC 3′-UTR are illustrated in Figure 2A. Four potential miR-203 target sites were found in the 3′-UTR of the SRC mRNA sequence. The minimum free energy values of these hybrids were −16.9, −20.1, −15.8, and −18.9 kcal/mol, respectively, which are well within the range of genuine miRNA-target pairs. Moreover, perfect base pairing occurred between the seed region (the core sequence that encompasses the first 2–8 bases of the mature miRNA) and the cognate targets. Furthermore, the miR-203 binding sequences in the SRC 3′-UTR are highly conserved across species.
Figure 2. Detection of an inverse correlation between the miR-203 and SRC levels in lung cancer tissue samples.
(A) Schematic description of the hypothetical duplexes formed by the interactions between the binding sites in the SRC 3′-UTR (top) and miR-203 (bottom). The predicted free energy value of each hybrid is indicated. The seed recognition sites are denoted, and all nucleotides in these regions are highly conserved across species, including human, mouse, and rat. (B) Quantitative RT-PCR analysis of the expression levels of miR-203 (in the form of the miRNA/U6 ratio) in six pairs of CL and NL samples. (C) Pearson's correlation scatter plot of the fold change of the levels of miR-203 and SRC protein in human lung cancer tissues. (D) Pearson's correlation scatter plot of the fold change of the levels of miR-203 and SRC mRNA in human lung cancer tissues. * P<0.05; ** P<0.01.
miRNAs are generally thought to have expression patterns that are opposite to that of their targets –. We next investigated whether miR-203 was inversely correlated with SRC in lung cancer. After determining the levels of miR-203 in the same six pairs of lung cancer tissues and the corresponding noncancerous tissues, we showed that the miR-203 levels were consistently downregulated in lung cancer tissues (Figure 2B). The inverse correlation between miR-203 levels and SRC protein levels (Figure 2C) and the disparity between miR-203 levels and SRC mRNA levels (Figure 2D) were further illustrated using Pearson's correlation scatter plots. As observed, an inverse correlation between the miR-203 levels and SRC protein levels, but not mRNA levels, was observed in human lung cancer tissues. Likewise, miR-203 level was also lower in A549 cells compared to that in HLF cells (Figure S1D in File S1). The results strongly indicated that a typical, miRNA-mediated, post-transcriptional regulation mechanism was involved in SRC repression.
The correlation between miR-203 and SRC was further examined by evaluating SRC expression in human lung adenocarcinoma A549 cells after overexpression of miR-203. In these experiments, miR-203 overexpression was achieved by transfecting the cells with pre-miR-203 (a synthetic RNA oligonucleotide duplex mimicking the miR-203 precursor). As anticipated, miR-203 levels in A549 cells were increased more than 300-fold when these cells were transfected with pre-miR-203 (Figure 3A). To validate that transfection of miR-203 was successful, one of the representative target genes of miR-203, PKCalpha (REF), was assessed and it was shown that PKCalpha was significantly downregulated in A549 cells after transfection of miR-203 (Figure S2 in File S1). Likewise, the expression of the SRC protein was reduced by the introduction of miR-203 in A549 cells (Figure 3, B and C). To determine the level at which miR-203 influenced SRC expression, we repeated the above-mentioned experiments and examined the expression of SRC mRNA after transfection. Although the intracellular level of miR-203 was significantly altered after treatment with pre-miR-203, the overexpression of miR-203 did not affect SRC mRNA stability (Figure 3D). On the other hand, the correlation between miR-203 and SRC was also examined by evaluating SRC expression in normal lung fibroblast HLF cells after knockdown of miR-203 with anti-miR-203 (chemically modified antisense oligonucleotides designed to specifically target mature miR-203) (Figure 3A). Knocking down miR-203 in HLF cells resulted in the increase of SRC protein (Figure 3, B and C) but not mRNA (Figure 3D) levels. These results demonstrate that miR-203 specifically regulates SRC protein expression at the post-transcriptional level, which is a typical animal miRNA-mediated regulation mechanism. To demonstrate the robustness of the test, we repeated the above-mentioned experiments in two additional lung adenocarcinoma cell lines, HCC827 and H1975. Likewise, the miR-203 levels were significantly increased when HCC827 and H1975 cells were transfected with pre-miR-203 (Figure S3, A and E in File S1). Consequently, the SRC protein levels were suppressed when HCC827 and H1975 cells were treated with pre-miR-203 (Figure S3, B, C, F and G in File S1), but the SRC mRNA levels were not influenced by such treatment (Figure S3, D and H in File S1).
Figure 3. Direct regulation of SRC expression by miR-203 at the posttranscriptional level.
(A) Quantitative RT-PCR analysis of the miR-203 levels in A549 cells treated with pre-miR-control or pre-miR-203 and in HLF cells treated with anti-miR-control or anti-miR-203. (B and C) Western blot analysis of SRC protein levels in A549 cells treated with pre-miR-control or pre-miR-203 and in HLF cells treated with anti-miR-control or anti-miR-203. B: representative image; C: quantitative analysis. (D) Quantitative RT-PCR analysis of SRC mRNA levels in A549 cells treated with pre-miR-control or pre-miR-203 and in HLF cells treated with anti-miR-control or anti-miR-203. (E) Firefly luciferase reporters containing wild-type (WT) or mutant (MUT) miR-203 binding sites in the SRC 3′-UTR were co-transfected into A549 cells with pre-miR-control or pre-miR-203 and into HLF cells with anti-miR-control or anti-miR-203. Twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit. The results are calculated as the ratio of firefly luciferase activity in the pre-miR-203- or anti-miR-203-transfected cells normalized to the control cells. * P<0.05; ** P<0.01.
To determine whether the negative regulatory effects that miR-203 exerted on SRC expression were mediated through the binding of miR-203 to the presumed sites in the 3′-UTR of the SRC mRNA, the full length SRC 3′-UTR containing the four presumed miR-203 binding sites was fused downstream of the firefly luciferase gene in a reporter plasmid. The resulting plasmid was transfected into A549 cells along with a transfection control plasmid (β-gal) and pre-miR-203. As expected, the overexpression of miR-203 resulted in a more than 50% reduction of luciferase reporter activity compared with cells treated with the negative control RNA (Figure 3E). Furthermore, we introduced point mutations into the corresponding complementary sites in the SRC 3′-UTR to eliminate the predicted miR-203 binding sites (all of the four binding positions were mutant). This mutated luciferase reporter was unaffected by the overexpression of miR-203 (Figure 3E). In contrast, when the resulting plasmid was transfected into HLF cells along with a transfection control plasmid (β-gal) and anti-miR-203, the knockdown of miR-203 resulted in a more than 75% increase of luciferase reporter activity compared with cells treated with the negative control RNA, while the mutated luciferase reporter was unaffected by the knockdown of miR-203 (Figure 3E). This finding suggests that the binding sites strongly contribute to the miRNA:mRNA interaction mediating the post-transcriptional repression of SRC expression. In conclusion, our results demonstrate that miR-203 directly recognizes and binds to the 3′-UTR of the SRC mRNA transcript and inhibits SRC translation.
