Global patterns of tree wood density.

Wood density is a fundamental property related to tree biomechanics and hydraulic function while playing a crucial role in assessing vegetation carbon stocks by linking volumetric retrieval and a mass estimate. This study provides a high-resolution map of the global distribution of tree wood density at the 0.01° (~1 km) spatial resolution, derived from four decision trees machine learning models using a global database of 28,822 tree-level wood density measurements. An ensemble of four top-performing models combined with eight cross-validation strategies shows great consistency, providing wood density patterns with pronounced spatial heterogeneity. The global pattern shows lower wood density values in northern and northwestern Europe, Canadian forest regions and slightly higher values in Siberia forests, western United States, and southern China. In contrast, tropical regions, especially wet tropical areas, exhibit high wood density. Climatic predictors explain 49%-63% of spatial variations, followed by vegetation characteristics (25%-31%) and edaphic properties (11%-16%). Notably, leaf type (evergreen vs. deciduous) and leaf habit type (broadleaved vs. needleleaved) are the most dominant individual features among all selected predictive covariates. Wood density tends to be higher for angiosperm broadleaf trees compared to gymnosperm needleleaf trees, particularly for evergreen species. The distributions of wood density categorized by leaf types and leaf habit types have good agreement with the features observed in wood density measurements. This global map quantifying wood density distribution can help improve accurate predictions of forest carbon stocks, providing deeper insights into ecosystem functioning and carbon cycling such as forest vulnerability to hydraulic and thermal stresses in the context of future climate change.

Canopy height affects the allocation of photosynthetic carbon and nitrogen in two deciduous tree species under elevated CO2.

Down-regulation of leaf N and Rubisco under elevated CO2 (eCO2) are accompanied by increased non-structural carbohydrates (NSC) due to the sink-source imbalance. Here, to investigate whether the canopy position affects the down-regulation of Rubisco, we measured leaf N, NSC and N allocation in two species with different heights at maturity [Fraxinus rhynchophylla (6.8 ± 0.3 m) and Sorbus alnifolia (3.6 ± 0.2 m)] from 2017 to 2019. Since 2009, both species were grown at three different CO2 concentrations in open-top chambers: ambient CO2 (400 ppm; aCO2); ambient CO2 × 1.4 (560 ppm; eCO21.4); and ambient CO2 × 1.8 (720 ppm; eCO21.8). Leaf N per unit mass (Nmass) decreased under eCO2, except under eCO21.8 in S. alnifolia and coincided with increased NSC. NSC increased under eCO2 in F. rhynchophylla, but the increment of NSC was greater in the upper canopy of S. alnifolia. Conversely, Rubisco content per unit area was reduced under eCO2 in S. alnifolia and there was no interaction between CO2 and canopy position. In contrast, the reduction of Rubisco content per unit area was greater in the upper canopy of F. rhynchophylla, with a significant interaction between CO2 and canopy position. Rubisco was negatively correlated with NSC only in the upper canopy of F. rhynchophylla, and at the same NSC, Rubisco was lower under eCO2 than under aCO2. Contrary to Rubisco, chlorophyll increased under eCO2 in both species, although there was no interaction between CO2 and canopy position. Finally, photosynthetic N content (Rubisco + chlorophyll + PSII) was reduced and consistent with down-regulation of Rubisco. Therefore, the observed Nmass reduction under eCO2 was associated with dilution due to NSC accumulation. Moreover, down-regulation of Rubisco under eCO2 was more sensitive to NSC accumulation in the upper canopy. Our findings emphasize the need for the modification of the canopy level model in the context of climate change.

Improving the Performance of the ToGoFET Probe: Advances in Design, Fabrication, and Signal Processing.

We report recent improvements of the tip-on-gate of field-effect-transistor (ToGoFET) probe used for capacitive measurement. Probe structure, fabrication, and signal processing were modified. The inbuilt metal-oxide-semiconductor field-effect-transistor (MOSFET) was redesigned to ensure reliable probe operation. Fabrication was based on the standard complementary metal-oxide-semiconductor (CMOS) process, and trench formation and the channel definition were modified. Demodulation of the amplitude-modulated drain current was varied, enhancing the signal-to-noise ratio. The I-V characteristics of the inbuilt MOSFET reflect the design and fabrication modifications, and measurement of a buried electrode revealed improved ToGoFET imaging performance. The minimum measurable value was enhanced 20-fold.

Down-regulation of photosynthesis and its relationship with changes in leaf N allocation and N availability after long-term exposure to elevated CO2 concentration.

