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We estimate the effects of employer tenure on wages using the instrumental variable (IV) methodology by analyzing data from the Japan Household Panel Survey and Keio Household Panel Survey from 2004 to 2014. We find that the estimates based on the IV method, which corrects for omitted variable bias due to individuals’ unobserved abilities and unobserved matching qualities, are significantly lower than those found using the ordinary least squares methodology. These results are robust across subsamples and specifications and cannot be rejected using an alternative estimation method. Our findings suggest that the recent returns on employer tenure in the Japanese labor market are likely to be lower than those discussed in previous studies.

Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.

The litter size of mouse strains is determined by the number of oocytes naturally ovulated. Many attempts have been made to increase litter sizes by conventional superovulation regimens (e.g., using equine or human gonadotropins, eCG/hCG but had limited success because of unexpected decreases in the numbers of embryos surviving to term. Here, we examined whether rat-derived anti-inhibin monoclonal antibodies (AIMAs) could be used for this purpose. When C57BL/6 female mice were treated with an AIMA and mated, the number of healthy offspring per mouse increased by 1.4-fold (11.9 vs. 8.6 in controls). By contrast, treatment with eCG/hCG or anti-inhibin serum resulted in fewer offspring than in nontreated controls. The overall efficiency of production based on all females treated (including nonpregnant ones) was improved 2.4 times with AIMA compared with nontreated controls. The AIMA treatment was also effective in ICR mice, increasing the litter size from 15.3 to 21.2 pups. We then applied this technique to an in vivo genome-editing method (improved genome-editing via oviductal nucleic acid delivery, i-GONAD) to produce C57BL/6 mice deficient for tyrosinase. The mean litter size following i-GONAD increased from 4.8 to 7.3 after the AIMA treatment and genetic modifications were confirmed in 80/88 (91%) of the offspring. Thus, AIMA treatment is a promising method for increasing the litter size of mice and may be applied for the easy proliferation of mouse colonies as well as in vivo genetic manipulation, especially when the mouse strains are sensitive to handling. Summary Sentence Treatment of female mice with an anti-inhibin monoclonal antibody increased the litter size after natural mating and could be applied to an in vivo genome-editing method (improved genome-editing via oviductal nucleic acid delivery) for the efficient generation of knockout mice.

This study investigates microbial growth, community composition, and histamine production in freshly caught mackerel stored at 4°C and 10°C. Fish were obtained directly from fishermen and immediately placed in low-temperature storage to minimize external contamination. Microbial activity was assessed using total viable count, amplicon sequencing, and HPLC analysis. The total bacterial count exceeded 5 log CFU/mL after 5 days at 4°C and 2 days at 10°C, indicating the presence of histamine-producing bacteria (HPB), particularly under psychrophilic conditions. Photobacterium was identified as the dominant bacterial genus at both temperatures, suggesting its role as a key psychrophilic HPB. Histamine levels increased progressively, reaching 5.11 ± 2.43 ppm at 4°C and 11.18 ± 4.67 ppm at 10°C, while histidine content declined. Despite cold storage and strict contamination control, naturally occurring bacteria in fish continued to grow and produce histamine. These findings highlight the importance of temperature control in preventing histamine accumulation and provide new insights into microbial dynamics and food safety risks in fresh mackerel.

The purpose of this study was to investigate the abundance and distribution of psychrophilic microorganisms associated with spoilage in beef slaughterhouse environments after cleaning. The processing lines and equipment used in slaughtering and boning were swabbed, and the microbial count was determined using a TSA and MRS medium and Chromocult ® Coliform agar incubated at 15ºC and 37ºC, respectively. As a result, the brisket saw (handle side) and trolley hook were the most heavily contaminated with microorganisms, with each having a microbial adhesion rate of 66.7%. The microbial adhesion rates of the apron and milling cutter (edge side) were 50%, respectively, and those of the foot cutter (edge and handle side), splitting saw (edge side), and knife (handle side) were 33.3%, respectively. Next, four colonies were randomly isolated from the petri dish used for the bacterial count measurement to identify the predominant microbial species of the microorganisms attached to each equipment. As a result of Sanger sequencing analysis, yeasts such as Candida zeylanoides and Rhodotorula sp. and bacteria including Pseudomonas sp. and Rhodococcus sp. were identified from the equipment used in the slaughtering line, and it was assumed that these microorganisms were of environmental origin. In contrast, only Pseudomonas sp. and Candida zeylanoides were isolated from the boning line. Despite the use of cleaning operations, this study identified some equipment was contaminated with microorganisms. Since this equipment frequently comes into direct contact with the carcass, it is critical to thoroughly remove the microorganisms through accurate cleaning to prevent the spread of microbial contamination on the carcasses.

