Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Identification of astrocyte regulators by nucleic acid cytometry.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.

Mismatch-introduced DNA probes constructed on the basis of thermodynamic analysis enable the discrimination of single nucleotide variants.

Genotyping of single nucleotide variants (SNVs) has enabled the assessment of disease-related risk factors and significantly improved the potency of diagnosis and prognosis. Meanwhile, genotyping of SNVs is challenging due to the high sequence similarity between wild-type (WT) and SNV. To increase the discrimination between WT and SNV, probes are modified with nucleic acid analogues such as locked nucleic acid (LNA), or deliberate mismatches are introduced to the probe sequence. However, nucleic acid analogues have limitation in high cost and complexity in their synthesis. And a generalized methodology has not been proposed for determining the position and type of deliberate mismatches at the designated experimental conditions to the best of our knowledge. Herein, we propose a reliable workflow for designing mismatch-introduced probes (MIPs) based on nucleic acid thermodynamic analysis and rejection sampling. The theoretical hybridization state of MIP was calculated using nucleic acid thermodynamics, and the detectability was estimated by rejection sampling that simulates the errors from experimental environments. We fabricated MIPs for SNVs in epidermal growth factor receptor, and experimentally demonstrated optimized detectability. The detectability increased up to 7.19-fold depending on the position and type of mismatch; moreover, the optimized MIP showed higher detectability than the LNA probe. This indicates that the workflow can be broadly applied to the optimization of probe sequence for the detection of various disease-related SNVs.

Improved Sensitivity of Intramolecular Strand Displacement Based on Localization of Probes.

Effective intermolecular interaction is required between probe and target molecules for successful detection of biomarkers. Here, we demonstrate that localization of probes on DNA nanostructures improves detection sensitivity and reaction rate. The structural flexibility of DNA nanostructures enabled frequent intramolecular interactions among the localized probes. The Smoluchowski coagulation method and the coarse-grained molecular dynamic software oxDNA were used for theoretical estimation of inter- and intramolecular behaviors of the DNA nanostructures as well as adequate experiments verifying the improvements in sensitivity with probe localization. Remarkably, the probe-localized DNA nanostructure had an increased sensitivity up to 274 times higher than that of the same probes without localization. We believe this achievement represents a wide applicability as a potential design strategy for robust, reliable, and sensitive biosensors.

Protein Nanoparticle Fabrication for Optimized Reticuloendothelial System Evasion and Tumor Accumulation.

Nanoparticles (NPs) of protein-based materials have become one of the most promising candidates for drug carriers in drug-delivery systems because of their in vivo nontoxicity, biodegradability, compatibility with hydrophilic drugs, and adaptability to the human body. Many studies have investigated the fabrication of protein NPs from human serum albumin (HSA) as a new drug carrier. It is important for these NPs to remain in the blood until they reach their therapeutic target to achieve the desired effect; the quicker the clearance of drugs from the body, the shorter is the residence time of drugs in the body, which eventually reduces drug efficacy. Macrophage uptake is a major mechanism for clearance of NPs from the body, so, reducing the degree of macrophage uptake is a major challenge in drug-delivery systems. Original studies of HSA NP uptake by macrophages showed that denatured HSA and HSA NPs synthesized with 80% (v/v) ethanol showed a high degree of macrophage uptake. We found that HSA NPs synthesized with lower ethanol content at pH 7 showed lower macrophage uptake in in vitro macrophage cellular uptake experiments. The effects of the preparation parameters of ethanol concentration, pH, and glutaraldehyde on the macrophage uptake of NPs were thoroughly studied. This newly developed protein NP with lower macrophage uptake has potential application as a drug carrier for many delivery systems.

Revealing the Presence of a Symbolic Sequence Representing Multiple Nucleotides Based on K-Means Clustering of Oligonucleotides.

In biological systems, a few sequence differences diversify the hybridization profile of nucleotides and enable the quantitative control of cellular metabolism in a cooperative manner. In this respect, the information required for a better understanding may not be in each nucleotide sequence, but representative information contained among them. Existing methodologies for nucleotide sequence design have been optimized to track the function of the genetic molecule and predict interaction with others. However, there has been no attempt to extract new sequence information to represent their inheritance function. Here, we tried to conceptually reveal the presence of a representative sequence from groups of nucleotides. The combined application of the K-means clustering algorithm and the social network analysis theorem enabled the effective calculation of the representative sequence. First, a "common sequence" is made that has the highest hybridization property to analog sequences. Next, the sequence complementary to the common sequence is designated as a 'representative sequence'. Based on this, we obtained a representative sequence from multiple analog sequences that are 8⁻10-bases long. Their hybridization was empirically tested, which confirmed that the common sequence had the highest hybridization tendency, and the representative sequence better alignment with the analogs compared to a mere complementary.