Down-regulation of photosynthesis under elevated CO2 (eCO2) concentrations could be attributed to the depletion of nitrogen (N) availability after long-term exposure to eCO2 (progressive nitrogen limitation, PNL) or leaf N dilutions due to excessive accumulation of nonstructural carbohydrates. To determine the mechanism underlying this down-regulation, we investigated N availability, photosynthetic characteristics, and N allocation in leaves of Pinus densiflora (shade-intolerant species, evergreen tree), Fraxinus rhynchophylla (intermediate shade-tolerant species, deciduous tree), and Sorbus alnifolia (shade-tolerant species, deciduous tree). The three species were grown under three different CO2 concentrations in open-top chambers, i.e., ambient 400 ppm (aCO2); ambient × 1.4, 560 ppm (eCO21.4); and ambient × 1.8, 720 ppm (eCO21.8), for 11 years. Unlike previous studies that addressed PNL, after 11 years of eCO2 exposure, N availability remained higher under eCO21.8, and chlorophyll and photosynthetic N use efficiency increased under eCO2. In the case of nonstructural carbohydrates, starch and soluble sugar showed significant increases under eCO2. The maximum carboxylation rate, leaf N per mass (Nmass), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were low under eCO21.8. The ratio of RuBP regeneration to the carboxylation rate as well as that of chlorophyll N to Rubisco N increased with CO2 concentrations. Based on the reduction in Nmass (not in Narea) that was diluted by increase in nonstructural carbohydrate, down-regulation of photosynthesis was found to be caused by the dilution rather than PNL. The greatest increases in chlorophyll under eCO2 were observed in S. alnifolia, which was the most shade-tolerant species. This study could help provide more detailed, mechanistically based processes to explain the down-regulation of photosynthesis by considering two hypotheses together and showed N allocation seems to be flexible against changes in CO2 concentration.

Capacitive Measurements of SiO2 Films of Different Thicknesses Using a MOSFET-Based SPM Probe.

We utilized scanning probe microscopy (SPM) based on a metal-oxide-silicon field-effect transistor (MOSFET) to image interdigitated electrodes covered with oxide films that were several hundred nanometers in thickness. The signal varied depending on the thickness of the silicon dioxide film covering the electrodes. We deposited a 400- or 500-nm-thick silicon dioxide film on each sample electrode. Thick oxide films are difficult to analyze using conventional probes because of their low capacitance. In addition, we evaluated linearity and performed frequency response measurements; the measured frequency response reflected the electrical characteristics of the system, including the MOSFET, conductive tip, and local sample area. Our technique facilitated analysis of the passivation layers of integrated circuits, especially those of the back-end-of-line (BEOL) process, and can be used for subsurface imaging of various dielectric layers.

Analysis of the Immune Responses in the Ileum of Gnotobiotic Pigs Infected with the Recombinant GII.p12_GII.3 Human Norovirus by mRNA Sequencing.

Norovirus genogroup II (NoV GII) induces acute gastrointestinal food-borne illness in humans. Because gnotobiotic pigs can be infected with human norovirus (HuNoV) GII, they are frequently used to analyze the associated pathogenic mechanisms and immune responses, which remain poorly understood. Recently, mRNA sequencing analysis (RNA-Seq) has been used to identify cellular responses to viruses. In this study, we investigated the host immune response and possible mechanisms involved in virus evasion in the ileum of gnotobiotic pigs infected with HuNoV by RNA-Seq. HuNoV was detected in the feces, blood, and tissues of the jejunum, ileum, colon, mesenteric lymph node, and spleen of pigs infected with HuNoV. In analysis of mRNA sequencing, expression of anti-viral protein genes such as OAS1, MX1, and MX2 were largely decreased, whereas type I IFN was increased in pigs infected with HuNoV. In addition, expression of TNF and associated anti-inflammatory cytokine genes such as IL10 was increased in HuNoV-infected pigs. Expression of genes related to natural killer (NK) cell cytotoxicity and CD8+ T cell exhaustion was increased, whereas that of MHC class I genes was decreased. Expression profiles of selected genes were further confirmed by qRT-PCR and Western blot. These results suggest that infection with HuNoV induces NK cell-mediated cytotoxicity but suppresses type I IFN- and CD8+ T cell-mediated antiviral responses.

The Effects of Manufacturing Inaccuracies in an Underwater Acoustic Projector Array on the Acoustic Interactions.