We next analyzed the biological consequences of the decreased SRC expression caused by miR-203 in lung cancer cells. As a potent oncogene, SRC regulates the proliferation and motility of cancer cells by affecting the FAK, EGFR, and Ras/ERK signaling pathways . Therefore, we investigated whether the suppression of SRC expression due to miR-203 regulates the activities of the molecules in the SRC/Ras/ERK signaling pathway. First, we validated the effect of SRC activity on the Ras/ERK signal pathway in A549 cells. In agreement with previous reports , , knockdown of SRC by siRNA-mediated RNA interference resulted in decreased SRC mRNA and protein levels (Figure 4, A–C), which, in turn, led to decreased Ras-GTP and phosphorylated-ERK1/2 (Figure 4, A and B). In contrast, overexpression of SRC resulted in increased SRC mRNA and protein levels (Figure 4, A–C), which, in turn, led to increased Ras-GTP and phosphorylated-ERK1/2 (Figure 4, A and B). Next, we investigated if miR-203 induction could mimic the SRC reduction in suppressing Ras/ERK signaling pathway. As anticipated, overexpression of miR-203 in A549 cells downregulated the SRC protein and resulted in less active Ras-GTP, which, in turn, led to less phosphorylated ERK1/2 (Figure 4, D and E). Taken together, the results suggest that Ras/ERK, as the downstream signaling pathway of SRC, may be tightly controlled by the miR-203/SRC regulatory pathway.
Figure 4. miR-203 represses the SRC/Ras/ERK signal pathway by silencing SRC.
(A and B) Western blotting analysis of the protein levels of SRC, Ras-GTP, total Ras, phosphorylated ERK1/2, and ERK1 in A549 cells transfected with control siRNA, SRC siRNA, control vector, or SRC vector. A: representative image; B: quantitative analysis. (C) Quantitative RT-PCR analysis of the SRC mRNA levels in A549 cells treated with control siRNA, SRC siRNA, control vector, or SRC vector. (D and E) Western blotting analysis of the protein levels of SRC, Ras-GTP, total Ras, phosphorylated ERK1/2, and ERK1 in A549 cells transfected with pre-miR-control or pre-miR-203. D: representative image; E: quantitative analysis. * P<0.05; ** P<0.01.
We next focused on studying the role of miR-203 in regulating SRC. Because SRC is known to be involved in the regulation of cell proliferation, apoptosis, and migration, we investigated whether miR-203 would regulate SRC to modulate cell proliferation, apoptosis, and migration in A549 cells. In support of the notion that SRC is essential in promoting proliferation , A549 cells transfected with SRC siRNA showed inhibition of cell proliferation (Figure S4A in File S1); in contrast, transfection with the SRC-overexpressing plasmid, which specially expresses the full-length open reading frame (ORF) of SRC without the miR-203–responsive 3′-UTR, had an opposite effect on cell proliferation (Figure S4B in File S1). Subsequently, we assessed the role of miR-203 on cell proliferation. As expected, A549 cells transfected with pre-miR-203 had decreased SRC protein levels (Figure 5, A and B) and proliferated at a significantly lower rate (Figure 5C). Moreover, compared to cells transfected with pre-miR-203, those transfected with pre-miR-203 and the SRC-overexpressing plasmid had recovered SRC protein levels (Figure 5, A and B) and exhibited significantly higher proliferation rates (Figure 5C), suggesting that miR-203-resistant SRC is sufficient to rescue the suppression of SRC by miR-203 and attenuate the anti-proliferative effect of miR-203 on lung cancer cells. Taken together, the results indicate that miR-203 might inhibit cell proliferation by silencing SRC.
Figure 5. The role of miR-203 targeting SRC in the regulation of proliferation, migration, and apoptosis of lung cancer cells.
(A and B) Western blotting analysis of the protein levels of SRC in A549 cells transfected with pre-miR-control, pre-miR-203, or pre-miR-203 plus the SRC-overexpressing vector. A: representative image; B: quantitative analysis. (C) The MTT viability assay was performed 12, 24, 36 and 48 h after the transfection of A549 cells with pre-miR-control, pre-miR-203, or pre-miR-203 plus the SRC-overexpressing vector. (D and E) Transwell analysis of A549 cells treated with equal doses of pre-miR-control, pre-miR-203, or pre-miR-203 plus the SRC-overexpressing vector. D: representative image; E: quantitative analysis. (F and G) A549 cells were transfected with equal doses of pre-miR-control, pre-miR-203, or pre-miR-203 plus the SRC-overexpressing vector. Cell apoptosis profiles were analyzed by flow cytometry. The biparametric histogram shows cells in early (bottom right quadrant) and late apoptotic states (upper right quadrant). Viable cells are double negative (bottom left quadrant). F: representative image; G: quantitative analysis. * P<0.05; ** P<0.01.
Next, we assessed the effect of miR-203 and SRC on the migration ability of A549 cells. Transfection of SRC siRNA remarkably reduced the number of A549 cells that passed through the Transwell chamber, whereas transfection of the SRC-overexpressing plasmid increased the migration rate (Figure S4, C and D in File S1). Additionally, the percentage of migrated cells was significantly lower in A549 cells transfected with pre-miR-203 (Figure 5, D and E). However, when A549 cells were co-transfected with pre-miR-203 and the SRC-overexpressing plasmid, SRC dramatically attenuated the anti-migration effect of miR-203 (Figure 5, D and E). The results indicate that miR-203 might inhibit cell migration by silencing SRC.
Finally, we investigated cell apoptosis using flow cytometry analysis. In apoptosis assay, apoptosis was induced by serum starvation in the culture media (Figure S5 in File S1). Transfection of SRC siRNA remarkably increased the percentage of apoptotic cells when compared to cells transfected with control siRNA, whereas transfection of the SRC-overexpressing plasmid decreased cell apoptosis (Figure S4, E and F in File S1). In addition, significantly more apoptotic cells were observed in A549 cells transfected with pre-miR-203 compared with those transfected with the pre-miR-control (Figure 5, F and G). Furthermore, when A549 cells were simultaneously transfected with pre-miR-203 and the SRC-overexpressing plasmid, SRC dramatically attenuated the pro-apoptotic effect of miR-203 (Figure 5, F and G). Taken together, these results indicate that miR-203 might modulate cell apoptosis by downregulating SRC in lung cancer cells.