Polymeric Nanocomplex Encapsulating Iron Oxide Nanoparticles in Constant Size for Controllable Magnetic Field Reactivity.

The magnetic properties of nanoparticles make them ideal for using in various applications, especially in biomedical applications. However, the magnetic force generated by a single nanoparticle is low. Herein, we describe the development of nanocomplexes (size of 100 nm) of many iron oxide nanoparticles (IONPs) encapsulated in poly(lactic- co-glycolic acid) (PLGA) using the simple method of emulsion solvent evaporation. The response of the IONP-encapsulated PLGA nanocomplexes (IPNs) to an external magnetic field could be controlled by modifying the amount of IONPs loaded into each nanocomplex. In a constant size of IPNs, larger loading numbers of IONPs resulted in more rapid responses to a magnetic field. In addition, nanocomplexes were coated with a silica layer to facilitate the addition of fluorescent dyes. This allowed visualization of the responses of the IPNs to an applied magnetic field corresponding to the IONP loading amount. We envision that these versatile, easy-to-fabricate IPNs with controllable magnetism will have important potential applications in diverse fields.

Cationic Surfactant-Induced Formation of Uniform Gold Nanoparticle Clusters with High Efficiency of Photothermal Conversion under Near-Infrared Irradiation.

A novel and simple method for the fabrication of gold nanoparticle (AuNP) clusters was introduced for use as an efficient near-infrared (NIR) photothermal agent. Cationic surfactants were employed to assemble AuNPs into clusters, during which polyvinylpyrrolidone (PVP) was used to stabilize the AuNP clusters. Through this manner, AuNP clusters with a uniform shape and a narrow size distribution (55.4 ± 5.0 nm by electron microscope) were successfully obtained. A mechanism for the formation of AuNP clusters was studied and proposed. Electrostatic interactions between AuNPs and cationic surfactants, hydrophobic interactions between hydrocarbon chains of cationic surfactants, and repulsive steric interactions of PVP were found to play an important role with regard to the formation mechanism. Photothermal effect in the NIR range of the AuNP clusters was demonstrated; results presented a highly efficient photothermal conversion (with a maximum η of 65%) of the AuNP clusters. The clusters could be easily coated by a silica layer, enabling their biocompatibility and colloidal stability in physiological fluids. The easy-to-fabricate AuNP clusters showed high potential of use as an NIR photothermal agent for cancer therapy.

Differences in DNA Probe-Mediated Aggregation Behavior of Gold Nanomaterials Based on Their Geometric Appearance.

Nanoparticles are used extensively to detect nucleic acid biomarkers due to their analytical applicability and sensitivity. Systems employing the surface plasmon resonance of gold nanomaterials are overwhelmingly considered to be candidates. The aggregation of gold nanomaterials mediated by the hybridization of target DNA at the interface causes a change in the surface plasmon resonance inherent in gold nanomaterials. Such changes can be measured by spectroscopy or even visualized by the naked eye, enabling effective and positive detection. The optical properties of gold nanoparticles are affected by their shape. The geometric appearance of the nanoparticles also affects their colloidal stability and aggregation behavior. In this study, we examined the effect of the geometric appearance of gold nanomaterials on DNA-mediated aggregation behavior through comparative experiments. Experimental and theoretical methods were used concurrently to derive accurate results and to support the hypotheses. Coarse-grained molecular dynamics simulations were performed with a large-scale atomic/molecular massively parallel simulator to understand the aggregation of nanoparticles with the same surface area and various aspect ratios. As a result, we confirmed that the aggregation sensitivity of nanoparticles was affected by the shape of the contact point with the gold nanomaterials. This study demonstrates that the design of a detection system should be accompanied by an in-depth review of the morphology of the nanoparticle.

Interrelationship between tetracycline resistance determinants, phylogenetic group affiliation and carriage of class 1 integrons in commensal Escherichia coli isolates from cattle farms.

