High-Throughput 3D Tumor Spheroid Array Platform for Evaluating Sensitivity of Proton-Drug Combinations.

Proton beam therapy (PBT) is a critical treatment modality for head and neck squamous cell carcinoma (HNSCC). However, not much is known about drug combinations that may improve the efficacy of PBT. This study aimed to test the feasibility of a three-dimensional (3D) tumor-spheroid-based high-throughput screening platform that could assess cellular sensitivity against PBT. Spheroids of two HNSCC cell lines-Fadu and Cal27-cultured with a mixture of Matrigel were arrayed on a 384-pillar/well plate, followed by exposure to graded doses of protons or targeted drugs including olaparib at various concentrations. Calcein staining of HNSCC spheroids revealed a dose-dependent decrease in cell viability for proton irradiation or multiple targeted drugs, and provided quantitative data that discriminated the sensitivity between the two HNSCC cell lines. The combined effect of protons and olaparib was assessed by calculating the combination index from the survival rates of 4 × 4 matrices, showing that Cal27 spheroids had greater synergy with olaparib than Fadu spheroids. In contrast, adavosertib did not synergize with protons in both spheroids. Taken together, we demonstrated that the 3D pillar/well array platform was a useful tool that provided rapid, quantitative data for evaluating sensitivity to PBT and drug combinations. Our results further supported that administration of the combination of PBT and olaparib may be an effective treatment strategy for HNSCC patients.

Isolation of Scalarane-Type Sesterterpenoids from the Marine Sponge Dysidea sp. and Stereochemical Reassignment of 12-epi-Phyllactone D/E.

The chemical investigation of the marine sponge Dysidea sp., which was collected from Bohol province in the Philippines, resulted in the identification of 15 new scalarane-type sesterterpenoids (1-14, 16), together with 15 known compounds. The chemical structures of the new compounds were elucidated based on NMR spectroscopy and HRMS. The structure of 12-epi-phyllactone D/E (15) isolated during this study was originally identified in 2007. However, careful inspection of our experimental 13C NMR spectrum revealed considerable discrepancies with the reported data at C-9, C-12, C-14, and C-23, leading to the correction of the reported compound to the C-12 epimer of 15, phyllactone D/E. The biological properties of compounds 1-16 were evaluated using the MDA-MB-231 cancer cell line. Compound 7, which bears a pentenone E-ring, exhibits significant cytotoxicity with a GI50 value of 4.21 µM.

Manganese Ferrite Nanoparticles Enhance the Sensitivity of Hepa1-6 Hepatocellular Carcinoma to Radiation by Remodeling Tumor Microenvironments.

We evaluated the effect of manganese ferrite nanoparticles (MFN) on radiosensitization and immunologic responses using the murine hepatoma cell line Hepa1-6 and the syngeneic mouse model. The clonogenic survival of Hepa1-6 cells was increased by hypoxia, while being restricted by ionizing radiation (IR) and/or MFN. Although MFN suppressed HIF-1α under hypoxia, the combination of IR and MFN enhanced apoptosis and DNA damage in Hepa1-6 cells. In the Hepa1-6 syngeneic mouse model, the combination of IR and MFN notably limited the tumor growth compared to the single treatment with IR or MFN, and also triggered more frequent apoptosis in tumor tissues than that observed under other conditions. Increased expression of PD-L1 after IR was not observed with MFN alone or the combination of IR and MFN in vitro and in vivo, and the percentage of tumor-infiltrating T cells and cytotoxic T cells increased with MFN, regardless of IR, in the Hepa1-6 syngeneic mouse model, while IR alone led to T cell depletion. MFN might have the potential to overcome radioresistance by alleviating hypoxia and strengthening antitumor immunity in the tumor microenvironment.

Therapeutic Potential of (-)-Agelamide D, a Diterpene Alkaloid from the Marine Sponge Agelas sp., as a Natural Radiosensitizer in Hepatocellular Carcinoma Models.