We report on the importance of considering manufacturing inaccuracies in underwater acoustic projectors by elucidating how small variations affect the response characteristics of a projector array in the presence of mutual-loading effects. A wave-based distributed mechanical model accurately calculates the changes arising from small variations, so rapid changes occurring in the vicinity of the transducer resonance can be simulated. The results showed the effects of mutual loading between projector units and confirmed that the changes can be drastically intensified in the presence of manufacturing inaccuracies. A voltage adjustment method to compensate for these changes is also demonstrated as a solution. This framework could guide the design of projector arrays in sound navigation and ranging (SONAR) systems for a variety of applications and, in particular, may contribute significantly to determining manufacturing tolerances.

Size-dependent variation in leaf functional traits and nitrogen allocation trade-offs in Robinia pseudoacacia and Cornus controversa.

Tree species vary in how they invest resources to different functions throughout their life histories, and investigating the detailed patterns of ontogenetic changes in key functional traits will aid in predicting forest dynamics and ecosystem processes. In this context, we investigated size-dependent changes in key leaf functional traits and nitrogen (N) allocation trade-offs in black locust (Robinia pseudoacacia L., an N-fixing pioneer species) and giant dogwood (Cornus controversa Hemsl., a mid-successional species), which have different life-history strategies, especially in their light use. We found that the leaf mass per area and leaf carbon concentrations increased linearly with tree size (diameter at breast height, DBH), whereas leaf N concentrations decreased nonlinearly, with U- and hump-shaped patterns in black locust and giant dogwood, respectively. We also discovered large differences in N allocation between the two species. The fraction of leaf N invested in cell walls was much higher in black locust than in giant dogwood, while the opposite was true for the light harvesting N fraction. Furthermore, these fractions were related to DBH to varying degrees: the cell wall N fraction increased with DBH for both species, whereas the light harvesting N fraction of giant dogwood decreased nonlinearly and that of black locust remained constant. Instead, black locust reduced the fraction of leaf N invested in other N pools, resulting in a smaller fraction compared to that of giant dogwood. On the other hand, both species had similar fraction of leaf N invested in ribulose-1,5-bisphosphate carboxylase/oxygenase across tree size. This study indicated that both species increased leaf mechanical toughness through characteristic changes in N allocation trade-offs over the lifetimes of the trees.

Potential use of transgenic domestic pigs expressing recombinant human erythropoietin in diabetes translation research.

Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n = 16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24 h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3 K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2 h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.

The critical warning sign of real-time brainstem auditory evoked potentials during microvascular decompression for hemifacial spasm.

OBJECTIVE: The aim of this study was to define the critical warning sign of real-time brainstem auditory evoked potential (BAEP) for predicting hearing loss (HL) after microvascular decompression (MVD) for hemifacial spasm (HFS). METHODS: Nine hundred and thirty-two patients with HFS who underwent MVD with intraoperative monitoring (IOM) of BAEP were analyzed. We used a 43.9 Hz/s stimulation rate and 400 averaging trials to obtain BAEP. To evaluate HL, pure-tone audiometry and speech discrimination scoring were performed before and one week after surgery. We analyzed the incidence for postoperative HL according to BAEP changes and calculated the diagnostic accuracy of significant warning criteria. RESULTS: Only 11 (1.2%) patients experienced postoperative HL. The group showing permanent loss of wave V showed the largest percentage of postoperative HL (p < 0.001). No patient who experienced only latency prolongation (≥1 ms) had postoperative HL. Loss of wave V and latency prolongation (≥1 ms) with amplitude decrement (≥50%) were highly associated with postoperative HL. CONCLUSIONS: Loss of wave V and latency prolongation of 1 ms with amplitude decrement ≥50% were the critical warning signs of BAEP for predicting postoperative HL. SIGNIFICANCE: These findings elucidate the critical warning sign of real-time BAEP.

A scanning probe mounted on a field-effect transistor: Characterization of ion damage in Si.

We have examined the capabilities of a Tip-On-Gate of Field-Effect Transistor (ToGoFET) probe for characterization of FIB-induced damage in Si surface. A ToGoFET probe is the SPM probe which the Field Effect Transistor(FET) is embedded at the end of a cantilever and a Pt tip was mounted at the gate of FET. The ToGoFET probe can detect the surface electrical properties by measuring source-drain current directly modulated by the charge on the tip. In this study, a Si specimen whose surface was processed with Ga+ ion beam was prepared. Irradiation and implantation with Ga+ ions induce highly localized modifications to the contact potential. The FET embedded on ToGoFET probe detected the surface electric field profile generated by schottky contact between the Pt tip and the sample surface. Experimentally, it was shown that significant differences of electric field due to the contact potential barrier in differently processed specimens were observed using ToGOFET probe. This result shows the potential that the local contact potential difference can be measured by simple working principle with high sensitivity.