BACKGROUND: Carriage of antibiotic-resistant foodborne pathogens by food production animals is one of many contributors to treatment failure in health care settings, and it necessitates an integrated approach to investigate the carriage of resistant pathogens harboring integrons in food-producing animals. METHODS: Escherichia coli isolates with reduced susceptibility to tetracycline antibiotics (n = 92) were tested for associations between carriage of class1 integrons, phylogenetic group affiliation and tetracycline resistance determinants using the MIC method, PFGE analysis, PCR and sequencing. RESULTS: Phylogroups B1 and A were the most common (58.7 and 19.6%, respectively), followed by groups D (20.7%) and B2 (1.1%). All isolates carried at least one of the tet genes examined. In addition, 88 (95.7%) of all tetracycline-resistant isolates carried tet(A) or tet(B), while 47 (51.1%) and 41 (44.6%) harbored only tet(A) or tet(B), respectively. Likewise, isolates harboring these genes had a higher chance (P < 0.05) of carrying class 1 integrons. Of the tested isolates, 38 (41.3%) carried the intI1 gene. Classical integrons with complete genes (sul1 and qacE∆1) at the 3'-CS were recognized in 27 isolates. PCR screening and subsequent sequencing demonstrated that 84.2% (32/38) of the intI1-positive isolates harbored resistance gene cassettes. Overall, seven gene cassettes were identified, either solely or combined with another gene cassette. The most common gene was aadA1 (10 isolates), followed by a combination of aadA1-dfrA1 (seven isolates), aadA1-dfrA12 (six isolates) and aadA1-aadA2-dfrA12 (three isolates). Genetic typing using PFGE showed minimum clonal relatedness with 28 different clusters and 12-25 discernible DNA fragments. CONCLUSIONS: This study brings new insight into the relationships between the presence of integrons, phylogenetic group association and characteristics of tetracycline antibiotic resistance determinants in commensal E. coli strains.

Multiplexed labeling system for high-throughput cell sorting.

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.

X-shaped DNA potentiates therapeutic efficacy in colitis-associated colon cancer through dual activation of TLR9 and inflammasomes.

BACKGROUND: Immunotherapy has been extensively pursed as a promising strategy for the treatment of cancer. Pattern-recognition receptors (PRRs) play important roles in triggering activation of innate and adaptive immunity. Therefore, agents that stimulate PRRs could be useful for cancer immunotherapy. We developed two kinds of X-shaped double-stranded oligodeoxynucleotides (X-DNA), a single unit of X-DNA (XS-DNA) composed of four strands of DNA and a ligated X-DNA complex (XL-DNA) formed by crosslinking each XS-DNA to the other, and investigated if they had immunostimulatory activity and could be applied to anti-cancer immunotherapy. METHODS: Activation of MAPKs and NF-κB was determined by immunoblotting in bone marrow-derived primary dendritic cells (BMDCs). Immune cytokines and co-stimulatory molecules were measured by ELISA and flow cytometry analysis. Anti-cancer efficacy was examined in an azoxymethane/dextran sulfate sodium-induced colitis-associated colon cancer mouse model. Association of X-DNA and TLR9 was determined by co-immunoprecipitation followed by immunoblotting. The involvement of TLR9 and inflammasomes was determined using TLR9- or caspase-1-deficient BMDCs. Inflammasome activation was examined by degradation of pro-caspase-1 to caspase-1 and cleavage of pro-IL-1ß to IL-1ß in BMDCs. RESULTS: XL-DNA and XS-DNA induced activation of MAPKs and NF-κB and production of immune cytokines and co-stimulatory molecules in BMDCs. BMDCs stimulated by XL-DNA induced differentiation of naïve CD4(+) T cells to TH1 cells. Intravenous injection of XL-DNA into mice resulted in increased serum IFN-γ and IL-12 levels, showing in vivo efficacy of XL-DNA to activate TH1 cells and dendritic cells. XL-DNA greatly enhanced the therapeutic efficacy of doxorubicin, an anti-cancer drug, in colitis-associated colon cancer. XL-DNA directly associated with TLR9. In addition, immunostimulatory activities of X-DNA were abolished in TLR9-deficient dendritic cells. Furthermore, X-DNA induced caspase-1 degradation and IL-1ß secretion in BMDCs, which were abolished in caspase-1-deficient cells. CONCLUSIONS: X-DNA induced the activation of dendritic cells as shown by the expression of immune-cytokines and co-stimulatory molecules, resulting in the differentiation of TH1 cells, mediated through dual activation of TLR9 and inflammasomes. X-DNA represents a promising immune adjuvant that can enhance the therapeutic efficacy of anti-cancer drugs by activating PRRs.

