The Purpose of Rac2 in Eosinophil Superoxide Release and Allergic

The Position of Rac2 in Eosinophil Superoxide Release and Allergic Airway Responses Andrea N. Lo, Troy Mitchell, Melanie Abel, James Dooley, Harissios Vliagoftis, David A. Williams, Marc E. Rothenberg, Gary Eitzen, Nives Zimmermann, Paige Lacy, Pulmonary Investigation Group, Department of Medication, and Department of Cell Biology, University of Alberta, Edmonton, AB, Division of Experimental Hematology and Division of Allergy and Immunology, Cincinnati Childrens Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH Background, Superoxide manufacturing from eosinophils undergoing respiratory burst correlates with asthma severity and it is considered to contribute to allergic signs and symptoms by triggering edema and tissue irritation.

Superoxide generation is dependent on activation of NADPH oxidase by a GTP bound Rho related guanosine triphosphatase, Rac1 selleck Dinaciclib or its homolog Rac2. While neutrophils express mainly Rac2, and Rac2 is the dominant protein that activates NADPH oxidase, it truly is not regarded no matter if Rac1 or Rac2 preferentially activates the oxidase in eosinophils. Our earlier research indicated that Rac2 is required for eotaxin 2 induced chemotaxis in eosinophils, demonstrating functional consequences in eosinophils. Here we deter mined whether Rac2 is actually a central regulator of mediator release and immune perform in eosinophils. Goals, To determine irrespective of whether Rac2 regulates the manufacturing of superoxide release from eosinophils and whether or not Rac2 mediates irritation and airway hyperresponsiveness.

Approaches, We isolated splenic eosinophils selleckchem MK-0752 from CD2 IL 5 transgenic mice and Rac2 deficient mice bred towards the CD2 IL 5 transgenic background and in contrast their capability to release superoxide in response to phorbol myristate acetate. To determine allergic inflammatory responses, we subjected mice to intraperitoneal sensitization with ovalbumin and alum followed by intranasal OVA challenge or intranasal sensitization to cockroach allergens and in contrast the responses of WT C57Bl 6 mice with Rac2 KO mice. Responses were determined by bronch oalveolar lavage cell counts and Penh measurements for AHR. Final results, Full spleen and MACS purified splenic eosinophils from Rac2 KO IL 5 Tg mice showed a reduction of superoxide release in comparison to WT mice. This was related to Rac2 KO neutrophils, which exhibit a deficiency in super oxide release. In each designs of airway irritation and AHR, Rac2 KO mice developed eosinophilia in BAL samples and hyperresponsiveness that was comparable to manage wild sort mice.

TNF a dependent and IL six dependent Preliminary clinical trial

TNF a dependent and IL 6 dependent. Preliminary clinical trial working with humanized anti IL 17 mAb has proven an improvement of clinical indications and symptoms of RA. It truly is nonetheless unknown, however, whether inhibi tion of IL 17 prevents joint destruction in RA. To even more verify the hypothesis, the present research was undertaken to clarify a role of IL 17 in RA working with our just lately established ex vivo human cellular model, the place rheumatoid ST derived inflammatory cells spon taneously build pannus like tissue in vitro and osteo clastic bone resorption. Elements and strategies Reagents IL 17 was bought from PeproTech. PGE1 was obtained from Sigma Aldrich. Indomethacin was obtained from Wako. Synovial tissue specimens ST specimens were obtained from individuals who fulfilled the American School of Rheumatology criteria for RA who underwent knee joint substitute.

In compliance with institutional policies, informed consent was obtained from all individuals. The examine was accepted by the ethics committee of every institution. In vitro reconstruction of inflammatory tissue by ST derived inflammatory cells ST derived inflammatory get more information cells were prepared as pre viously described. In quick, ST specimens had been minimize into smaller pieces and cultured in one hundred mm dishes consist of ing RPMI 1640 with 10% fetal calf serum and 1,000 U ml penicillin G sodium streptomycin sulfate. Immediately after one to 3 days incubation, tissue was removed and single cells were collected by vigorous pipetting. Cell suspensions were washed when, and viable cells have been collected into Lymphocyte Separation Medium.