The cellular tyrosine kinase, SRC, is frequently overexpressed or aberrantly activated in a range of cancers . For example, the increased expression of SRC has been reported in 60–80% of adenocarcinomas and 50% of squamous cell carcinomas from lung cancer patients . Additionally, high levels of SRC activity have been reported in lung cancer, particularly adenocarcinomas, and the degree of kinase activity correlates with tumor size . SRC can interact with many genetic and signaling pathways, including the FAK, EGFR, Ras/ERK, JAK/STAT, PI3K/AKT, and VEGF pathways , , . These diverse substrate interactions link SRC with a broad range of oncogenic mechanisms, including cell proliferation, migration, apoptosis, and angiogenesis . In this study, we showed that silencing SRC expression using siRNA could inhibit cell proliferation and migration and promote apoptosis in lung cancer cells, while overexpressing SRC had opposite effects, validating its role as an essential oncogene during lung tumorigenesis. Interestingly, we identified discordance between the SRC protein and mRNA levels in human lung cancer tissue samples and cultured cells. The results suggest a post-transcriptional regulation mechanism involved in SRC repression. One centrally important mode of post-transcriptional regulation is the repression of mRNA transcripts by miRNAs (25). Therefore, we searched for miRNAs that could target SRC, and experimentally validated miR-203 as a direct regulator of SRC. Interesting, SRC has been reported as a target gene of miR-203 in bladder cancer . Taken together, our results identified a miRNA as a link between the SRC regulatory pathway and the pathogenesis of lung cancer.
In this study, we further showed that ectopic expression of miR-203 could inhibit the translation of SRC in lung cancer cells, which, in turn, was associated with a reduction in the levels of Ras-GTP and phosphorylated-ERK1/2. Thus, Ras/ERK signaling is also regulated by the miR-203/SRC regulatory pathway. The results reveal a new possible target for regulating Ras/ERK activity in lung cancer, which can be accomplished by affecting the miR-203 level. As the downstream signal transduction pathway of SRC, activation of Ras/ERK pathway is associated with cell transformation and tumor progression . Consistent with this, we lastly showed that miR-203 could suppress SRC expression and, in turn, inhibit proliferation and migration and promote apoptosis of lung cancer cells. The results reveal a critical role for miR-203 as a tumor suppressor in lung carcinogenesis through the repression of SRC translation. Actually, miR-203 has been reported to act as a tumor-suppressive miRNA in many cancer types (30–34), and it was shown that miR-203 expression was downregulated in various prostate cancer cell lines . The ectopic expression of miR-203 in prostate cancer cell lines could influence proliferation, apoptosis, and migration , , and the overexpression of miR-203 in laryngeal carcinoma cells reduced cell viability and led to a cell cycle arrest in G1 phase . Additionally, expression of miR-203 suppressed cell proliferation and migration in human triple-negative breast cancer cells . These studies, in summary, reveal the essential role of miR-203 as a tumor suppressor gene. Interestingly, it is noted that restoration of SRC expression can successfully attenuate the anti-proliferative, anti-migration, and pro-apoptotic effects of miR-203 on lung cancer cells, although miR-203 has many other targets. The results suggest that targeting SRC is a major mechanism by which miR-203 exerts its tumor-suppressive function. Therefore, the modulation of SRC by miR-203 might explain, at least in part, why the downregulation of miR-203 during lung carcinogenesis can accelerate lung cancer progression.
Taken together, this study delineates a novel regulatory network employing miR-203, SRC, and downstream signaling factors to fine-tune cell proliferation, migration, and apoptosis in lung cancer cells. This study may provide a potential novel target for future lung cancer therapy.
Table S1, Figure S1–S5. Table S1. Clinical features of lung cancer patients; Figure S1. the expression level of SRC and miR-203 in normal lung fibroblast cell line HLF cells and lung cancer cell lines A549 cells; Figure S2. miR-203 regulates PKCalpha expression in A549 cells; Figure S3. miR-203 directly regulates SRC expression at the post-transcriptional level; Figure S4. The role of SRC in the regulation of proliferation, migration, and apoptosis of lung cancer cells; Figure S5. The apoptosis of full-serum cultured A549 cells and serum-starvation cultured A549 cells.
Conceived and designed the experiments: DL XC. Performed the experiments: NW HL YZ CW. Analyzed the data: SZ YP YW XY. Contributed reagents/materials/analysis tools: JZ CYZ KZ. Contributed to the writing of the manuscript: JZ CYZ KZ DL XC.
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Do you feel bored of your cluttered and untidy living room? Then you need to keep your living room tidy with some living room storage. There several living room storage solutions that you can use. Here are some of them. Expect living room furnishings to do double duty. A storage ottoman offers comfort and beauty while providing hidden storage beneath the seat for stowing magazines, games, or throws. Add a fabric skirt to a console table to create hidden storage. Tuck baskets and bins behind the curtain for sorting DVDs, CDs, and video games. If your home's entryway opens directly into the living room, the space beneath a skirted console can also conceal footwear, winter gear, and sporting goods. Look for an occasional, coffee, or console table with shelves and/or drawers to augment living room storage. Choose one that fits where you need it, complements your style, and offers ample surface for beverages, books, and baubles.
Declutter existing shelves by adding bins, baskets, and decorative boxes for concealing small items. If your living room lacks shelves and the vertical space for hanging them, consider adding shelving up high, even above windows. On the other hand, you can also try this living room storage solution: Design a built-in to instill your living room with architectural interest while providing a beautiful bank of cabinetry, drawers, and open shelves.
Label: storage ideas living room, living room storage design, living room storage inspiration, storage solutions living room, storage for living room, .


Product was easy to setup and use. User friendly and not to technically challenging for new users. Great customer service department if you enter any issues.
Clean and simple interface, great customer service We've been using Freshsales for the past few months and it's by far the easiest CRM we've worked with. A lot of other software is bulky and takes way to long to setup. We were up and running the first day with Freshsales. It's so Intuitive that our sales staff doesn't even need to be trained on it. If it doesn't do what you want, just ask. Their team is great at responding to feedback.
We've been using Freshsales for the past few months and it's by far the easiest CRM we've worked with.
A lot of other software is bulky and takes way to long to setup.
We were up and running the first day with Freshsales.
