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In this study, we investigated the brittle fracture behavior of Sn-3.0Ag-0.5Cu (SAC305) solder joints with a Direct Electroless Gold (DEG) surface finish, formed using laser-assisted bonding (LAB) and mass reflow (MR) techniques. Commercial SAC305 solder balls were used to ensure consistency. LAB increases void fractions and coarsens the primary β-Sn phase with higher laser power, resulting in a larger eutectic network area fraction. In contrast, MR produces solder joints with minimal voids and a thicker intermetallic compound (IMC) layer. LAB-formed joints exhibit higher high-speed shear strength and lower brittle fracture rates compared to MR. The key factor in the reduced brittle fracture in LAB joints is the thinner IMC layer at the joint interface. This study highlights the potential of LAB in enhancing the mechanical reliability of solder joints in advanced electronic packaging applications.

Background Livestock, particularly cattle, are crucial for biotechnology fields, such as genetic breeding, infectious diseases, bioreactors, and specific disease models. However, genetic engineering in cattle has lagged due to long gestation periods, single embryo pregnancies, and high rearing costs. Additionally, the slow validation of germline transmission and the absence of germline-competent embryonic stem cells hinder progress. With the development of genome editing technologies like ZFN , TALEN , and CRISPR-Cas9 , recent advancements have shown that Cas9 -expressing pigs and chickens have been successfully produced. We hypothesize that generating CRISPR/Cas9 -expressing cattle and their resources will provide a powerful resource for bovine genome editing, advancing our understanding of bovine genetics and disease resistance. Results In this study, two types of Cas9 -expressing cattle were successfully produced: Cas9 - RFP -fatty acid dehydrogenase I ( FatI ), Cas9 - GFP -sgRNA for the prion protein (sg PRNP ). Somatic cells from these cattle were induced to mutate multiple target genes when single-guide RNAs (sgRNAs) were transfected into the somatic cells. Additionally, semen from Cas9 expressing male cattle was frozen and used to fertilize wild-type oocytes, successfully transmitting the transgene ( Cas9 , reporter genes, FatI ), and sg PRNP ) to the next generation. Furthermore, the gene editing capabilities of Cas9 , including knockout and high-efficiency knock-in, were confirmed in embryos derived from F1 semen through in vitro production. Conclusion These data demonstrate, for the first time, that Cas9 -expressing cattle were successfully born, and this transgene was transmitted to the next-generation calves (F1) and F2 embryos. In addition, somatic and germ cells derived from F0 and F1generations were used to evaluate the potential for gene editing (knockout and knock-in) in multiple genes. PRNP -mutated F1 cattle are currently being raised as a resistance model for bovine spongiform encephalopathy. These transgenic bovine models and their derivatives will serve as a valuable resource for both in vitro and in vivo genome editing, advancing our genetic understanding of bovine genomics and diseases.

In this study, an interconnection was formed between a Cu/SnAg pillar bump and an Ni-less surface-treated Cu pad through laser-assisted bonding (LAB), and its bonding characteristics were evaluated. The LAB process influences the bond quality and mechanical strength based on the laser irradiation time and laser power density. The growth of the intermetallic compound (IMC) in the joint cross-section was observed via FE-SEM analysis. Under optimized LAB conditions, minimal IMC growth and high bonding strength were achieved compared to conventional thermo-compression bonding (TCB) and mass reflow (MR) processes. As the laser irradiation time and laser power density increased, solder splashing was observed at bump temperatures above 300 °C. This is hypothesized to be due to the rapid temperature rise causing the flux to vaporize explosively, resulting in simultaneous solder splashing. With increasing laser power density, the failure mode transitioned from the solder to the IMC.

Yeast trehalose phosphate synthase(TPS1) gene was introduced into the tobacco chloroplast ornuclear genomes to study resultant phenotypes. PCR and Southern blots confirmedstable integration of TPS1 into the chloroplast genomes ofT1, T2 and T3 transgenic plants. Northern blotanalysis of transgenic plants showed that the chloroplast transformantexpressed169-fold more TPS1 transcript than the best survivingnuclear transgenic plant. Although both the chloroplast and nuclear transgenicplants showed significant TPS1 enzyme activity, no significant trehaloseaccumulation was observed in T0/T1 nuclear transgenicplants whereas chloroplast transgenic plants showed 15–25 fold higheraccumulation of trehalose than the best surviving nuclear transgenic plants.Nuclear transgenic plants (T0) that showed even small amounts oftrehalose accumulation showed stunted phenotype, sterility and otherpleiotropiceffects whereas chloroplast transgenic plants (T1, T2,T3) showed normal growth and no pleiotropic effects. Transgenicchloroplast thylakoid membranes showed high integrity under osmotic stress asevidenced by retention of chlorophyll even when grown in 6% PEG whereaschloroplasts in untransformed plants were bleached. After 7 hrdrying, chloroplast transgenic seedlings (T1, T3)successfully rehydrated while control plants died. There was no differencebetween control and transgenic plants in water loss during dehydration butdehydrated leaves from transgenic plants (not watered for 24 days) recoveredupon rehydration turning green while control leaves dried out. Theseobservations suggest that trehalose functions by protecting biologicalmembranesrather than regulating water potential. In order to prevent escape of droughttolerance trait to weeds and associated pleiotropic traits to related crops, itmay be desirable to engineer crop plants for drought tolerance via thechloroplast genome instead of the nuclear genome.