Single suspensions of ST derived inflammatory cells were seeded at a density of five × 105 nicely in 48 properly culture plates and cultured in Dulbeccos modified Eagles medium containing 10% FCS, a hundred U ml penicillin G sodium and a hundred ug ml streptomycin sulfate. The culture was observed for morphologic alterations beneath an inverted phase contrast microscope twice every week for four weeks. reversible Aurora Kinase inhibitor When cultured in DMEM and 10% FCS within the absence or presence of IL 17 or indomethacin, ST derived inflammatory cells begun to aggregate, forming foci within a couple of days. Additional cul turing resulted in three dimensional development, which in the long run created macroscopic tissue 2 mm in size inside of four weeks. Morphologic changes had been semiquantita tively scored on a scale of 0 to 4, in accordance on the degree of tissue improvement, where 0 was no cellular foci or aggregations, one was the formation of cellular foci or aggregation, two was even further growth of cellular aggrega tions, three was even more three D growth which has a multilayered construction, and four was the growth of macroscopic tis sue. Cumulative tissue growth score was calculated through the total sum of your tissue growth scores obtained twice weekly for 4 weeks of culture.

Neutralization of TGF B in conditioned medium from human metast

Neutralization of TGF B in conditioned medium from human metastatic MDA MB 231 breast cancer cells permitted the di?erentiation BGB324 of osteoblasts in culture, suggesting that TGF B negatively a?ects osteoblasts while advertising growth from the metastatic cells. In the presence of cancer cells, osteoblasts boost expression of pro in?ammatory cytokines this kind of as IL 6, monocyte chemotactic protein one, macrophage in?ammatory protein two, keratinocyte chemoattractant and VEGF. These molecules not merely enable support tumor cells, but additionally are osteoclastogenic. When BGB324 the bone reduction is comprehensive, the osteoblasts are absent from your lesion. Thus, in the program on the osteolytic procedure, the osteo blasts are not able to ful?ll their position as bone setting up cells.

selleck Breast cancer is usually compared with prostate cancer, which BKM120 metastasizes for the skeleton selleck inhibitor by using a equivalent frequency. In contrast to breast cancer, prostate bone metastasis typically results in osteoblastic lesions. While the outcome is predominantly osteoblastic, it is actually known that prostate cancer lesions display each blastic and lytic traits early while in the method. There may be evidence that osteoblastic metastases form at web-sites of osteolytic lesions, suggesting an general boost of bone remodeling Accelerated osteoblastogenesis is often stimulated by aspects secreted by prostate cancer cells, such as endothelin one, TGF B, and ?broblast development aspect. These molecules lead to osteoblasts not only to form new bone but additionally to release RANKL together with other osteoclastic mediators.

Whilst the mechanisms of osteoteoblastic and osteolytic responses will not be absolutely understood, it truly is clear that lots of BKM120 elements concerned in osteolytic breast cancer bone metastasis also regulate the osteolytic aspects of prostate cancer. Akech and colleagues just lately reported that Runx2 is made through the very metastatic prostate cancer cell Computer three, and positively correlates on the severity of osteolytic sickness. There may be also evidence that molecules in conditioned medium from Pc three cells alone, or from the two Computer 3 cells and MC3T3 E1 osteoblasts, market osteoclastogenesis. Although a lot of the development variables developed by breast and prostate cancers may be di?erent, ultimately they engage the bone re modeling process. The bone remodeling microenvironment can be a complicated technique during which the cell functions are managed by multifunctional transcription aspects, cytokines and growth variables. The dynamics of this procedure are inter rupted when metastatic breast cancer cells are intro duced, including yet another layer of lively molecules for the bone surroundings.

These concentrations are achievable in patients and also have pre

These concentrations are achievable in sufferers and also have previously been shown to inhibit MAP kinase signalling. To verify this observation, we also discovered that low doses of Iressa inhibited signalling with the MAP kinase pathway. To ascer tain irrespective of whether this sensitivity was inherent to other BLBC cell lines we repeated the same experiment in HCC1937 cells, and relatively surprisingly these cells had been still in a position to kind colonies in anchorage independent situations while in the pres ence of as much as 2M Iressa. Similarly, the MDA MB 468 basal like breast cancer cells are insensitive to Iressa at first but might be sensitized by targeting PI3 kinase with LY294002, an observation that we independently confirmed. Within a separate review, LY294002 has been proven to inhibit phosphorylation of YB 1.