It's so Intuitive that our sales staff doesn't even need to be trained on it.
If it doesn't do what you want, just ask. Their team is great at responding to feedback.
I am very happy with many of the features of Freshsales.
Seamless integration with our googlemail and calendar, seamless integration with our ticket solution. Phone integration is the best I expirienced in my 20 year career (but most of the other also where pretty bad). Integration on the events from the website A list view of over 25 leads could easily been done via reports – still I would love to also have this option in the regular view – this is mainly an issue for an manager who tries to look into all the leads. Like mentioned it still works but could be easier. Easy for us to implement and good daily expirience. The whole sales team likes me more now (and that is no joke). So far the best I experienced on the market for non super complex use-cases.
Seamless integration with our googlemail and calendar, seamless integration with our ticket solution.
A list view of over 25 leads could easily been done via reports – still I would love to also have this option in the regular view – this is mainly an issue for an manager who tries to look into all the leads. Like mentioned it still works but could be easier.
Easy for us to implement and good daily expirience. The whole sales team likes me more now (and that is no joke).
So far the best I experienced on the market for non super complex use-cases.
I wouldn't say this product has the most features, but it offers everything your sales team needs to get to work for a more than competitive price. The email tracking, for instance, is really handy if you have team members sharing multiple accounts. I've seen other products offer similar features for much higher monthly rates, so you're really getting a deal. I also like the user-friendly interface, which really helps when you're trying to get your sales team onboard with using a new tool. It would be great if the software was a bit more customizable but if you have a traditional sales team with traditional sales needs then you should have no issues adapting to this product. I believe a 30 day trial is available for this product and I recommend trying it out. If you are trying to find a professional solution at a competitive price I think you will find that this product fits your needs.
I wouldn't say this product has the most features, but it offers everything your sales team needs to get to work for a more than competitive price. The email tracking, for instance, is really handy if you have team members sharing multiple accounts.
I've seen other products offer similar features for much higher monthly rates, so you're really getting a deal. I also like the user-friendly interface, which really helps when you're trying to get your sales team onboard with using a new tool.
It would be great if the software was a bit more customizable but if you have a traditional sales team with traditional sales needs then you should have no issues adapting to this product.
I believe a 30 day trial is available for this product and I recommend trying it out. If you are trying to find a professional solution at a competitive price I think you will find that this product fits your needs.
Freshsales is a sales-oriented CRM platform produced by the company behind Freshworks, the customer engagement platform. In 2012, burdened by a major vendor's price for a CRM that had support for APIs, and using valuable developer resources simply to maintain their system of additional integrated platforms, Freshdesk built their own CRM with the built-in features their salespeople preferred.
Freshdesk was founded in 2010 by current CEO Girish Mathrubootham in Chennai, India.
Prices reflect monthly prices per user when billed month-to-month and when billed annually.
Freshsales offers 24 x 7 email support, which can be reached via support@freshsales.io.
They also offer 24 x 5 phone support for all plans. Their contact number in the US is +1 (866) 832-3090.
Freshsales is geared towards high velocity sales teams – rather than a few big clients, these teams would have a large number of moderately-sized clients. With so much activity on different accounts, combined with the modern data needs and responsibilities of a salesperson, sales teams need a platform that combines multiple applications into one. The platform that's easiest to use and saves users the most clicks per task wins out.
Towards this end, Freshsales is a competitive sales-oriented CRM – it's got an open, straightforward UI, integrated phone and email, lead management, a deals pipeline, event tracking, a mobile app – all the features you'd expect for a sales CRM. The pricing is competitive; while users can get a barebones version of the platform for free up to ten users, the main functionality comes from email tracking, templates, notifications and Google calendar integration.
The '360 degree customer view' presents comprehensive customer details in one place – their contact information, correspondence history, and sales history. The deals pipeline can be customized by stages and can automatically arrange leads by progress and allows manual arrangement through drag and dropping leads. Leads can be imported through CSV files, web forms or emails, but unfortunately, Freshsales does not automatically pull contacts in from Google, which is a handy feature of other CRMs like Insightly. Aside from the importation options, contacts can be added manually, but starting with the Garden plan users can enjoy Auto Profile Enrichment which draws information and photos automatically from social media.
The Garden plan is Freshsales' most popular plan. Users get the email tracking features with associated metrics, the email templates, the configurable lead assignment by size, industry and territory. It falls short of the in-app event tracking and website events tracking. Therefore the Garden plan seems best suited for sales teams selling services or a products without a need (or desire) for a highly nuanced online sales or marketing presence.
Note that some CRMs with built-in phone features offer unlimited calling for certain plans; Freshsales does not. Users must pay $1 per phone line and then calling rates apply. In contrast, an even more heavily sales-oriented CRM such as Close.io – which was also developed as an internal solution for salespeople – is more expensive but allows unlimited calling with more automated features out of the box. Companies that spent a lot of time on the phone may prefer the predictable albeit higher fees of Close.io to those of Freshsales, which would fluctate depending on phone activity. On the other hand, Freshsales offers more 'traditional' CRM features than Close.io, and notably offers a mobile app, which Close does not.
Freshsales integrates with Freshdesk, Segment, and Google Calendar. Zapier and access to the developer API can handle many other integrations.
If sales is a major part of your business and you're looking for a lightweight but fairly functional CRM, Freshsales offers nice functionality for the price. It's not highly customizable, but it's easy to setup, friendly on the eyes, and incorporates enough email functions – templates, scheduling, metrics, logging – for a nice balance between phone and email sales correspondence. They've got a 30-day free trial, no credit card required, which is more than most CRMs. If you're in the market for a sales CRM solution, it's worth giving a shot.
What Do You Think of Freshsales?


text:
Historical Romance Review: One True Knight by Dana D'Angelo
This book has been reviewed by History Undressed reviewer, Morgan Wyatt...
ABOUT THE BOOK...
When the beautiful yet feisty Rowena de Belleville discovers her father's plan to remarry, she fears the worst and flees her home — only to be thrust into the arms of a stranger.
Desperate to hide her identity from her pursuers, she embraces the dark stranger. But her reckless act backfires as it awakens a passion buried deep within her soul, while igniting the fuse of her mysterious benefactor.
Unable to escape destiny, their paths cross yet again. This time she learns the handsome man is Jonathan d'Abelard - the Iron Hawk, a legendary knight feared by all save one faceless killer bent on making his life a living hell…
Will her chance encounter draw them together, or ensnare her in a dangerous game of seduction, feverish desire and vengeance?
MORGAN'S REVIEW...