Gnotobiotic Miniature Pig Interbreed Somatic Cell Nuclear Transfer for Xenotransplantation.

Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos.

Well-defined differentiation of hESC-derived hemangioblasts by embryoid body formation without enzymatic treatment.

Human hemangioblasts exist only during the early embryonic developmental stage thereby limiting the adult cellular source from which to obtain such cells for study. To overcome this, hemangioblast studies have focused on utilizing human embryonic stem cell (hESC) derivatives but current methods are cell-line dependent. Single cell dissociation of a hESC colony quickly led to cell death in most hESC lines due to enzyme treatment which, in turn, reduced induction potential and hemangioblast differentiation efficiency. Therefore, we sought to effectively improve the process of cell dissociation that is adaptable to various hESC lines and increase the initial induction potential of embryoid body (hEB). As a result, we determined an effective cell dissociation method through a comparison study involving various reagents which demonstrated successful dissociation regardless of cell line and enhanced hemangioblast differentiation efficiency.

Immunogenicity of bivalent human papillomavirus DNA vaccine using human endogenous retrovirus envelope-coated baculoviral vectors in mice and pigs.

Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.

Proteins associated with reproductive disorders in testes of human erythropoietin gene-harboring transgenic boars.

To investigate reproductive disorder in human erythropoietin (EPO)-expressing pig, we performed comparative proteomic analyses of testicular tissues from human erythropoietin (hEPO) gene-harboring transgenic pigs and wild type pigs born from natural conception. In hEPO TG pigs, we found relatively low sperm motility and higher death rate indicating impaired sperm development. Consistently, plasma concentration of testosterone was significantly lower in the transgenic post-pubertal boars compared with wild type boars. Normalized protein spots showing higher than 2-fold differential expression intensity in two-dimensional polyacrylamide gel electrophoresis were selected for matrix associated laser desorption/ionization time-to-flight mass spectrometry analysis. Specific proteins were identified by searching the NCBI protein sequence databases. Among 55 proteins selected, 12 proteins were identified as those differentially expressed between transgenic and wild type pigs. Three downregulated proteins (ß-globin, carbonyl reductase 1, and peroxiredoxin 6) and nine upregulated proteins (cytoskeletal ß-actin, α 2,3-sialyltransferase, apolipoprotein A-I, tubulin α-1A chain, tropomodulin 3, thioredoxin, heat shock Protein 70.2, ch4/domains of swine IgM, and albumin), all of which are closely related to apoptosis and cytoskeletal development, were found in the transgenic boar testes. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay confirmed the increased occurrence of apoptosis in the transgenic boar testes compared with the wild type boar testes. Reproductive defects of the hEPO-expressing transgenic pigs may be caused by the abnormal expression of the genes identified in this study.

Production of recombinant human von Willebrand factor in the milk of transgenic pigs.

Von Willebrand factor (vWF), a large multimeric glycoprotein present in blood plasma, is a blood protein of the coagulation system. It is defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura-hemolytic uremic syndrome and heyde's syndrome. We have developed a line of transgenic swine harboring recombinant human von Willebrand factor (rhvWF) cDNA through microinjection of fertilized one-cell pig zygotes. Expression of rhvWF in the mammary gland and secretion of rhvWF into the milk of the transgenic swine were confirmed by immunohistochemical and western blot analyses, respectively, and rhvWF proteins were detected in milk from all lactating founder females at concentrations that were 28- to 56-folds greater than that in circulating human plasma. The amino acid sequence of rhvWF protein in the transgenic pig milk matched that of vWF produced from human blood plasma. This study provides evidence that production of rhvWF from transgenic pig milk is a potentially valuable technology and can be used as a cost-effective alternative in clinical applications.