full report This can be in preserving with our former scientific studies demonstrating that YB 1 is phosphorylated by Akt in response to PI3 kinase activation. We for that reason questioned regardless of whether knocking down YB 1 in HCC1937 cells before treating with Iressa can be helpful at decreasing the capacity of these cells to increase in soft agar. The suppression of YB one alone induced a 42% reduction while in the amount of colonies compared with handle, but there was even more significant decreases in colony amount with all the addition of as very little as 0. 25M Iressa. Consequently, our scientific studies indicate that despite the fact that some BLBC cells may be delicate to Iressa, for other people the inhibition of YB 1 can be required to sensitize the cells to drug. We have been rather amazed the SUM149 cells have been so sen sitive to the drug.

the full report An clear explanation can be that these cells express activating mutations in EGFR that will make them delicate to Iressa, as has been described for lung can cer. We consequently sequenced EGFR but unexpectedly did not obtain such mutations. All 28 exons coding for this gene have been amplified by PCR and sequenced. Activating mutations such as L858R or delL747 P753insS which have previously been reported to get linked with Iressa sensitivity weren’t located. Having said that, we did recognize five single nucleotide poly morphisms in exons 12, 13, 15 and 20.There was 1 homozygous non translated SNP, three heterozygous synonymous SNPs, and 1 heter ozygous non synonymous SNP .These dbSNPs are previously recognized for EGFR, even though their functional significance just isn’t nonetheless identified. The SNP of most interest is R521K, situated on exon 13, because it results in an amino acid modify positioned inside the extracellular domain of the receptor. We concluded that irrespective of activating mutations in EGFR, Iressa inhibits the development of basal like breast cancer cells.

Cytoplasmic staining of variable intensity was observed in the

Cytoplasmic staining of variable intensity was observed while in the tumors and 14% lacked IGFBP seven staining, 20% had minimal staining, 32% intermediate staining and 34% sturdy staining. Reduced IGFB 7 was related with substantial cyclin E expression, retinoblastoma protein inactivation, low bcl 2 and poorly differentiated tumors. There was more a substantially impaired prognosis for individuals with minimal IGFB seven protein tumors. Interestingly, IGFB 7 was strongly and inversely associated with proliferation in estrogen recep tor adverse tumors, suggesting a crucial cell cycle regulatory function for IGFBP seven separate in the interac tion with all the estrogen receptor pathway. Growth of acquired resistance towards antiestrogen therapy can be a significant problem in human breast cancer, and expertise of alterations resulting in resistance is important for collection of additional remedy.

To mimic the clinical scenario we’ve established a series of MCF seven human breast cancer cell lines by long-term remedy using the antiestrogens tamoxifen, ICI 164,384, and ICI 182,780. Prevalent for these cell lines is often a decreased expression inhibitor Amuvatinib from the estrogen receptor . In human breast cancer, lack of response to endocrine treatment is often linked with decreased expression of your estrogen receptor and increased expression of epider mal growth issue receptor and or HER 2 neu. Our antiestrogen resistant cell lines didn’t express altered levels of EGFR, HER two neu, ErbB 3 and ErbB four. Estrogen and antiestrogen regulation of HER 2 neu expression was in essence similar in parent and resistant MCF 7 cells.

Treatment with antibodies to HER two neu didn’t have an impact on development PTC124 structure of MCF seven cells or resistant cells, indicating that within this in vitro model technique, acquired antiestrogen resistance isn’t going to emerge from activation from the HER two neu signalling pathway. Nonetheless, addition of heregulin1 ?one abolished the inhibitory exercise of ICI 182,780 on MCF seven cells, demonstrating that activation with the HER 2 neu receptor signalling pathway can override the development inhibitory impact of ICI 182,780. The effect of heregulin1 ?one may very well be abrogated by Herceptin. It has been advised in various studies of breast cancer that overexpression of the development component receptor erbB2 is related with much less advantage from certain adjuvant remedies. The mechanisms are usually not totally understood. The erbB2 recep tor activates quite a few signal pathways which includes the phos phatidyl inositol three kinase Akt pathway, which is implicated in cell survival. This pathway has proven to get a target with the tumor suppressor PTEN.