One True Knight by Dana D'Angelo is a medieval romance that highlights the differences in the freedoms enjoyed by men and women of the period. Rowena's ability to be her own woman, and manage the castle grind to an unexpected halt when her father, Sir Phillip's announcement that he will marry. Ten years ago, when his beloved wife died he swore to never remarry. He also chose to ignore Rowena because he blamed her for the plague her mother contacted from nursing her. Her impulsive nature forces her into action.
Jonathan is more than a wandering knight he is a man on a mission. His first mission is vengeance against the Grey Knight. The nefarious knight is so bold to send him messages and taunts causing Jonathan to scurry across the country in search of the knight. His cousin has asked him to vet Sit Phillip before she marries. His cousin a widow finds herself forced to choose between her two neighbors to be able to afford some type of protection for her people.
Rowena escapes the castle disguises as a simple servant. She isn't quite sure of her plans, but she realizes a new mistress will change her role in the house, and maybe even push her out of it. Jonathan disguises himself as a peasant too in an effort to find out more about Sir Phillip. In an effort to escape her father's guards, she chooses to kiss Jonathan as a distraction, sparking an attraction between the two of them. She flees refusing even to leave a name with the besotted knight.
One True Knight has a great deal going on, besides romance including intrigue and betrayal. There is also the double disguise of Jonathan and Rowena pretending to be other people, always a handy plot device. The chemistry between the two works well. The characterization of the cousin being a pawn in the games of men is accurate for the period. This is a good debut novel, but there are areas where it could be stronger.
More detail would give me a sense of being there. Historical references were rather vague, specific ones as a tie-in could help in getting a feel for the period, and the people. In the beginning, Rowena acts more like a spoiled brat caring more about herself, than anyone else. She doesn't care about her father's happiness; she's just upset because his marriage might mess her life up. She sets up her own nursemaid to get in trouble so she can run the country in disguise. Rowena improves as the book goes on maturing with the chapters.
One True Knight is a good bet for medieval romance fans. Its short length makes it a quick read too.
Labels: Dana D'Angelo, One True Knight
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redpajama_set_name: RedPajamaCommonCrawl


text:
«Ередіано» () — футбольний клуб з міста Ередія, Коста-Рика, в даний момент виступає в Прімері, найсильнішому дивізіоні Коста-Рики.

Основні кольори клубу — жовтий і червоний.

Історія
Клуб заснований 12 червня 1921 року, і є одним з найстаріших в країні. Своє перше чемпіонство команда завоювала в перший рік свого існування. За свою історію клуб завоював 26 чемпіонських кубків, що є третім за титулування показником в Коста-Риці. Клуб жодного разу не залишав вищий дивізіон країни.

Стадіон

Домашні матчі проводить на стадіоні «Еладіо Росабаль Кордеро», що вміщує 8700 глядачів. Стадіон був побудований в 1951 році і названий на честь одного із засновників клубу — костариканського футболіста Еладіо Росабаля Кордеро.

Досягнення
 Чемпіон Коста-Рики (26): 1921, 1922, 1924, 1927, 1930, 1931, 1932, 1933, 1935, 1937, 1947, 1948, 1951, 1955, 1961, 1978, 1979, 1981, 1985, 1987, 1992/93, Літ.. 2012, Літ. 2013, Літ. 2015, Літ. 2017
 Переможець Ліги КОНКАКАФ: 2018

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Футбольні клуби, засновані 1921
Футбольні клуби Коста-Рики
1921 у костариканському спорті
засновані у Коста-Риці 1921

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After the most successful season in franchise history, the Columbus Blue Jackets find themselves shrouded in a bizarre haze surrounding the future of their goaltending heading into the offseason.
On the one hand, Sergei Bobrovsky is almost certainly going to win his 2nd Vezina Trophy as the league's top goaltender next month. They have a fair amount of quality depth in the pipeline, between Joonas Korpisalo and Anton Forsberg, to say nothing of the recently acquired Matiss Kivlanieks, or the international excellence of Elvis Merzlikins.
On the other, Bobrovsky was abjectly bad in these playoffs, Korpisalo could easily be poached by Vegas next month (and even if he isn't, wasn't spectacular this past season), Forsberg has never looked comfortable in his brief stints in the NHL, Kivlanieks is, at this moment, nowhere near ready for the NHL, and Merzlikins' contract situation is cloudy. So, please allow me to try to paint a clearer portrait of the Blue Jackets current state of goaltending.
Bobrovsky went from hero to zero in a matter of weeks. Definitively the top goalie in the league this regular season, Bob underwhelmed dramatically in the playoffs. The concern doesn't seem to be that he was bad, more so that this spell of poor playoff performance doesn't appear to be an anomaly; he's never quite looked like the same goalie in the Stanley Cup Playoffs, and hiss numbers bear that out. Still, he's unquestionably the Blue Jackets top goalie heading into next season, and, aside from the fact that he's got a No Movement-Clause (NMC) in his contract, the Jackets wouldn't dream of exposing him to Vegas. So, for the next two years at least, he'll undeniably be the Blue Jackets starting goaltender.
The young Finn was underwhelming this past season when compared to his first stint with the club in 15-16. Still, he shows promise to be an everyday NHL goaltender. He has basically three different immediate fates: one is that he is the player poached next month by Vegas as their Columbus selection. Route two, perhaps the most likely of the three, is that he is resigned (he's an RFA) and retains his current position as backup goalie behind Bobrovsky. Option three is that the Blue Jackets, spooked by Bobrovsky's leaky playoff performance, look to upgrade the backup position. If that's the case, Korpisalo would re-join Cleveland in the AHL, assuming he's not picked by Vegas, or traded.
The 24-year-old Swede played in 51 games this season for the AHL Monsters, and went 27-17-4-2 with a .926 sv% and a 2.28 GAA. Forsberg is a solid AHL player who has frankly underwhelmed in his three stints in the NHL. Still, he's seen as a valued player in the organization, and is clearly one of the better goalie prospects in the AHL. He's an RFA but I'd expect the Blue Jackets to keep him around next season, regardless of what happens in Vegas.
The 23-year-old may be the wild card in this whole group. He's clearly ready for the next level after consistently putting up spectacular numbers in the top Swiss league with Lugano. The Latvian also has shown well, repeatedly, at the international stage. Look no further than this past month at the IIHF World Championships, where he played in six games and managed an impressive .937 sv%, including allowing just 2 goals to eventual World Champion Team Sweden. Or his play in the Spengler Cup earlier this season, where he took his team to the finals before losing to Team Canada. He was named to the tournament's all-star team for posting a 2.14 GAA and a .940 sv%.