Radiation therapy (RT) is an effective local treatment for unresectable hepatocellular carcinoma (HCC), but there are currently no predictive biomarkers to guide treatment decision for RT or adjuvant systemic drugs to be combined with RT for HCC patients. Previously, we reported that extracts of the marine sponge Agelas sp. may contain a natural radiosensitizer for HCC treatment. In this study, we isolated (-)-agelamide D from Agelas extract and investigated the mechanism underlying its radiosensitization. (-)-Agelamide D enhanced radiation sensitivity of Hep3B cells with decreased clonogenic survival and increased apoptotic cell death. Furthermore, (-)-agelamide D increased the expression of protein kinase RNA-like endoplasmic reticulum kinase/inositol-requiring enzyme 1α/activating transcription factor 4 (PERK/eIF2α/ATF4), a key pathway of the unfolded protein response (UPR) in multiple HCC cell lines, and augmented radiation-induced UPR signaling. In vivo xenograft experiments confirmed that (-)-agelamide D enhanced tumor growth inhibition by radiation without systemic toxicity. Immunohistochemistry results showed that (-)-agelamide D further increased radiation-induced ATF4 expression and apoptotic cell death, which was consistent with our in vitro finding. Collectively, our results provide preclinical evidence that the use of UPR inducers such as (-)-agelamide D may enhance the efficacy of RT in HCC management.

Oxygenated Theonellastrols: Interpretation of Unusual Chemical Behaviors Using Quantum Mechanical Calculations and Stereochemical Reassignment of 7α-Hydroxytheonellasterol.

A total of eight new oxygenated 4-exo-methylene sterols, 1-8, together with one artifact 9 and six known sterols 11-16, were isolated from the marine sponge Theonella swinhoei collected from the Bohol province in Philippines. Structures of sterols 1-8 were determined from 1D and 2D NMR data. Among the sterols, 8α-hydroxytheonellasterol (4) spontaneously underwent an allylic 1,3-hydroxyl shift to produce 15α-hydroxytheonellasterol (9) as an artifact; this was rationalized by quantum mechanical calculations of the transition state. In addition, the 1,2-epoxy alcohol subunit of 8α-hydroxy-14,15-ß-epoxytheonellasterol (5) was assigned using the Gauge-Independent Atomic Orbital (GIAO) NMR chemical shift calculations and subsequent DP4+ analysis. Finally, comparison of the 13C chemical shifts of isolated 7α-hydroxytheonellasterol (6) with the reported values revealed significant discrepancies at C-6, C-7, C-8, and C-14, leading to reassignment of the C-7 stereochemistry in the known structure.

Fucoidan-Manganese Dioxide Nanoparticles Potentiate Radiation Therapy by Co-Targeting Tumor Hypoxia and Angiogenesis.

Tumor hypoxia is a major mechanism of resistance to radiation therapy (RT), which is associated with poor prognosis in affected cancer patients. Various approaches to treat hypoxic and radioresistant cancers, including pancreatic cancer, have shown limited success. Fucoidan, a polysaccharide from brown seaweed, has antitumor and antiangiogenesis activities. Here, we discuss the development of fucoidan-coated manganese dioxide nanoparticles (Fuco-MnO2-NPs) and testing of the therapeutic potential with RT using pancreatic cancer models. In vitro data showed that Fuco-MnO2-NPs generated oxygen efficiently in the presence of H2O2 and substantially suppressed HIF-1 expression under a hypoxic condition in human pancreatic cancer cells. Fuco-MnO2-NPs reversed hypoxia-induced radioresistance by decreasing clonogenic survival and increasing DNA damage and apoptotic cell death in response to RT. In a BxPC3 xenograft mouse model, the combination treatment with Fuco-MnO2-NPs and RT resulted in a greater tumor growth delay than RT alone. Fucoidan-coated NPs, but not naked ones, further suppressed tumor angiogenesis, as judged by immunohistochemistry data with diminished expression of phosphorylated vascular endothelial growth factor receptor 2 (VEGFR2) and CD31. These data suggest that Fuco-MnO2-NPs may potentiate the effects of RT via dual targeting of tumor hypoxia and angiogenesis, and they are of great clinical potential in the treatment of hypoxic, radioresistant pancreatic cancer.

Induction of galectin-1 by TGF-ß1 accelerates fibrosis through enhancing nuclear retention of Smad2.

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-ß1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-ß1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-ß1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.