Eighteen of these compounds are actually authorized by the US Foo

Eighteen of those compounds happen to be authorized from the US Food and Drug Administration, which include five for breast cancer. Phase I clinical trials are ongoing for 7 compounds, phase II trials are underway for 7 com pounds, such as 6 for breast cancer, and one com pound is presently currently being tested in a phase III trial. Hence even further validation of signatures may very well be achievable while in the close to long term. Robust predictors of drug response are observed whatsoever amounts of the genome With 7 data varieties accessible on the single set of samples, we were properly positioned to assess regardless of whether distinct tech nologies or molecular information sorts continually out perform other individuals while in the prediction of drug sensitivity. To acquire a ranking of your significance from the molecular datasets, we in contrast prediction effectiveness of classifiers constructed on in dividual information sets and their blend for 29 common cell lines.

Importantly, no single information sort performed effectively for all com pounds, with each and every data kind carrying out finest for some com pounds. Table S6a,c in Extra file three exhibits the ranking in the datasets accord ing towards the independent classifiers obtained with LS SVM and RF, respectively. For that LS SVM classifiers, RNAseq performed most effective for 22 compounds, exon array for selleck chemical twenty compounds, SNP6 for 18, U133A for 17 and methylation information for 12 compounds. Related benefits had been confirmed with all the RF technique. Despite the fact that it had varying overall performance for individual compounds, on the whole, RNAseq appreciably outperformed all other data forms across the comprehensive panel of 90 compounds.

SNP6 copy number data resulted in drastically worse predictive energy compared to all other data forms. Moreover, exon array outperformed U133A, with a P worth of 0. 0002. In Table S6b,d in Supplemental file 3, a distinction is SRT1720 Sirtuin inhibitor manufactured in between two groups of compounds, compounds for which all datasets execute similarly effectively versus compounds for which success with one particular dataset are much better than obtained with any on the other datasets, defined as an AUC improve of no less than 0. 1. One example is, exon array worked most effective for VX 680, RNAseq for carbopla tin, and RPPA for bortezomib. Data sort specificity was generally not linked to therapeutic compound class, while there have been some exceptions for LS SVM with RNAseq doing effectively for polyamine an alogs and mitotic inhibitors, SNP6 for ERBB2 epidermal growth element receptor inhibitors, and methylation for CDK1 inhibitors. The full mixture of genome wide datasets yielded a larger AUC worth compared to the ideal performing person dataset for only a limited amount of compounds. The complete combin ation signatures, even so, generally ranked closely to your ideal signatures based mostly on individual information forms.

This signaling module presents a direct feed into the Raf Mek Erk

This signaling module gives a direct feed in to the Raf Mek Erk pathway, suggesting that these cell lines have an choice route accessible for Erk activation. This interaction might aid to describe why these basal cell lines are specifically aggressive. Pak1 plays a pivotal role inside the network versions In our model, Pak1 is required to the activation of Mek and Erk. Particularly, Pak1 phosphorylates Mek, which in turn facilitates signaling along the Raf Mek Erk cascade. It follows, then, that network versions with Pak1 omitted through the initial state fail to activate Erk. Throughout the cell lines, the distribution of Pak1 transcript levels is extremely skewed, so our discretization algorithm yields two clusters, a substantial group centered at 0. 26, and also a tiny group centered at two. sixteen.

Pak1 is existing during the initial state of your cell lines with higher expression and absent from your other folks. The four cell lines with substantial Pak1 transcript amounts, MDAMB134, 600MPE, SUM52PE and SUM44PE, are all of luminal origin. Based selleck chemical CX-4945 over the observations that Pak1 right regulates MAPK signaling, and that its expression pattern shows significant variation in breast cancers, we hypothesized that Pak1 vary entially regulates MAPK signaling across our panel of cell lines. We tested this hypothesis experimentally. The 1st situation we addressed was whether Pak1 protein ranges differ across the cell lines. We observed highly variable expression of complete Pak1 protein. Exclusively, three with the four cell lines with elevated Pak1 transcript amounts have concordantly higher Pak1 protein levels.

