Role of fumarate reductase on the fermentation properties of Lactobacillus delbrueckii ssp. bulgaricus.

Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus are symbiotic starters widely used in yogurt fermentation. They exchange metabolites to meet their nutritional demands during fermentation, promoting mutual growth. Although S. thermophilus produces fumaric acid, and the addition of fumaric acid has been shown to promote the growth of L. bulgaricus monoculture, whether fumaric acid produced by S. thermophilus is used by L. bulgaricus during coculture remains unclear. Furthermore, the importance of fumaric acid metabolism in the growth of L. bulgaricus is yet to be elucidated. Therefore, in this study, we investigated the importance of fumaric acid metabolism in L. bulgaricus monocultures and coculture with S. thermophilus. We deleted the fumarate reductase gene (frd), which is responsible for the metabolism of fumaric acid to succinic acid, in L. bulgaricus strains 2038 and NCIMB 701373. Both Δfrd strains exhibited longer fermentation times than their parent strains, and fumaric acid was metabolized to malic acid rather than succinic acid. Coculture of Δfrd strains with S. thermophilus 1131 also resulted in a longer fermentation time, and the accumulation of malic acid was observed. These results indicated that fumaric acid produced by S. thermophilus is used by L. bulgaricus as a symbiotic substance during yogurt fermentation and that the metabolism of fumaric acid to succinic acid by fumarate reductase is a key factor determining the fermentation ability of L. bulgaricus.

Effect of fumaric acid on the growth of Lactobacillus delbrueckii ssp. bulgaricus during yogurt fermentation.

Yogurt is traditionally fermented by a symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. These bacteria exchange metabolites with each other to meet their nutritional demands during protocooperation, resulting in a shorter fermentation time. In this study, we investigated whether fumaric acid functions as a symbiotic agent to promote the growth of Lb. bulgaricus by evaluating 8 strains of Lb. bulgaricus and 7 strains of Strep. thermophilus. All the tested Lb. bulgaricus strains metabolized the added fumaric acid into succinic acid during monoculture in milk, and 6 strains (75%) showed shorter fermentation time compared with the control. The addition of malic acid showed similar trends as that of fumaric acid, indicating that the reverse tricarboxylic acid cycle was functioning in Lb. bulgaricus. All 7 Strep. thermophilus strains tested produced fumaric acid during monoculture in milk. Further, in Lb. bulgaricus 2038, the gene expression of fumarate reductase that converts fumaric acid to succinic acid, was higher in the coculture with Strep. thermophilus 1131 than in the monoculture. These findings indicate that fumaric acid produced by Strep. thermophilus can function as a symbiotic substance during yogurt fermentation to stimulate the growth of Lb. bulgaricus.

Effect of lactose hydrolysis on the milk-fermenting properties of Lactobacillus delbrueckii ssp. bulgaricus 2038 and Streptococcus thermophilus 1131.

Yogurt is a well-known nutritious and probiotic food and is traditionally fermented from milk using the symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, yogurt consumption may cause health problems in lactose-intolerant individuals, and the demand for lactose-free yogurt has been increasing. The standard method to prepare lactose-free yogurt is to hydrolyze milk by lactase; however, this process has been reported to influence the fermentation properties of starter strains. This study aimed to investigate the fermentation properties of an industrial starter culture of L. bulgaricus 2038 and S. thermophilus 1131 in lactose-hydrolyzed milk and to examine the metabolic changes induced by glucose utilization. We found that the cell number of L. bulgaricus 2038, exopolysaccharide concentration, and viscosity in the coculture of L. bulgaricus 2038 and S. thermophilus 1131 was significantly increased in lactose-hydrolyzed milk compared with that in unhydrolyzed milk. Although the cell number of S. thermophilus 1131 showed no difference, production of formic acid and reduction of dissolved oxygen were enhanced in lactose-hydrolyzed milk. Further, in lactose-hydrolyzed milk, S. thermophilus 1131 was found to have increased the expression of NADH oxidase, which is responsible for oxygen reduction. These results indicated that glucose utilization promoted S. thermophilus 1131 to rapidly reduce the dissolved oxygen amount and produce a high concentration of formic acid, presumably resulting in the increased cell number of L. bulgaricus 2038 in the coculture. Our study provides basic information on the metabolic changes in starter strains in lactose-hydrolyzed milk, and demonstrates that lactose-free yogurt with increased cell number of L. bulgaricus can be prepared without delay in fermentation and decrease in the cell number of S. thermophilus.

