High Resolution Melt Analysis (HRMA); a Viable Alternative to Agarose Gel Electrophoresis for Mouse Genotyping

Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort.This work was supported by NHMRC (http://www.nhmrc.gov.au) grants 366476 and 1003648 (to R.M.A.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Cortactin is necessary for E-cadherin–mediated contact formation and actin reorganization

Classical cadherin adhesion molecules are key determinants of cell–cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. We now report that cortactin is recruited to cell–cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin–actin cooperation that supports productive contact formation

Cortactin is necessary for E-cadherin–mediated contact formation and actin reorganization

Classical cadherin adhesion molecules are key determinants of cell–cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. We now report that cortactin is recruited to cell–cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin–actin cooperation that supports productive contact formation

Rac is a dominant regulator of cadherin-directed actin assembly that is activated by adhesive ligation independently of Tiam1

Classic cadherins function as adhesion-activated cell signaling receptors. On adhesive ligation, cadherins induce signaling cascades leading to actin cytoskeletal reorganization that is imperative for cadherin function. In particular, cadherin ligation activates actin assembly by the actin-related protein (Arp) 2/3 complex, a process that critically affects the ability of cells to form and extend cadherin-based contacts. However, the signaling pathway(s) that activate Arp2/3 downstream of cadherin adhesion remain poorly understood. In this report we focused on the Rho family GTPases Rac and Cdc42, which can signal to Arp2/3. We found that homophilic engagement of E-cadherin simultaneously activates both Rac1 and Cdc42. However, by comparing the impact of dominant-negative Rac1 and Cdc42 mutants, we show that Rac1 is the dominant regulator of cadherin-directed actin assembly and homophilic contact formation. To pursue upstream elements of the Rac1 signaling pathway, we focused on the potential contribution of Tiam1 to cadherin-activated Rac signaling. We found that Tiam1 or the closely-related Tiam2/STEF1 was recruited to cell-cell contacts in an E-cadherin-dependent fashion. Moreover, a dominant-negative Tiam1 mutant perturbed cell spreading on cadherin-coated substrata. However, disruption of Tiam1 activity with dominant-negative mutants or RNA interference did not affect the ability of E-cadherin ligation to activate Rac1. We conclude that Rac1 critically influences cadherin-directed actin assembly as part of a signaling pathway independent of Tiam1

High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping.

Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort

Assay conversion.

<p>Amplicon sizes are approximate. Assays were performed in the HRMA reaction conditions and examined by gel electrophoresis and scored as follows: +++; both amplicons produced in equivalent amounts, +; both amplicons produced but one is favoured, −; one or both of the amplicons is not produced. Assays that did not score +++ were optimized with altered primer ratios and again assessed by electrophoresis. NT: not tested, NMP: Normalized melting peak, FDC: Fluorescence difference curve.</p

Small changes in fragment length or sequence are sensitively detected by HRMA.

<p>(A) Agarose gel electrophoretic analysis of products derived from amplification of SSLP marker microsatellite D7Mit69 from 5 inbred strains of mice. The predicted allele sizes are: FVB; 230 bp, BL/6 and BALB/b; 236 bp, C3H and 129; 238 bp. (B) High Resolution Melt Analysis of the same products showing that the three allele sizes are readily discriminated using either Shifted melting curve or Difference curve analysis. Blue: FVB, Red: C3H and 129, Grey: BALB/b and BL/6. SM: 1 Kb+ DNA ladder, NTC: No template control.</p

Analisis perhitungan harga pokok produksi menggunakan metode job order costing sebagai dasar penentuan harga jual produk: Studi kasus pada PT. Larasati Multi Sentosa Pasuruan periode januari 2018

INDONESIA : Dalam perusahaan manufaktur, siklus akuntansi biaya diawali dengan pencatatan harga pokok produksi. Harga Pokok Produksi sangat penting bagi perusahaan untuk menetapkan harga jual dan menentukan laba. Penelitian ini bertujuan untuk mengetahui perhitungan harga pokok produksi produk Decking pada PT. Larasati Multi Sentosa dengan menggunakan metode job order costing. Penelitian ini menggunakan metode kualitatif dengan jenis pendekatan deskriptif. Penelitian ini dilakukan dengan empat tahapan yaitu, (1) pengumpulan data dilakukan dengan wawancara, observasi, dan dokumentasi , (2) reduksi data, (3) penyajian data, (4) analisis data dan penarikan kesimpulan. Hasil penelitian menunjukkan bahwa terdapat perbedaan perhitungan harga pokok produksi perusahaan dengan metode job order costing. Pada perhitungan menurut perusahaan harga pokok produksinya sebesar Rp. 5.961.133.750 , sedangkan harga pokok produksi berdasarkan metode job order costing adalah Rp. 1.548.477.000. Selisih disebabkan karena harga pokok produksi perusahaan menggunakan perhitungan angka estimasi serta tidak memasukkan biaya depresiasi gedung dan juga mesin ke dalam perhitungan harga pokok produksinya. Untuk laba yang diinginkan karena perusahaan tidak menetapkan target labanya, dalam penentuan harga jual dengan asumsi laba 30% juga mendapatkan hasil yang sama, yaitu terlalu rendah dari harga jual menurut perusahaan. ENGLISH : In manufacturing companies, the cost accounting cycle is began with the cost of production. Cost of Production is very important for the company to set the selling price and determine the profit. The research aims at determining the calculation of cost of production of Decking products at PT. Larasati Multi Sentosa by using job order costing method. The research used qualitative method with descriptive approach type. The research was conducted with four stages: (1) data collection was done by interview, observation, and documentation, (2) data reduction, (3) data presentation, (4) data analysis and drawing conclusion. The research results showed that there are differences in the calculation of the cost of production of the company with job order costing method. In the calculation according to the company, the cost of production was Rp. 5.961.133.750, while the cost of production based on job order costing method was Rp. 1,548,477,000. The difference was caused by the cost of production of the company used the calculation of estimation numbers and did not include the cost of depreciation of the building and the machine into the calculation of the cost of production. For the desired profit due to the company did not set the profit target, in determining the selling price with the assumption of 30% profit also got the same result, it was lower than the selling price according to the company

A murine Zic3 transcript with a premature termination codon evades nonsense-mediated decay during axis formation

SUMMARY The ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-containing transcripts might be targeted for nonsense-mediated decay (NMD). NMD efficiency is known to vary greatly between transcripts, tissues and individuals and it is possible that differences in survival of PTC-containing transcripts partially explain the striking phenotypic variability that characterizes ZIC3-associated congenital defects. For example, the PTC-containing transcripts might encode a C-terminally truncated protein that retains partial function or that dominantly interferes with other ZIC family members. Here we describe the katun (Ka) mouse mutant, which harbours a mutation in the Zic3 gene that results in a PTC. At the time of axis formation there is no discernible decrease in this PTC-containing transcript in vivo, indicating that the mammalian Zic3 transcript is relatively insensitive to NMD, prompting the need to re-examine the molecular function of the truncated proteins predicted from human studies and to determine whether the N-terminal portion of ZIC3 possesses dominant-negative capabilities. A combination of in vitro studies and analysis of the Ka phenotype indicate that it is a null allele of Zic3 and that the N-terminal portion of ZIC3 does not encode a dominant-negative molecule. Heterotaxy in patients with PTC-containing ZIC3 transcripts probably arises due to loss of ZIC3 function alone