To evaluate gene expressions mostly engaged in early development of apple fruit, we performed the identification of transcripts differentially expressed in young fruit by using microarrays spotted with 6,253 cDNAs collected from young and mature apple fruits of the cultivar Fuji (Malus domestica Borkh. cv. Fuji). A total of 3,484 cDNAs out of 6,253 were selected after quality control of microarray spots and analyzed for differential gene expression patterns between young fruit and other tissues (mature fruit, leaf and flower). Among them, 192 cDNAs displayed a signal value higher than twofold in young fruit compared with other tissues. Blast analysis of the 192 cDNA clones identified 88 non-redundant groups encoding proteins with known function and 50 non-redundant groups with unknown function. The putative protein products were classified into the following categories: photosynthesis (16.7%), protein synthesis (12.3%), cell proliferation and differentiation (10.9%), cell enlargement (5.8%), metabolism (8.0%), stress response (7.2%), others (2.9%), and unknown functions (32.2%). Furthermore, confirming the microarray data by reverse transcription-polymerase chain reaction revealed that the wide range of transcripts differentially expressed in young fruit was expressed in other organs but not in the mature fruit. The data presented suggested that apple fruit development depends on the tight regulation of the expression of a number of genes, which are also expressed in other organs.

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.

From database comparisons of 1,117 expressed sequence tags (ESTs) generated from ripened Fuji apple fruits, we identified ten ubiquitin (Ub)-related genes. RNA gel-blot analysis suggests that these Ub-related genes are induced by at least four distinct signaling pathways in fruits. In this study, we analyzed structure and expression of MdFBCP1, encoding an F-box-containing protein 1, in Fuji apples. MdFBCP1 transcript was predominantly expressed in the fully ripened climacteric fruits, in which serge of ethylene production occurred. The MdFBCP1 gene was also activated effectively in response to exogenous ethylene treatment, with the induction pattern being comparable to those of ACC oxidase and β-cyanoalanine synthase. Thus, it seems likely that the expression of MdFBCP1 is closely associated with a climacteric ethylene production and ACC oxidase activity and, hence, MdFBCP1 may play a role in the ripening process of Fuji apple fruits. Yeast two hybrid and in vitro pull-down assays revealed that MdFBCP1 physically interacted with MdSkp1 and N-terminal F-box motif was essential for this interaction. These results suggest that MdFBCP1 indeed functions as an F-box-containing protein and participates in the formation of SCF complex, which acts as E3 Ub ligase. Genomic Southern blot analysis showed that MdFBCP1 exhibited different pattern of restriction enzyme digestion in three cultivars (Tsugaru, Golden Delicious and Fuji) that produce different amount of ethylene, suggesting that the MdFBCP1 gene is organized in a cultivar specific manner. Collectively, our data suggest that Ub degradation pathway may play an important role in the ripening of Fuji apple fruits.

Livestock, particularly cattle, are crucial for biotechnology fields, such as genetic breeding, infectious diseases, bioreactors, and specific disease models. However, genetic engineering in cattle has lagged due to long gestation periods, single embryo pregnancies, and high rearing costs. Additionally, the slow validation of germline transmission and the absence of germline-competent embryonic stem cells hinder progress. With the development of genome editing technologies like ZFN, TALEN, and CRISPR-Cas9, recent advancements have shown that Cas9-expressing pigs and chickens have been successfully produced. We hypothesize that generating CRISPR/Cas9-expressing cattle and their resources will provide a powerful resource for bovine genome editing, advancing our understanding of bovine genetics and disease resistance. In this study, two types of Cas9-expressing cattle were successfully produced: Cas9-RFP-fatty acid dehydrogenase I (FatI), Cas9-GFP-sgRNA for the prion protein (sgPRNP). Somatic cells from these cattle were induced to mutate multiple target genes when single-guide RNAs (sgRNAs) were transfected into the somatic cells. Additionally, semen from Cas9 expressing male cattle was frozen and used to fertilize wild-type oocytes, successfully transmitting the transgene (Cas9, reporter genes, FatI), and sgPRNP) to the next generation. Furthermore, the gene editing capabilities of Cas9, including knockout and high-efficiency knock-in, were confirmed in embryos derived from F1 semen through in vitro production. These data demonstrate, for the first time, that Cas9-expressing cattle were successfully born, and this transgene was transmitted to the next-generation calves (F1) and F2 embryos. In addition, somatic and germ cells derived from F0 and F1generations were used to evaluate the potential for gene editing (knockout and knock-in) in multiple genes. PRNP-mutated F1 cattle are currently being raised as a resistance model for bovine spongiform encephalopathy. These transgenic bovine models and their derivatives will serve as a valuable resource for both in vitro and in vivo genome editing, advancing our genetic understanding of bovine genomics and diseases.