Targeting Na-H exchanger 1 overcomes nuclear factor kappa B-mediated tumor resistance to radiotherapy.

Intrinsic or acquired radioresistance often limits the efficacy of radiation therapy (RT), thereby leading to local control failure. Cancerous cells have abnormal pH dynamics due to high metabolic demands, but it is unclear how pH dynamics contribute to radioresistance. In this study, we investigated the role of Na-H exchange 1 (NHE1), the major intracellular pH (pHi) regulator, in RT response. We observed that RT increased NHE1 expression and modulated pHi in MDA-MB-231 human breast cancer cells. When combined with RT, pharmacological NHE1 inhibition by 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) reduced pHi and clonogenic survival. EIPA attenuated radiation-damaged DNA repair, increasing G2/M cell cycle arrest. The combination of EIPA and RT increased apoptotic cell death while decreasing phosphorylation of NF-κB p65. Similarly, the knockdown of NHE1 increased radiosensitivity with lower pHi and increased apoptosis. Consistent with in vitro data, the EIPA plus RT inhibited the growth of MDA-MB-231 xenograft tumors in mice to a greater extent than either EIPA or RT alone. EIPA abrogated the RT-induced increase in NHE1 and phospho-NF-κB p65 expression in tumor tissues. Such coincidence of increased NHE1 level, pHi, and NF-κB activation was also found in radioresistant MDA-MB-231 cells, which were reversed by EIPA treatment. Bioinformatics analysis of RNA sequencing data revealed that inhibiting NHE1 reversed three core gene networks that were up-regulated in radioresistant cells and correlated with high NHE1 expression in patient samples: NF-κB, senescence, and extracellular matrix. Taken together, our findings suggest that NHE1 contributes to RT resistance via NF-κB-mediated signaling networks, and NHE1 may be a promising target for improving RT outcomes.

Influence of dietary organic trace minerals on enteric methane emissions and rumen microbiota of heat-stressed dairy steers.

Ruminants are the main contributors to methane (CH4), a greenhouse gas emitted by livestock, which leads to global warming. In addition, animals experience heat stress (HS) when exposed to high ambient temperatures. Organic trace minerals are commonly used to prevent the adverse effects of HS in ruminants; however, little is known about the role of these minerals in reducing enteric methane emissions. Hence, this study aimed to investigate the influence of dietary organic trace minerals on rumen fermentation characteristics, enteric methane emissions, and the composition of rumen bacteria and methanogens in heat-stressed dairy steers. Holstein (n=3) and Jersey (n=3) steers were kept separately within a 3×3 Latin square design, and the animals were exposed to HS conditions (Temperature-Humidity Index [THI], 82.79 ± 1.10). For each experiment, the treatments included a Control (Con) consisting of only basal total mixed rations (TMR), National Research Council (NRC) recommended mineral supplementation group (NM; TMR + [Se 0.1 ppm + Zn 30 ppm + Cu 10 ppm]/kg dry matter), and higher concentration of mineral supplementation group (HM; basal TMR + [Se 3.5 ppm + Zn 350 ppm + Cu 28 ppm]/kg dry matter). Higher concentrations of trace mineral supplementation had no influence on methane emissions and rumen bacterial and methanogen communities regardless of breed (p > 0.05). Holstein steers had higher ruminal pH and lower total volatile fatty acid (VFA) concentrations than Jersey steers (p < 0.05). Methane production (g/d) and yield (g/kg dry matter intake) were higher in Jersey steers than in Holstein steers (p < 0.05). The relative abundances of Methanosarcina and Methanobrevibacter olleyae were significantly higher in Holstein steers than in Jersey steers (p < 0.05). Overall, dietary organic trace minerals have no influence on enteric methane emissions in heat-stressed dairy steers; however, breed can influence it through selective alteration of the rumen methanogen community.

Effect of organic mineral supplementation in reducing oxidative stress in Holstein calves during short-term heat stress and recovery conditions.