Evaluation of the formation of a junctional DNA nanostructure through annealing curve analysis.

During the self-assembly of different numbers of oligonucleotides comprising junctional DNA nanostructures, a change in environmental variables (e.g., temperature or salt concentration) has a substantial influence on the final products. Further, distinctive annealing temperatures of oligonucleotides are observed depending on the state of hybridization. Here, we present an evaluation of the annealing characteristics of oligonucleotides for the formation of a simple junctional DNA nanostructure using an annealing curve analysis. This method may be useful for analyzing the formation of complex junctional DNA nanostructures.

mRNA-Producing Pseudo-nucleus System.

A pseudo-eukaryotic nucleus (PEN) system consisting of a gene-containing DNA hydrogel encapsulated in a liposome is fabricated. Owing to the structural characteristics of gene-containing DNA hydrogel, mRNA transcription efficiency is promoted 2.57-fold. Through the use of PEN as a platform for mRNA delivery to the cytosol, prolonged protein translation is achieved.

Prevalence of Antimicrobial Resistance and Transfer of Tetracycline Resistance Genes in Escherichia coli Isolates from Beef Cattle.

The aim of this study was to investigate the prevalence and transferability of resistance in tetracycline-resistant Escherichia coli isolates recovered from beef cattle in South Korea. A total of 155 E. coli isolates were collected from feces in South Korea, and 146 were confirmed to be resistant to tetracycline. The tetracycline resistance gene tet(A) (46.5%) was the most prevalent, followed by tet(B) (45.1%) and tet(C) (5.8%). Strains carrying tet(A) plus tet(B) and tet(B) plus tet(C) were detected in two isolates each. In terms of phylogenetic grouping, 101 (65.2%) isolates were classified as phylogenetic group B1, followed in decreasing order by D (17.4%), A (14.2%), and B2 (3.2%). Ninety-one (62.3%) isolates were determined to be multidrug resistant by the disk diffusion method. MIC testing using the principal tetracyclines, namely, tetracycline, chlortetracycline, oxytetracycline, doxycycline, and minocycline, revealed that isolates carrying tet(B) had higher MIC values than isolates carrying tet(A). Conjugation assays showed that 121 (82.9%) isolates could transfer a tetracycline resistance gene to a recipient via the IncFIB replicon (65.1%). This study suggests that the high prevalence of tetracycline-resistant E. coli isolates in beef cattle is due to the transferability of tetracycline resistance genes between E. coli populations which have survived the selective pressure caused by the use of antimicrobial agents.

X-DNA origami-networked core-supported lipid stratum.

DNA hydrogels are promising materials for various fields of research, such as in vitro protein production, drug carrier systems, and cell transplantation. For effective application and further utilization of DNA hydrogels, highly effective methods of nano- and microscale DNA hydrogel fabrication are needed. In this respect, the fundamental advantages of a core-shell structure can provide a simple remedy. An isolated reaction chamber and massive production platform can be provided by a core-shell structure, and lipids are one of the best shell precursor candidates because of their intrinsic biocompatibility and potential for easy modification. Here, we demonstrate a novel core-shell nanostructure made of gene-knitted X-shaped DNA (X-DNA) origami-networked gel core-supported lipid strata. It was simply organized by cross-linking DNA molecules via T4 enzymatic ligation and enclosing them in lipid strata. As a condensed core structure, the DNA gel shows Brownian behavior in a confined area. It has been speculated that they could, in the future, be utilized for in vitro protein synthesis, gene-integration transporters, and even new molecular bottom-up biological machineries.

Effects of germanium biotite supplement on immune responses of vaccinated mini-pigs to foot-and-mouth disease virus challenge.