Although essential oils exhibit antimicrobial properties, their application is limited, owing to their strong volatility and poor water solubility. Emulsification is a valid strategy for improving chemical stability. In this study, we prepared a mustard oil (MO) emulsion with egg yolk lecithin and evaluated its antimicrobial activity against Listeria monocytogenes in vitro and in cheese curd. The particle size of the MO emulsion was approximately 0.19μm and remained stable for 30 days of storage. The MO emulsion showed strong antimicrobial activity against L. monocytogenes in vitro. Moreover, 40 ppm of MO was sufficient to inhibit the growth of L. monocytogenes in culture, and the addition of 160 ppm of MO decreased the population of L. monocytogenes. When 50 ppm of emulsified MO was added to milk during cheese curd production and it was stored at 10°C for 10 days, the growth of L. monocytogenes was suppressed. When the cheese curd with MO emulsion was stored at 4°C, the bacterial count was significantly decreased (P < 0.05), and no bacterial growth was observed after 14 days of storage. Furthermore, the sensory characteristics of cheese curd with the MO emulsion were acceptable. These results indicate MO emulsions may be useful in controlling the growth of L. monocytogenes in fresh cheese. •MO emulsion (40 ppm) was sufficient to inhibit L. monocytogenes growth in vitro.•L. monocytogenes did not grow well in cheese curd made by adding MO emulsion to milk.•MO emulsion did not have an effect on the sensory characteristics of fresh cheese.

When harmful bacteria are detected in the final product at a food manufacturing plant, it is necessary to identify and eliminate the source of contamination so that it does not occur again. In the current study, the source of contamination was tracked using core genome multilocus sequence typing (cgMLST) analysis in cases where Escherichia coli was detected in the final product at a food manufacturing plant. cgMLST analysis was performed on 40 strains of E . coli collected from the environment [floor (26 strains), drainage ditch (5 strains), container (4 strains), post-heating production line (1 strain)] and products [final product (3 strains) and intermediate product (1 strain)]. In total, 40 E . coli isolates were classified into 17 genogroups by cgMLST analysis. The 4 E . coli strains isolated from the intermediate and final products were classified into two genogroups (I and II). Certain isolates collected from the environment also belonged to those genogroups, it was possible to estimate the transmission of E . coli in the manufacturing plant. Thus, the dynamics of E . coli in the food manufacturing location were clarified by using cgMLST analysis. In conclusion, our results indicate that cgMLST analysis can be effectively used for hygiene management at food manufacturing locations.

A bstract We evaluate the Λ-parameter in the M S ¯ scheme for the pure SU(3) gauge theory with the twisted gradient flow (TGF) method. A running coupling constant g TGF 2 (1/L) is defined in a finite volume box with size of L 4 with the twisted boundary condition. This defines the TGF scheme. Using the step scaling method for the TGF coupling with lattice simulations, we can evaluate the Λ-parameter non-perturbatively in the TGF scheme. In this paper we determine the dimensionless ratios, Λ T G F / σ and r 0 Λ TGF together with the Λ-parameter ratio Λ SF /Λ TGF on the lattices numerically. Combined with the known ratio Λ M S ¯ / Λ S F , we obtain Λ M S ¯ / σ = 0.5315 81 − 48 + 269 and r 0 Λ M S ¯ = 0.6062 92 − 52 + 309 , where the first error is statistical one and the second is our estimate of systematic uncertainty.

Background Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called “pronuclear injection-based targeted transgenesis” (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT ( i -PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre -mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre- LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. Results To enable the i -PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. Conclusions Simultaneous use of PhiC31 and FLP in i -PITT approach allows insertion of donor plasmids containing Cre- loxP -based conditional expression cassettes.

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1.sup.H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1.sup.H46R ;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.