BACKGROUND: This study investigated the effects of inorganic and organic minerals on physiological responses, oxidative stress reduction, and rumen microbiota in Holstein bull calves (123.81 ± 9.76 kg; 5 months old) during short-term heat stress (HS) and recovery periods. Eight Holstein calves were randomly assigned to four treatment groups: no mineral supplementation (Con), inorganic minerals (IM), organic minerals (OM), and high-concentration organic minerals (HOM) and two thermal environments (HS and recovery) using 4 × 2 factorial arrangement in a crossover design of four periods of 35 d. Calves were maintained in a temperature-controlled barn. The experimental period consisted of 14 d of HS, 14 d of recovery condititon, and a 7-d washing period. RESULTS: Body temperature and respiration rate were higher in HS than in the recovery conditions (P < 0.05). Selenium concentration in serum was high in the HOM-supplemented calves in both HS (90.38 µg/dL) and recovery periods (102.00 µg/dL) (P < 0.05). During the HS period, the serum cortisol was 20.26 ng/mL in the HOM group, which was 5.60 ng/mL lower than in the control group (P < 0.05). The total antioxidant status was the highest in the OM group (2.71 mmol Trolox equivalent/L), followed by the HOM group during HS, whereas it was highest in the HOM group (2.58 mmol Trolox equivalent/L) during the recovery period (P < 0.05). Plasma malondialdehyde and HSP70 levels were decreased by HOM supplementation during the HS and recovery periods, whereas SOD and GPX levels were not significantly affected (P > 0.05). The principal coordinate analysis represented that the overall rumen microbiota was not influenced by mineral supplementation; however, temperature-induced microbial structure shifts were indicated (PERMANOVA: P < 0.05). At the phylum level, Firmicutes and Actinobacteria decreased, whereas Fibrobacteres, Spirochaetes, and Tenericutes increased (P < 0.05), under HS conditions. The genus Treponema increased under HS conditions, while Christensenella was higher in recovery conditions (P < 0.05). CONCLUSION: HOM supplementation during HS reduced cortisol concentrations and increased total antioxidant status in Holstein bull calves, suggesting that high organic mineral supplementation may alleviate the adverse effects of HS.

Effects of Italian ryegrass silage-based total mixed ration on rumen fermentation, growth performance, blood metabolites, and bacterial communities of growing Hanwoo heifers.

This study utilized Italian ryegrass silage (IRGS) - based total mixed ration (TMR) as feedstuff and evaluated its effects on rumen fermentation, growth performance, blood parameters, and bacterial community in growing Hanwoo heifers. Twenty-seven Hanwoo heifers (body weight [BW], 225.11 ± 10.57 kg) were randomly allocated to three experimental diets. Heifers were fed 1 of 3 treatments as follows: TMR with oat, timothy, and alfalfa hay (CON), TMR with 19% of IRGS (L-IRGS), and TMR with 36% of IRGS (H-IRGS). Feeding high levels of IRGS (H-IRGS) and CON TMR to heifers resulted in a greater molar proportion of propionate in the rumen. The impact of different TMR diets on the BW, average daily gain, dry matter intake, and feed conversion ratio of Hanwoo heifers during the growing period did not differ (p > 0.05). Furthermore, the blood metabolites, total protein, albumin, aspartate aminotransferase, glucose, and total cholesterol of the heifers were not affected by the different TMR diets (p > 0.05). In terms of rumen bacterial community composition, 264 operational taxonomic units (OTUs) were observed across the three TMR diets with 240, 239, and 220 OTUs in CON, L-IRGS, and H-IRGS, respectively. IRGS-based diets increased the relative abundances of genera belonging to phylum Bacteroidetes but decreased the abundances of genus belonging to phylum Firmicutes compared with the control. Data showed that Bacteroidetes was the most dominant phylum, while Prevotella ruminicola was the dominant species across the three TMR groups. The relative abundance of Ruminococcus bromii in the rumen increased in heifers fed with high inclusion of IRGS in the TMR (H-IRGS TMR). The relative abundance of R. bromii in the rumen significantly increased when heifers were fed H-IRGS TMR while P. ruminicola increased in both L-IRGS and H-IRGS TMR groups. Results from the current study demonstrate that the inclusion of IRGS in the TMR is comparable with the TMR containing high-quality forage (CON). Thus, a high level of IRGS can be used as a replacement forage ingredient in TMR feeding and had a beneficial effect of possibly modulating the rumen bacterial community toward mainly propionate-producing microorganisms.