Moreover, a handful of other cell lines also show above expression of Pak1 protein. Pak1 transcript and protein ranges are considerably correlated. Even though this romance is largely dependent on the cell lines that hugely express selleckchem Pak1, it none theless supports the idea that elevated transcript ranges have an impact on protein expression amounts. Focal adjustments in copy quantity are thought to convey a selective advantage for tumor development, so we following asked no matter whether Pak1 is amplified in any of our cell lines. The four cell lines that more than express Pak1 display higher level amplification of your Pak1 amplicon, none of your other cell lines show this amplification. Also to Pak1 amplification, 3 of those cell lines also show amplifi cation at CCND1, although in all instances you will find distinct peaks at every single locus. If Pak1 without a doubt regulates MAPK signaling, we’d count on to locate a correlation in between Pak1 and phospho Mek ranges. To address this, we quantified isoform particular phospho Mek ranges in our cell lines. We identified a small but considerable correlation concerning complete Pak1 and per cent Mek1 S298.

For cultures of renal cortical tissue, a piece of cortical tissue

For cultures of renal cortical tissue, a piece of cortical tissue was weighed and minced extensively with a razor blade. Glomeruli or cortical tissues have been suspended in DMEM supplemented with 0. one U mL insulin, one hundred U mL penicillin and 100 ug mL strepto mycin at a density of 2000 glomeruli mL and 10 mg mL, respectively. Immediately after 48 h incubation at 37 C 5% CO2, super natants have been harvested and stored at ?twenty C till further analysis. TGF B1 content of culture supernatant was mea sured following acid activation, utilizing a commercially available enzyme linked immunosorbent assay kit according for the manufac turers instructions. TIMP 1 ranges were analyzed utilizing another commercially offered ELISA kit. Fibronectin was measured with a modified aggressive ELISA, in accordance to published techniques.

Three samples from each and every rat were analyzed. Quantitation of tubulointerstitial mRNA expression Cortical total RNA was extracted with Trizol reagent according for the manu facturers instructions. The mRNA expression was deter mined by a two step reverse transcription polymerase chain response. A cDNA copy was created with reverse a knockout post transcriptase from RNA PCR Core kit. True time PCR was carried out applying the LightCycler Program and SYBR Green I as dsDNA binding dye. The next primer pairs had been made use of, PDGF A. Our data were not usually distributed. Statistical analysis between groups was performed by Kruskal Wallis and subsequent Mann Whitney U testing. A p value decrease than 0. 05 was regarded major.

Success Entire body fat, food and drug intake On the finish of your experiment, animals imply body weights had been 601 68 g during the two K Handle, 544 34 g in the 1 K Management, 523 forty g inside the cGS and 497 thirty g inside the cGS Imatinib Amuvatinib 850879-09-3 group, respectively. Suggest food and water intakes did not considerably vary among the groups through out the experiment. Proteinuria, blood stress and renal function Just before the commence of treatment, nephritic animals had been strati fied to start with equal amounts of pre remedy proteinuria inside the two diseased groups. Urinary protein reduction enhanced steadily in untreated diseased ani mal groups through the experiment. Administration of Imatinib slowed the deterioration of urinary protein excretion. In week twenty, proteinuria was substantially reduced within the Imatinib treated animals. As proven in Figure 1B, systolic blood pressure was in creased slightly throughout the disease progression while in the anti thy1 induced chronic glomerulosclerosis model. In week 20, treatment method with Imatinib lowered systolic blood stress substantially. As proven in Table one, animals with chronic anti thy1 glomerulosclerosis showed important increases in blood creatinine and urea concentrations and decrease in creatinine clearances, indicating chronic renal insufficiency.

Around 1 ug of complete RNA from every single sample was synthesi

Somewhere around 1 ug of total RNA from every single sample was synthesized into cDNA following the manu facturers guidelines making use of the Transcriptor Very first Strand cDNA Synthesis Kit obtained from Roche Diagnostics. We made use of the following sequences for your forward and reverse primers, Reverse transcription polymerase chain response was set up applying the following PCR cycle, 95 C for five min, × 35 cycles, and 72 C for 10 min. PCR merchandise have been resolved on the 1. 6% agarose gel, visualized, and photograph graphed below UV light. Western blot examination U251 and 5310 cells were transfected with SV sh, M sh, U sh, M fl and U fl for 72 hrs. Cells were collected and lysed in RIPA buffer containing 1 mM sodium orthovanadate, 0. 5 mM PMSF, ten ug mL aprotinin, 10 ug mL leupeptin and resolved via SDS Page.