The Blue Jackets won the rare recruiting war for Kivlanieks. The USHL Player and Goaltender of the Year posted obscene numbers, going 36-7-4-2 with a 1.85 GAA, a .932 sv%, and 5 shutouts in 49 games for the Sioux City Musketeers. Without knowing the results of the Vegas expansion draft, it seems likely that the 20 year old will be in Cleveland (AHL) in some capacity next year, but there's a chance he'll play in the ECHL to get more games under his belt. It's fairly rare for a prospect to be signed to an NHL contract straight from the USHL, but the Latvian clearly has upside. He was previously committed to Minnesota State.
Thome played on 3 different USHL teams this year, most recently backstopping the Waterloo Black Hawks. The 20-year-old will be going to the University of North Dakota and will compete as a freshman to back up senior Cam Johnson. UND's backup this year, Matej Tomek, will be leaving the NCAA to play in the USHL, coincidentally for Waterloo, in a de facto trade. Thome was drafted in the 6th round, 155th overall in 2016, out of Aberdeen of the NAHL. Though he is still seen as a long-term play, the Blue Jackets are hopeful to see what Thome can do at a powerhouse like North Dakota.
Once seen as a potential starting NHL goaltender, Dansk may be at the end of his rope with the organization. He's been on loan the past two seasons to Rogle of the Sweden's top league, who finished 2nd to last in the league. He split time with former NHLer Anders Lindback, so there's no doubting his skill. Consistency is an issue for Dansk, and I get the sense that the Blue Jackets will not be in his future. He will be an RFA this summer.
In less than a month, the NHL entry draft and expansion draft will be in the rear-view mirror, and this depth chart could need a complete overhaul. What changes, if any will occur? Do they look to draft a goalie? Does that decision hinge on if Korpisalo is taken by Vegas? If he is taken by Vegas, does Columbus look to upgrade their backup position externally? With so many teams jockeying to not lose prized assets in June for free, there should be backup options available. The question, then, becomes at what price, and who?


Later, Ammaji asks the priest to find a lucky muhurat for them to get married, as ganga and sagar excitedly sit. he says that 32 out of 36 compatibility tests they pass already. they are happy. all are very happy as he talks about a date, 30 days from now, which is perfect. ganga and sagar are thoroughly amused. ammaji sets the date as that itself. the screen freezes on ganga and sagar's happy face.
Precap: Pulkit starts arranging for the wedding. He arranges for the furniture, chairs and fans etc. Prabha says that they should get table fans too, instead of only ceiling ones. he asks her not to worry, as its all taken care of. after he leaves, prabha thinks that he has no clue that the fan brought on rent shall cause such havoc in the family.


text:
Steve Whidden re-elected to fourth term as Hendry County sheriff
Devan Patel
Hendry County Sheriff Steve Whidden will serve a fourth term in office after coasting to victory in Tuesday's election.
Whidden, the Republican nominee, received 7,520 out of 12,768 votes cast, or 58.9%, in defeating Democratic challenger Johnny Jackson and no-party affiliated candidate Ricky Garcia.
Jackson finished in second-place after receiving 3,571 votes, or 28%, while Garcia received 1,677 votes, or 13.1%
Whidden advanced to the general election in November after receiving nearly 80% of the vote in the August primary, when he defeated Lee County Sheriff's Office Capt. Pete Hedrick.
More:Dozens of deputies lied, cheated and stole. This Florida sheriff hired them anyway.
More:Hendry County sheriff posts his account of May shooting that wounded two Black men; attorneys say he's wrong
His victory set up a rematch against Jackson and Garcia, who both were unsuccessful in trying to unseat Whidden in 2016.
When Jackson challenged Whidden, who has since switched political alliances, in the Democratic primary in 2016, Whidden narrowly escaped defeat. The margin of victory in the 2016 primary was 67 votes with Whidden receiving 1,209 votes to 1,142 for Jackson.
Support for Whidden in 2020 closely mirrored the results in the 2016 general election when he received 59.8% of the 10,875 votes cast.
Connect with reporter Devan Patel: @DevanJPatel (Twitter) or devan.patel@naplesnews.com


text:
Richard N.
Vibra Specialty Hospital of Portland
On January 7th, Richard Nystrom underwent open heart surgery at Yakima Regional Medical Center. Unfortunately, he suffered post-operative complications that effected his heart, kidneys, lungs, and digestive system. In the weeks that followed, Richard's condition spiraled downward. Perilously ill with multi-system failure, Richard was transferred to Vibra Specialty Hospital in Portland due to Vibra's well-earned reputation for treating complex medical problems.
When he arrived at Vibra, Richard was met by an interdisciplinary team of clinicians including Dr. Mary Ann Arndt, one of Vibra's nephrologists. Dr. Arndt meticulously reviewed the medications that Richard had been prescribed, and she recognized one as being particularly toxic to the kidneys. "I remember Dr. Arndt saying that the drug was poisoning my father," recalls Valarie, Richard's daughter. As soon as the medication in question was discontinued, Richard's kidneys began to recover. The improvement was dramatic.
With Richard's kidneys functioning properly, physicians at Vibra could now turn their attention to Richard's other problems, most notably his seriously impaired digestive system. So many disorders were occurring simultaneously that some thought Richard might not survive. But through every phase of his recovery was one of Vibra's tenured hospitalists, Dr. June Jia, who became Richard's champion. "She was incredible," said Valarie. "She irrigated his stomach, gave him blood transfusions, managed his abdominal wound, and so much more, all while keeping our family informed every step of the way."
Richard remembers little of this time, suffering from a brain dysfunction syndrome caused by the failure of his body's other organs. "But I remember my family visiting me," said Richard. "My granddaughter, Valarie-Jean, set framed pictures of my whole family along the window sill, and those were the first things I'd see when I woke up, and the last things I'd see when I fell asleep." Concentrating on those pictures fortified his will to persevere. "I'd better stick around," he would remind himself. "I've got a lot of family all pulling for me."
"Stick around" he did. Richard's will to live, coupled with the steadfast attention of Vibra's doctors, nurses, and wound specialist, finally lead to the long-awaited change in the trajectory of his health. Richard's belowaverage lab values began to rise, and the above-average lab values began to fall. Richard was getting better.
Working collaboratively with the clinicians, Vibra's team of physical therapists targeted their efforts on increasing Richard's ability to sit, stand, and walk independently, while the occupational therapists focused on self-care tasks, improving motor skills, and increasing activity tolerance and safety. Though Richard tired easily, he worked hard to achieve the independence he needed to safely leave the hospital. Two months after he arrived, Richard was ready to bid a warmhearted farewell to Vibra's many caregivers who helped him progress along the continuum of care.