Higher Concentration of Dietary Selenium, Zinc, and Copper Complex Reduces Heat Stress-Associated Oxidative Stress and Metabolic Alteration in the Blood of Holstein and Jersey Steers.

This study investigated the influence of high concentrations of dietary minerals on reducing heat stress (HS)-associated oxidative stress and metabolic alterations in the blood of Holstein and Jersey steers. Holstein steers and Jersey steers were separately maintained under a 3 × 3 Latin square design during the summer conditions. For each trial, the treatments included Control (Con; fed basal TMR without additional mineral supplementation), NM (NRC recommended mineral supplementation group; [basal TMR + (Se 0.1 ppm + Zn 30 ppm + Cu 10 ppm) as DM basis]), and HM (higher than NRC recommended mineral supplementation group; [basal TMR + (Se 3.5 ppm + Zn 350 ppm + Cu 28 ppm) as DM basis]). Blood samples were collected at the end of each 20-day feeding trial. In both breeds, a higher superoxide dismutase concentration (U/mL) along with lower HSP27 (µg/L) and HSP70 (µg/L) concentrations were observed in both mineral-supplemented groups compared to the Con group (p < 0.05). The HM group had significantly higher lactic acid levels in Jersey steers (p < 0.05), and tended to have higher alanine levels in Holstein steers (p = 0.051). Based on star pattern recognition analysis, the levels of succinic acid, malic acid, γ-linolenic acid, 13-methyltetradecanoic acid, and tyrosine decreased, whereas palmitoleic acid increased with increasing mineral concentrations in both breeds. Different treatment groups of both breeds were separated according to the VIP scores of the top 15 metabolites through PLS−DA analysis; however, their metabolic trend was mostly associated with the glucose homeostasis. Overall, the results suggested that supplementation with a higher-than-recommended concentration of dietary minerals rich in organic Se, as was the case in the HM group, would help to prevent HS-associated oxidative stress and metabolic alterations in Holstein and Jersey steers.

Increasing buffering capacity enhances rumen fermentation characteristics and alters rumen microbiota composition of high-concentrate fed Hanwoo steers.

The buffering capacity of buffer agents and their effects on in vitro and in vivo rumen fermentation characteristics, and bacterial composition of a high-concentrate fed Hanwoo steers were investigated in this study. Treatments were comprised of CON (no buffer added), BC0.3% (low buffering capacity, 0.3% buffer), BC0.5% (medium buffering capacity, 0.5% buffer), and BC0.9% (high buffering capacity, 0.9% buffer). Four Hanwoo steers in a 4 × 4 Latin square design were used for the in vivo trial to assess the effect of treatments. Results on in vitro experiment showed that buffering capacity, pH, and ammonia-nitrogen concentration (NH3-N) were significantly higher in BC0.9% and BC0.5% than the other treatments after 24 h incubation. Individual and total volatile fatty acids (VFA) concentration of CON were lowest compared to treatment groups. Meanwhile, in vivo experiment revealed that Bacteroidetes were dominant for all treatments followed by Firmicutes and Proteobacteria. The abundances of Barnesiella intestinihominis, Treponema porcinum, and Vibrio marisflavi were relatively highest under BC0.9%, Ruminoccocus bromii and Succiniclasticum ruminis under BC0.5%, and Bacteroides massiliensis under BC0.3%. The normalized data of relative abundance of observed OTUs' representative families have grouped the CON with BC0.3% in the same cluster, whereas BC0.5% and BC0.9% were clustered separately which indicates the effect of varying buffering capacity of buffer agents. Principal coordinate analysis (PCoA) on unweighted UniFrac distances revealed close similarity of bacterial community structures within and between treatments and control, in which BC0.9% and BC0.3% groups showed dispersed community distribution. Overall, increasing the buffering capacity by supplementation of BC0.5% and and BC0.9% buffer agents enhanced rumen fermentation characteristics and altered the rumen bacterial community, which could help prevent ruminal acidosis during a high-concentrate diet.

Downregulation of Mcl-1 by Panobinostat Potentiates Proton Beam Therapy in Hepatocellular Carcinoma Cells.