Cloning and characterization of 5'-untranslated region of porcine beta casein gene (CSN2).

beta-Casein (CSN2) is a major milk protein in most mammals. The CSN2 gene is generally induced by lactogenic hormones bound to its promoter. The expression of this gene can be enhanced by signal transducers and activators of transcription (STAT) and glucocorticoid receptor (GR). Here, we analyzed the promoter and intron 1 regions of the porcine CSN2 gene. The porcine CSN2 promoter and intron 1 regions (-3098bp to +2446bp) were cloned into the pGL3-Basic vector containing the luciferase reporter gene (pCSN2-PEI). Lactogenic signals induced the transcription of porcine CSN2. By using AG490, a Janus kinase (JAK) inhibitor, we demonstrated that STAT5 positively regulates the transcription of porcine CSN2. Further, seven STAT mutants were generated by site-directed mutagenesis. By performing electrophoretic mobility shift assays (EMSAs), we located a critical element for pCSN2-PEI transcription bound to STAT5 in the -102bp to -84bp region. The construct containing only the promoter region (pCSN2-P), however, did not exert any promotive effects on transcription in two cell types-a mouse mammary epithelial cell line (HC11) and porcine mammary gland epithelial cells (PMECs). Thus, the construct containing intron 1 of porcine CSN2 exerts an elevating effect on transcription. We suggest that the transcription of porcine CSN2 is regulated by lactogenic signals via the STAT5 site (-102bp to -84bp) and intron 1.

Characterization of the bovine endogenous retrovirus beta3 genome.

We recently used degenerate PCR and locus-specific PCR methods to identify the endogenous retroviruses (ERV) in the bovine genome. Using the ovine ERV classification system, the bovine ERVs (BERVs) could be classified into four families. Here, we searched the most recently released bovine genome database with the partial nucleotide sequence of the pro/pol region of the BERV beta3 family. This allowed us to obtain and analyze the complete genome of BERV beta3. The BERV beta3 genome is 7666 nucleotides long and has the typical retroviral organization, namely, 5'-long terminal repeat (LTR)-gag-pro-pol-env-LTR-3'. The deduced open reading frames for gag, pro, pol and env of BERV Beta en- code 507, 271, 879 and 603 amino acids, respectively. BERV beta3 showed little amino acid similarity to other betaretroviruses. Phylogenetic analysis showed that it clusters with HERV-K. This is the first report describing the genetic structure and sequence of an entire BERV.

Identification and classification of endogenous retroviruses in cattle.

The aim of this study was to identify the endogenous retrovirus (ERV) sequences in a bovine genome. We subjected bovine genomic DNA to PCR with degenerate or ovine ERV (OERV) family-specific primers that aimed to amplify the retroviral pro/pol region. Sequence analysis of 113 clones obtained by PCR revealed that 69 were of retroviral origin. On the basis of the OERV classification system, these clones from degenerate PCR could be divided into the beta3, gamma4, and gamma9 families. PCR with OERV family-specific primers revealed an additional ERV that was classified into the bovine endogenous retrovirus (BERV) gamma7 family. In conclusion, here we report the results of a genome scale study of the BERV. Our study shows that the ERV family expansion in cattle may be somewhat limited, while more diverse family members of ERVs have been reported from other artiodactyls, such as pigs and sheep.

Natural protection from zoonosis by alpha-gal epitopes on virus particles in xenotransmission.

Clinical transplantation has become one of the preferred treatments for end-stage organ failure, and one of the novel approaches being pursued to overcome the limited supply of human organs involves the use of organs from other species. The pig appears to be a near ideal animal due to proximity to humans, domestication, and ability to procreate. The presence of Gal-alpha1,3-Gal residues on the surfaces of pig cells is a major immunological obstacle to xenotransplantation. Alpha1,3galactosyltransferase (alpha1,3GT) catalyzes the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-gal epitope) on the glycoproteins and glycolipids of non-primate mammals, but this does not occur in humans. Moreover, the alpha-gal epitope causes hyperacute rejection of pig organs in humans, and thus, the elimination of this antigen from pig tissues is highly desirable. Recently, concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of alpha-gal on their viral particles. In this study, transgenic cells expressing alpha1,3GT were selected using 1.25 mg/ml neomycin. The development of HeLa cells expressing alpha1,3GT now allows accurate studies to be conducted on the function of the alpha-gal epitope in xenotransmission. The expressions of alpha-gal epitopes on HeLa/alpha-gal cells were demonstrated by flow cytometry and confocal microscopy using cells stained with IB4-fluorescein isothiocyanate lectin. Vaccinia viruses propagated in HeLa/alpha-gal cells also expressed alpha-gal on their viral envelopes and were more sensitive to inactivation by human sera than vaccinia virus propagated in HeLa cells. Moreover, neutralization of vaccinia virus was inhibited in human serum by 10 mm ethylene glycol bis(beta-aminoethylether)tetraacetic acid (EDTA) treatment. Our data indicated that alpha-gal epitopes are one of the major barriers to zoonosis via xenotransmission.