Today, temporarily living in Portland near Valarie, Richard is happy to be alive. His experience has inspired him to write a book. "I really believe Vibra saved my life," says Richard. Valarie echoes that sentiment, saying expressively, "I want everyone at Vibra to know how grateful I am that you saved my dad."


text:
When does OJ get out of prison?
Admin March 25, 2021 Blog
1 When does OJ get out of prison?
2 How many kids does OJ Simpson and Nicole Brown have?
3 When will OJ be free?
4 Was Oj acquitted?
5 Who killed Nicole Brown Simpson?
OJ Simpson left prison on October 1, 2017. Since being released on parole, Simpson has been enjoying retirement in Las Vegas, where he lives in a friend's house in a gated community. InTouch Weekly reports that he is "keeping a low profile these days".
How many kids does OJ Simpson and Nicole Brown have?
O.J. Simpson and Nicole Brown Simpson had two kids together, Sydney Simpson and Justin Simpson. Both were grammar-school age during the time their father was on trial for murdering their mother.
Is O J Simpson still in prison?
In 2008, he was convicted and sentenced to 33 years' imprisonment, with a minimum of nine years without parole. He served his sentence at the Lovelock Correctional Center near Lovelock, Nevada. Simpson was granted parole on July 20, 2017. He was eligible for release from prison on October 1, 2017, and was released on that date.
When will OJ be free?
Doc: O.J. Simpson will never be free. If the hearing goes as expected, Simpson would be eligible to leave prison on Oct. 1.
Was Oj acquitted?
O.J. Simpson acquitted. At the end of a sensational trial, former football star O.J. Simpson is acquitted of the brutal 1994 double murder of his estranged wife, Nicole Brown Simpson, and her friend, Ronald Goldman .
Where did OJ kill Nicole?
Nicole was the ex-wife of OJ Simpson and she was killed at age 35 at her Brentwood home along with her friend, restaurant waiter Ronald Goldman . Her murder occurred on June 12, 1994. OJ met Nicole in 1977 when she was a waitress at Jack Hanson's Beverly Hills nightclub "The Daisy".
Who killed Nicole Brown Simpson?
O.J. Simpson was acquitted of murdering Nicole Brown Simpson and Ron Goldman, but the disgraced NFL player allegedly confessed to his agent that he did it! Mike Gilbert claims O.J. admitted to killing them, and that 'the Juice' tried to commit suicide before turning himself into the police!
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text:
Minimal pair
Find sources: "Minimal pair" – news · newspapers · books · scholar · JSTOR (November 2017) (Learn how and when to remove this template message)
This article contains IPA phonetic symbols. Without proper rendering support, you may see question marks, boxes, or other symbols instead of Unicode characters. For an introductory guide on IPA symbols, see Help:IPA.
In phonology, minimal pairs are pairs of words or phrases in a particular language, spoken or signed, that differ in only one phonological element, such as a phoneme, toneme or chroneme,[1] and have distinct meanings. They are used to demonstrate that two phones are two separate phonemes in the language.
Many phonologists in the middle part of the 20th century had a strong interest in developing techniques for discovering the phonemes of unknown languages, and in some cases, they set up writing systems for the languages. The major work of Kenneth Pike on the subject is Phonemics: a technique for reducing languages to writing.[2] The minimal pair was an essential tool in the discovery process and was found by substitution or commutation tests.[3]
As an example for English vowels, the pair "let" + "lit" can be used to demonstrate that the phones [ɛ] (in let) and [ɪ] (in lit) actually represent distinct phonemes /ɛ/ and /ɪ/. An example for English consonants is the minimal pair of "pat" + "bat". The following table shows other pairs demonstrating the existence of various distinct phonemes in English. All of the possible minimal pairs for any language may be set out in the same way.
IPA 1
pin bin /pɪn/ /bɪn/ initial consonant
rot lot /rɒt/ /lɒt/
thigh thy /θaɪ/ /ðaɪ/
zeal seal /ziːl/ /siːl/
bin bean /bɪn/ /biːn/ vowel
pen pan /pɛn/ /pæn/
cook kook /kʊk/ /kuːk/
hat had /hæt/ /hæd/ final consonant
mean meme /miːn/ /miːm/
Phonemic differentiation may vary between different dialects of a language so a particular minimal pair in one accent may be a pair of homophones in another. That means not that one of the phonemes is absent in the homonym accent but only that it is not contrastive in the same range of contexts.
1.1 Quantity
1.1.1 Syntactic gemination
1.2 Tone
1.3 Stress
1.4 Juncture
2 Minimal sets
4 In sign languages
In addition to the minimal pairs of vowels and consonants provided above, others may be found:
QuantityEdit
Many languages show contrasts between long and short vowels and consonants. A distinctive difference in length is attributed by some phonologists to a unit called a chroneme. Thus, Italian has the following minimal pair that is based on long and short /l/:
pala /ˈpala/ shovel
palla /ˈpalla/ ball
However, in such a case it is not easy to decide whether a long vowel or consonant should be treated as having an added chroneme or simply as a geminate sound with phonemes.
Classical Latin, German, some Italian dialects, almost all Uralic languages, Thai, and many other languages also have distinctive length in vowels. An example is the cŭ/cū minimal pair in the dialect that is spoken near Palmi (Calabria, Italy):
Dialect spoken in Palmi
Cŭ voli? /kuˈvɔːli/ short cŭ < lat. qu(is) ("who?") Quis vult? Chi vuole? Who wants?
Cū voli? /kuːˈvɔːli/ long cū < lat. qu(o) (ill)ŭ(m) ("for-what him?") Quō illum/illud vult? Per che cosa lo vuole? For what (reason) does he want him/it?
Syntactic geminationEdit
In some languages like Italian, word-initial consonants are geminated after certain vowel-final words in the same prosodic unit. Sometimes, the phenomenon can create some syntactic-gemination-minimal-pairs:
Italian sandhi
Meaning of the sample sentence
dà casa /dakˈkaza/ (he/she) gives (his/her) house Carlo ci dà casa. Carlo gives us his house.
da casa /daˈkaza/ from home Carlo uscì da casa. Carlo got out from home.
In the example, the graphical accent on dà is just a diacritical mark that does not change the pronunciation of the word itself. However, in some specific areas, like Tuscany, both phrases are pronounced /daˈkkaːza/ and so can be distinguished only from the context.