Epigenetic modulation by histone deacetylase (HDAC) inhibitors is an attractive anti-cancer strategy for diverse hematological and solid cancers. Herein, we explored the relative effectiveness of the pan-HDAC inhibitor panobinostat in combination with proton over X-ray irradiation in HCC cells. Clonogenic survival assays revealed that radiosensitization of Huh7 and Hep3B cells by panobinostat was more evident when combined with protons than X-rays. Panobinostat increased G2/M arrest and production of intracellular reactive oxygen species, which was further enhanced by proton irradiation. Immunofluorescence staining of γH2AX showed that panobinostat enhanced proton-induced DNA damage. Panobinostat dose-dependently decreased expression of an anti-apoptotic protein, Mcl-1, concomitant with increasing acetylation of histone H4. The combination of panobinostat with proton irradiation enhanced apoptotic cell death to a greater extent than that with X-ray irradiation. Depletion of Mcl-1 by RNA interference enhanced proton-induced apoptosis and proton radiosensitization, suggesting a potential role of Mcl-1 in determining proton sensitivity. Together, our findings suggest that panobinostat may be a promising combination agent for proton beam therapy in HCC treatment.

Kimchi cabbage (Brassica rapa L.) by-products treated with calcium oxide and alkaline hydrogen peroxide as feed ingredient for Holstein steers.

This study aimed to investigate the effects of Kimchi cabbage by-products either treated or untreated with calcium oxide (CaO) and alkaline hydrogen peroxide (AHP) as substitutional ingredient of total mixed ration (TMR) on in vitro fermentation, in situ disappearance and growth performance of Holstein steers. Cannulated Holstein (600 ± 47 kg) was used for both the in vitro and in situ experiments. The treatments used were TMR only (CON), TMR + 30% Kimchi cabbage by-products fresh matter (FM) basis (TC), TMR + 30% Kimchi cabbage by-products FM basis + 5% CaO FM basis (TCC), and TMR + 30% Kimchi cabbage by-products FM basis + 5% CaO FM basis + 3.22% AHP FM basis (TCCA). For in vivo experiment, thirty-four Holstein steers (273 ± 45 kg) were subjected to a 150-day feeding trial, divided into two groups: CON and TC. In the in vitro experiment, pH of TCCA was greatest (p < 0.05) among other treatments at all incubation times. Ammonia nitrogen and volatile fatty acid concentrations were not significantly different for each treatment. However, butyrate was greater (p < 0.05) in TCC and CON than in both TC and TCCA. During in situ experiment, the dry matter (DM) disappearance was greatest (p < 0.05) in TCCA among other treatments. Also, disappearance of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were observed greatest (p > 0.05) in TCCA treatment. In the in vivo experiment, average daily gain (ADG) did not differ between CON and TC. In blood profile analysis, alanine aminotransferase, aspartate aminotransferase, glucose, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and total protein concentration were not significantly different between treatments. But, creatinine concentration was greater (p < 0.05) in TC than in CON. Overall results suggest that Kimchi cabbage by-products either treated or untreated with CaO and AHP can be used as substitutional ingredient in TMR for Holstein steers.

Holstein and Jersey Steers Differ in Rumen Microbiota and Enteric Methane Emissions Even Fed the Same Total Mixed Ration.