Right after overnight transfer onto nitrocellulose membranes, blots have been blocked with 5% non unwanted fat dry milk in PBS and 0. 1% Tween 20. Blots had been then incubated with primary antibody, followed by incuba tion with HRP conjugated secondary antibody. Immunore active bands had been visualized employing chemiluminescence selleckchem ECL Western blotting detection reagents on Hyperfilm MP autoradiography film obtained from Amersham. GAPDH antibody was used to confirm that very similar quantities of protein were loaded in all lanes. FACS examination U251 and 5310 cells have been seeded on 100 mm tissue cul ture plates. Cells had been transfected with M fl, transfected with M fl and blocked with 9B1 antibody, taken care of with recombinant uPAR or taken care of with recombinant uPAR and blocked with 9B1 antibody.

48 72 kinase inhibitor Semagacestat hrs after transfec tion or one 2 hrs just after recombinant uPAR therapy, cells have been handled with 50 mM EDTA, washed with PBS, pelleted at 1000 rpm for 5 min, and re suspended in PBS in an appendorff tube at a concentration of one × 106 cells mL. Cells were then incubated with HRP conjugated iNOS antibody for 1 hr on ice, pelleted, and washed 3 times with PBS to eliminate excess primary antibody. Cells were then re suspended in one ml of PBS and incubated with Alexa Fluor 594 fluorescent labeled secondary antibody for one hr on ice. Following three additional washes in PBS, cell pellet was re suspended in 10% buffered formalin and analyzed on a Coulter EPICS XL AB6064 movement cytometer. Detection of NO in 5310 glioma cells DAF 2DA is really a non fluorescent cell permeable reagent which will measure free NO in living cells. Once within the cell, the diacetate groups of your DAF 2DA reagent are hydro lyzed by cytosolic esterases, therefore releasing DAF 2 and se questering the reagent within the cell. Production of NO while in the cell, if any, converts the non fluorescent dye, DAF two, to its fluorescent triazole derivative, DAF two T.

EGFR expression is shown for being regulated by lots of component

EGFR expression is shown to become regulated by many things that regulate growth and proliferation. In breast cancer, EGFR and ErbB2 expression was observed to be below management from the Y box transcription translation factor YB1 which is phosphorylated by Akt. Even so, YB1 continues to be shown to regulate each EGFR and ErbB2 expression. As we did not observe upregula tion of ErbB2 in our flotillin one knockdown cells, YB1 just isn’t incredibly more likely to be the reason behind EGFR upregulation upon flotillin 1 knockdown. Interestingly, earlier scientific studies have advised that ele vated flotillin 2 expression in gastric cancers correlates with ErbB2 levels, and flotillins are required to stabilize ErbB2 from the plasma membrane in SKBR3 breast cancer cells.

Depletion of either in the flotillin proteins resulted in enhanced endocytosis and degradation of ErbB2 in these details these cells, implicating that flotillins regulate ErbB2 trafficking. Furthermore, flotillins were observed to type complexes with ErbB2, which also contained the heat shock protein Hsp90. Even so, this seems to not be the situation in MCF7 cells by which the amount of ErbB2 was not altered upon flotillin depletion. So, it’s evident that flotillins exhibit various results on receptor trafficking and signaling in breast cancer cells of various origin. That is not surprising, thinking about the cell lines utilized are different regarding their genetic back ground and oncogenic mutations which can be current in these cells. Such as, according for the Sanger institute COS MIC database, MCF7 cells exhibit a mutation from the catalytic subunit of PI3K, whereas SKBR3 cells possess a WT PI3K.

Nonetheless, each cell lines express non mutated EGFR and Ras proteins. Yet another factor that may affect the results obtained in a variety of studies would be the indicates of knocking down flotil lin expression. For erismodegib msds instance, Lin et al. described that flotillin one knockdown in MCF7 cells minimizes cell viability and impairs tumorigenicity in MCF7 cells. In contrast to these information, we right here observed elevated MAPK signaling and greater cyclin D mRNA expression on flotillin 1 ablation. Additionally, Lin et al. detected a decreased AKT phosphorylation and concomitant upregulation of the forkhead transcription component Foxo3 and that is associ ated with decreased cell viability due to upregulation of apoptotic genes. Though Foxo3 expression was in creased in our flotillin one knockdown cells, we did not observe any evident impairment of AKT activation, in contrast to Lin et al.