Since the outbreaks of foot-and-mouth disease (FMD) in South Korea in 2010-2011, a trivalent vaccine has been used as a routine vaccination. Despite the high efficacy of the trivalent vaccine, low antibody formation was reported in the pig industry and there is considerable concern about the ability of the vaccine to protect against the Andong strain responsible for recent outbreaks in South Korea. To overcome these problems, immunostimulators have been widely used to improve vaccine efficacy in South Korea, although without any scientific evidence. Based on the current situation, the aim of this study was to investigate the effects of germanium biotite, a feed supplement used to enhance the immune system, on the immune responses to FMD vaccination through the Andong strain challenge experiment in trivalent vaccinated pigs. Following the challenge, the germanium biotite-fed pigs showed high levels of IL-8 in serum, and increased cellular immune responses to stimulation with the Andong strain antigen compared to nonsupplemented pigs. In addition, higher FMD virus (FMDV) neutralizing antibody titers were detected in the germanium biotite-fed group than in the nonsupplemented group before the challenge. The findings of this study indicate that germanium biotite supplement might enhance immune responses to the FMD vaccine in pigs.

Induction of immune responses in mice and pigs by oral administration of classical swine fever virus E2 protein expressed in rice calli.

Classical swine fever (CSF), caused by the CSF virus (CSFV), is a highly contagious disease in pigs. In Korea, vaccination using a live-attenuated strain (LOM strain) has been used to control the disease. However, parenteral vaccination using a live-attenuated strain still faces a number of problems related to storage, cost, injection stress, and differentiation of CSFV infected and vaccinated pigs. Therefore, two kinds of new candidates for oral vaccination have been developed based on the translation of the E2 gene of the SW03 strain, which was isolated from an outbreak of CSF in 2002 in Korea, in transgenic rice calli (TRCs) from Oriza sativa L. cv. Dongjin to express a recombinant E2 protein (rE2-TRCs). The expression of the recombinant E2 protein (rE2) in rE2-TRCs was confirmed using Northern blot, SDS-PAGE, and Western blot analysis. Immune responses to the rE2-TRC in mice and pigs were investigated after oral administration. The administration of rE2-TRCs increased E2-specific antibodies titers and antibody-secreting cells when compared to animals receiving the vector alone (p < 0.05 and p < 0.01). In addition, mice receiving rE2-TRCs had a higher level of CD8+ lymphocytes and Th1 cytokine immune responses to purified rE2 (prE2) in vitro than the controls (p < 0.05 and p < 0.01). Pigs receiving rE2-TRCs also showed an increase in IL-8, CCL2, and the CD8+ subpopulation in response to stimulation with prE2. These results suggest that oral administration of rE2-TRCs can induce E2-specific immune responses.

Supplementation of dietary germanium biotite enhances induction of the immune responses by foot-and-mouth disease vaccine in cattle.

BACKGROUND: After the recent outbreak of foot-and-mouth disease (FMD) in Korea, a vaccination policy has been applied to control the disease. In addition, several non-specific immune stimulators have been used without any scientific evidence that they would enhance the immune response after FMD vaccination and/or protect against FMD. Based on the current situation, the aim of this study was to evaluate the effect of the non-specific immune stimulator germanium biotite on FMD vaccination and immune responses in cattle. To achieve our goal, immune responses to FMD vaccination, such as levels of IgG and IgA, antibody duration, and virus-neutralizing titers were investigated after germanium biotite feeding. The PBMC typing and proliferative response after stimulation with mitogens, the cytokines expression level of PBMC, and the lysozyme activity in the serum were measured to evaluate the immune enhancing effects of germanium biotite following its administration. RESULTS: Following the first vaccination, high level of IgG (at 4 weeks) and IgA (at 2 and 31 weeks) titers in serum and saliva were observed in the germanium biotite-feeding group (p < 0.05). The germanium biotite group also showed high and longstanding inhibition percentage value in ELISA assay at 31 weeks (p < 0.05). Generally, higher virus-neutralizing antibody titers were observed in the feeding group at 20 and 31 weeks after vaccination. Following the feeding germanium biotite, the germanium biotite group showed increased subpopulation of CD4+ lymphocytes and MHC I+II+ cells in PBMCs at 23 week, responding to stimulation of ConA. The levels of IFN-γ (at 3 and 8 weeks), IL-1α (at 3, 11, and 23 weeks), IL-1ß (at 3, 8, and 11 weeks), and IL-4 (at 8 and 11 weeks) gene expression were also significantly increased in the feeding group (p < 0.01 and p < 0.05). Feeding with germanium biotite increased the lymphocytes' proliferative response to the stimulation of ConA and LPS at 23 weeks and lysozyme activity at 9 weeks after feeding. CONCLUSIONS: These results suggest that germanium biotite feeding could increase the protection against FMD virus infection via the induction of higher humoral and cellular immune responses in cattle.