Previous studies have focused on the rumen microbiome and enteric methane (CH4) emissions in dairy cows, yet little is known about steers, especially steers of dairy breeds. In the present study, we comparatively examined the rumen microbiota, fermentation characteristics, and CH4 emissions from six non-cannulated Holstein (710.33 ± 43.02 kg) and six Jersey (559.67 ± 32.72 kg) steers. The steers were fed the same total mixed ration (TMR) for 30 days. After 25 days of adaptation to the diet, CH4 emissions were measured using GreenFeed for three consecutive days, and rumen fluid samples were collected on last day using stomach tubing before feeding (0 h) and 6 h after feeding. CH4 production (g/d/animal), CH4 yield (g/kg DMI), and CH4 intensity (g/kg BW0.75) were higher in the Jersey steers than in the Holstein steers. The lowest pH value was recorded at 6 h after feeding. The Jersey steers had lower rumen pH and a higher concentration of ammonia-nitrogen (NH3-N). The Jersey steers had a numerically higher molar proportion of acetate than the Holstein steers, but the opposite was true for that of propionate. Metataxonomic analysis of the rumen microbiota showed that the two breeds had similar species richness, Shannon, and inverse Simpson diversity indexes. Principal coordinates analysis showed that the overall rumen microbiota was different between the two breeds. Both breeds were dominated by Prevotella ruminicola, and its highest relative abundance was observed 6 h after feeding. The genera Ethanoligenens, Succinivibrio, and the species Ethanoligenens harbinense, Succinivibrio dextrinosolvens, Prevotella micans, Prevotella copri, Prevotella oris, Prevotella baroniae, and Treponema succinifaciens were more abundant in Holstein steers while the genera Capnocytophaga, Lachnoclostridium, Barnesiella, Oscillibacter, Galbibacter, and the species Capnocytophaga cynodegmi, Galbibacter mesophilus, Barnesiella intestinihominis, Prevotella shahii, and Oscillibacter ruminantium in the Jersey steers. The Jersey steers were dominated by Methanobrevibacter millerae while the Holstein steers by Methanobrevibacter olleyae. The overall results suggest that sampling hour has little influence on the rumen microbiota; however, breeds of steers can affect the assemblage of the rumen microbiota and different mitigation strategies may be needed to effectively manipulate the rumen microbiota and mitigate enteric CH4 emissions from these steers.

Seasonal Influence on Rumen Microbiota, Rumen Fermentation, and Enteric Methane Emissions of Holstein and Jersey Steers under the Same Total Mixed Ration.

Seasonal effects on rumen microbiome and enteric methane (CH4) emissions are poorly documented. In this study, 6 Holstein and 6 Jersey steers were fed the same total mixed ration diet during winter, spring, and summer seasons under a 2 × 3 factorial arrangement for 30 days per season. The dry matter intake (DMI), rumen fermentation characteristics, enteric CH4 emissions and rumen microbiota were analyzed. Holstein had higher total DMI than Jersey steers regardless of season. However, Holstein steers had the lowest metabolic DMI during summer, while Jersey steers had the lowest total DMI during winter. Jersey steers had higher CH4 yields and intensities than Holstein steers regardless of season. The pH was decreased, while ammonia nitrogen concentration was increased in summer regardless of breed. Total volatile fatty acids concentration and propionate proportions were the highest in winter, while acetate and butyrate proportion were the highest in spring and in summer, respectively, regardless of breed. Moreover, Holstein steers produced a higher proportion of propionate, while Jersey steers produced a higher proportion of butyrate regardless of season. Metataxonomic analysis of rumen microbiota showed that operational taxonomic units and Chao 1 estimates were lower and highly unstable during summer, while winter had the lowest Shannon diversity. Beta diversity analysis suggested that the overall rumen microbiota was shifted according to seasonal changes in both breeds. In winter, the rumen microbiota was dominated by Carnobacterium jeotgali and Ruminococcus bromii, while in summer, Paludibacter propionicigenes was predominant. In Jersey steers, Capnocytophaga cynodegmi, Barnesiella viscericola and Flintibacter butyricus were predominant, whereas in Holstein steers, Succinivibrio dextrinosolvens and Gilliamella bombicola were predominant. Overall results suggest that seasonal changes alter rumen microbiota and fermentation characteristics of both breeds; however, CH4 emissions from steers were significantly influenced by breeds, not by seasons.

Growth performance and blood profiles of Hanwoo steers at fattening stage fed Korean rice wine residue.