ToneEdit
Minimal pairs for tone contrasts in tone languages can be established; some writers refer to that as a contrast involving a toneme. For example, Kono distinguishes high tone and low tone on syllables:[4][5]
high /kɔ́ɔ́/ 'to mature'
low /kɔ̀ɔ̀/ 'rice'
StressEdit
Languages in which stress may occur in different positions within the word often have contrasts that can be shown in minimal pairs, as in Greek and Spanish:
Greek ποτέ /poˈte/ ever
Greek πότε /ˈpote/ when
Spanish esta /ˈesta/ this
Spanish está /esˈta/ (he/she/it) is
JunctureEdit
English-speakers are able to hear the difference between, for example, "great ape" and "grey tape", but phonemically, the two phrases are identical: /ɡreɪteɪp/.[6][7] The difference between the two phrases, which constitute a minimal pair, is said to be one of juncture. At the word boundary, a "plus juncture" /+/ is posited and said to be the factor conditioning allophones to allow distinctivity: the result is that "great ape" has an /eɪ/ diphthong shortened by pre-fortis clipping and, since it is not syllable-initial, a /t/ with little aspiration (variously [t˭], [ɾ], [ʔt], [ʔ], etc., depending on dialect); meanwhile in "grey tape", the /eɪ/ has its full length and the /t/ is aspirated [tʰ].
Only languages with allophonic differences associated with grammatical boundaries have juncture as a phonological element. It is claimed that French does not have juncture as a phonological element[8] so, for example, "des petits trous" (little holes) and "des petites roues" (little wheels), phonemically both /depətitʁu/, are phonetically identical.
Minimal setsEdit
The principle of a simple binary opposition between the two members of a minimal pair may be extended to cover a minimal set in which a number of words differ from one another in terms of one phone in a particular position in the word.[9] For example, the vowels /a/, /e/, /i/, /o/, /u/ of Swahili are shown to be distinct by the following set of words: pata 'hinge', peta 'bend', pita 'pass', pota 'twist', puta 'thrash'.[10] However, establishing such sets is not always straightforward [11] and may require very complex study of multiple oppositions as expounded by, for example, Nikolai Trubetzkoy.[12]
TeachingEdit
Minimal pairs were an important part of the theory of pronunciation teaching during its development in the period of structuralist linguistics, particularly in the 1940s and 1950s, and minimal pair drills were widely used to train students to discriminate among the phonemes of the target language.[13] However, later writers have criticized the approach as being artificial and lacking in relevance to language learners' needs.[14]
Some writers have claimed that learners are likely not to hear differences between phones if the difference is not a phonemic one.[15][16] One of the objectives of contrastive analysis[17] of languages' sound systems was to identify points of likely difficulty for language learners that would arise from differences in phoneme inventories between the native language and the target language. However, experimental evidence for this claim is hard to find, and the claim should be treated with caution.[18]
In sign languagesEdit
In the past, signs were considered holistic forms without internal structure. However, the discovery in the mid-20th century that minimal pairs also exist in sign languages showed that sign languages have sublexical structure.[19] Signs consist of phonemes, which are specifications for location, movement, handshape, orientation, and non-manual elements. When signs differ in only one of these specifications, they form a minimal pair. For instance, the German Sign Language signs shoes and socks are identical in form apart from their handshapes.
Minimal pairs in Phoneme
^ Jones, Daniel (1944). "Chronemes and Tonemes". Cite journal requires |journal= (help)
^ Pike, Kenneth (1947). Phonemics.
^ Swadesh, M. (1934). "The Phonemic Principle". Cite journal requires |journal= (help)
^ Roach, Peter (2001). Phonetics. Oxford. p. 25. ISBN 978-0-19-437239-8.
^ Manyeh, Morie Komba (1983). Aspects of Kono Phonology (PhD). University of Leeds. p. 152.
^ Jones, D. (1931). The "Word" as a phonetic entity.
^ O'Connor and Tooley (1964). The perceptibility of certain word-boundaries.
^ O'Connor (1973). Phonetics.
^ Ladefoged, P. (2006). A Course in Phonetics. pp. 35–6.
^ Ladefoged, P. (2001). Vowels and Consonants. p. 26.
^ Fromkin and Rodman (1993). An Introduction to Language. pp. 218–220.
^ Trubetzkoy, N. (1969). Principles of Phonology.
^ Celce-Murcia; et al. (1996). Teaching Pronunciation. pp. 3–4.
^ Brown, Gillian (1990). Listening to Spoken English. pp. 144–6.
^ Lado, R. (1961). Language Testing. p. 15.
^ Pennington, M. (1996). Phonology in English Language Teaching. p. 24.
^ Lado, R. (1957). Linguistics across Cultures.
^ Celce-Murcia; et al. (1996). Teaching Pronunciation. pp. 19–20.
^ Stokoe, W. C. (2005-01-01). "Sign Language Structure: An Outline of the Visual Communication Systems of the American Deaf". Journal of Deaf Studies and Deaf Education. 10 (1): 3–37. doi:10.1093/deafed/eni001. ISSN 1465-7325. PMID 15585746.
BibliographyEdit
Brown, G. (1990) Listening to Spoken English, Longman
Celce-Murcia, M., D. Brinton and J. Goodwin (1996) Teaching Pronunciation, Cambridge University Press
Fromkin, V. and Rodman, R. (1993) An Introduction to Language, Harcourt Brace Jovanovich
Jones, Daniel (1931) 'ðə "wəːd" əz ə fonetik entiti' ['The "Word" as a phonetic entity'], Le Maître Phonétique, XXXVI, pp. 60–65.
Jones, Daniel (1944) 'Chronemes and Tonemes', Acta Linguistica, IV, Copenhagen, pp. 1–10.
Ladefoged, Peter (2001) Vowels and Consonants, Blackwell
Ladefoged, Peter (2006) A Course in Phonetics, Thomson
Lado, R. (1957) Linguistics across Cultures, University of Michigan Press
Lado, R. (1961) Language Testing, Longman
O'Connor, J.D. (1973) Phonetics, Penguin
O'Connor, J.D and Tooley, O. (1964) 'The perceptibility of certain word-boundaries', in Abercrombie et al. (eds) In Honour of Daniel Jones, Longman, pp. 171–6.
Pennington, M. (1996) Phonology in English Language Teaching, Longman
Pike, Kenneth (1947) Phonemics, University of Michigan Press
Roach, Peter (2009) English Phonetics and Phonology, Cambridge University Press
Swadesh, M. (1934) 'The Phonemic Principle', Language vol. 10, pp. 117–29
Trubetzkoy, N., translated by C. Baltaxe(1969) Principles of Phonology, University of California Press
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