The aim of this study was to investigate the effects of Korean rice wine residue (RWR) on the growth performance and blood profiles of Hanwoo steers in the fattening stage. In situ and in vivo experiments were conducted to analyze rumen fermentation characteristics and total tract digestibility, respectively. Three cannulated Hanwoo steers (mean body weight: 448 ± 30 kg) were used in both analyses. The growth performance of 27 experimental animals in the fattening stage (initial body weight: 353.58 ± 9.76 kg) was evaluated after 13 months of feeding. The animals were divided into three treatment groups (n = 9/group). The treatments comprised total mixed ration (TMR) only (CON), TMR + 10% RWR (10% RWR), and TMR + 15% RWR (15% RWR). The diets of equal proportions were fed daily at 08:00 and 18:00 h based on 2% of the body weight. The animals had free access to water and trace mineral salts throughout the experiment. Supplementation of 15% RWR significantly decreased (p < 0.05) the rumen fluid pH compared with the control treatment, but there was no significant difference in the total volatile fatty acid concentration. It also significantly increased (p < 0.05) dry matter digestibility compared with the other treatments. The total weight gain and average daily gain of the animals in the RWR-supplemented groups were significantly higher (p < 0.05) than those in the control group. Furthermore, the feed intake and feed efficiency of the RWR-supplemented groups were higher than those of the control group. Supplementation of RWR did not affect the alcohol, albumin, glucose, total cholesterol, triglyceride, and low-density lipoprotein concentrations, and aspartate aminotransferase and alanine transaminase activities in the blood; these parameters were within the normal range. The high-density lipoprotein and creatinine concentrations were significantly higher in the 15% RWR group, whereas the blood urea nitrogen concentration was significantly higher in the 10% RWR group than in the other groups. These results suggest that TMR with 15% RWR can serve as an alternate feed resource for ruminants.

Targeting DNA-dependent protein kinase sensitizes hepatocellular carcinoma cells to proton beam irradiation through apoptosis induction.

Recent studies have highlighted the implications of genetic variations in the relative biological effectiveness (RBE) of proton beam irradiation over conventional X-ray irradiation. Proton beam radiotherapy is a reasonable radiotherapy option for hepatocellular carcinoma (HCC), but the impact of genetic difference on the HCC RBE remains unknown. Here, we determined proton RBE in human HCC cells by exposing them to various doses of either 6-MV X-rays or 230-MeV proton beams. Clonogenic survival assay revealed variable radiosensitivity of human HCC cell lines with survival fraction at 2 Gy ranging from 0.38 to 0.83 and variable proton RBEs with 37% survival fraction ranging from 1.00 to 1.48. HCC cells appeared more sensitive to proton irradiation than X-rays, with more persistent activation of DNA damage repair proteins over time. Depletion of a DNA damage repair gene, DNA-PKcs, by siRNA dramatically increased the sensitivity of HCC cells to proton beams with a decrease in colony survival and an increase in apoptosis. Our findings suggest that there are large variations in proton RBE in HCC cells despite the use of a constant RBE of 1.1 in the clinic and targeting DNA-PKcs in combination with proton beam therapy may be a promising regimen for treating HCC.

Enhancing Butyrate Production, Ruminal Fermentation and Microbial Population through Supplementation with Clostridium saccharobutylicum.

Butyrate is known to play a significant role in energy metabolism and regulating genomic activities that influence rumen nutrition utilization and function. Thus, this study investigated the effects of an isolated butyrate-producing bacteria, Clostridium saccharobutylicum, in rumen butyrate production, fermentation parameters and microbial population in Holstein-Friesian cow. An isolated butyrate-producing bacterium from the ruminal fluid of a Holstein-Friesian cow was identified and characterized as Clostridium saccharobutylicum RNAL841125 using 16S rRNA gene sequencing and phylogenetic analyses. The bacterium was evaluated on its effects as supplement on in vitro rumen fermentation and microbial population. Supplementation with 106 CFU/ml Clostridium saccharobutylicum increased (p < 0.05) microbial crude protein, butyrate and total volatile fatty acids concentration but had no significant effect on NH3-N at 24 h incubation. Butyrate and total VFA concentrations were higher (p < 0.05) in supplementation with 106 CFU/ml Clostridium saccharobutylicum compared with control, with no differences observed for total gas production, NH3-N and propionate concentration. However, as the inclusion rate (CFU/ml) of C. saccharobutylicum was increased, reduction of rumen fermentation values was observed. Furthermore, butyrate-producing bacteria and Fibrobacter succinogenes population in the rumen increased in response with supplementation of C. saccharobutylicum, while no differences in the population in total bacteria, protozoa and fungi were observed among treatments. Overall, our study suggests that supplementation with 106 CFU/ml C. saccharobutylicum has the potential to improve ruminal fermentation through increased concentrations of butyrate and total volatile fatty acid, and enhanced population of butyrate-producing bacteria and cellulolytic bacteria F. succinogenes.