Impact of a prolonged interatrial conduction time for predicting the recurrence of atrial fibrillation after circumferential pulmonary vein isolation of persistent atrial fibrillation.

There are some cases that are difficult to cure with only circumferential pulmonary vein isolation (CPVI) of persistent atrial fibrillation (PerAF). Recently, prolonged interatrial conduction times (IACTs), which seem to be associated with progressive remodeled atria, have been reported as a predictor of new-onset AF. This study aimed to investigate the prognostic value of a prolonged IACT for predicting AF recurrences after CPVI of PerAF. One hundred thirteen patients who underwent CPVI without an empirical substrate modification of PerAF were retrospectively analyzed. The IACT was defined as the interval from the earliest P-wave onset on the ECG to the latest activation in the coronary sinus and was measured after achieving the CPVI and conversion to sinus rhythm. During a mean 22.7-month follow-up after the initial procedure, 56 patients (50%) had AF recurrences. Patients with AF recurrence had a longer IACT than those without AF recurrence (p < 0.001). The best discriminative cut-off value for the IACT was 123 ms (sensitivity 53%, specificity 85%). In a Cox multivariate analysis, a prolonged IACT of ≥ 123 ms was the only independent predictor (hazard ratio: 2.38; 95% confidence interval: 1.36-4.16, p = 0.002) of being associated with the incidence of an AF recurrence. Even after multiple CPVI procedures, patients with an IACT ≥ 123 ms had a higher AF recurrence rate than those with an IACT < 123 ms (p = 0.002). In conclusion, a prolonged IACT of ≥ 123 ms may be a useful marker for predicting AF recurrences after both initial and multiple CPVI procedures for PerAF.

Identification and functional analysis of variants of a cancer/testis antigen LEMD1 in colorectal cancer stem-like cells.

Colorectal cancer (CRC) is one of the most common malignancy, and the prognosis is not still satisfactory due to treatment resistance, recurrence and distant metastasis. Cancer stem cells (CSCs)/cancer-initiating cells (CICs) is endowed with higher tumor-initiating ability, self-renewal ability and differentiation ability, and CSCs/CICs are resistant to treatments. Thus, CSCs/CICs are thought to be responsible for recurrence and distant metastasis, and eradication of CSCs/CICs is essential to cure CRCs. However, the molecular mechanisms of CSCs/CICs are remain unknown, and we aimed to elucidate molecular aspects of CR-CSCs/CICs in this study. We screened the transcriptome data of primary human CR-CSCs/CICs that we previously established, and found that LEM domain containing 1 (LEMD1) is preferentially expressed in CR-CSCs/CICs. LEMD1 belongs to cancer-testis (CT) antigen, and has five transcript variants (variant 1 [V1] - variant 5 [V5]). We found that LEMD1 V1, V2 and V3 is expressed in testis and CR-CSCs/CICs, whereas LEMD1 V4 and V5 is ubiquitously expressed. LEMD1 gene knockdown experiments using siRNAs and gene overexpression experiments revealed that LEMD1 has a role in the maintenance of CR-CSCs/CICs. These observations indicate that CR-CSC/CIC-specific LEMD1 variants are reasonable target of CR-CSC/CIC-targeted therapy.

LY6/PLAUR domain containing 3 has a role in the maintenance of colorectal cancer stem-like cells.

Colorectal carcinoma (CRC) is one of the most frequently diagnosed cancers and the leading cause of cancer-related death for both men and women. Recent studies have revealed that a small sub-population of cancer cells, termed cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), are endowed with tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs are resistant to current therapies including chemotherapy and radiotherapy. Thus, CSCs/CICs are responsible for recurrence and metastasis, and eradication of CSCs/CICs is essential to cure cancer. In this study, we isolated CR-CSCs/CICs as sphere-cultured cells and found that a product derived from LY6/PLAUR domain containing 3 (LYPD3) is preferentially expressed in CSCs/CICs. Gene overexpression and gene knockdown experiments revealed that LYPD3 has a role in the maintenance of CR-CSCs/CICs. The findings provide a novel molecular insight into CR-CSCs/CICs.

A novel nuclear DnaJ protein, DNAJC8, can suppress the formation of spinocerebellar ataxia 3 polyglutamine aggregation in a J-domain independent manner.

Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

Dynamic Kinetic Resolution Approach for the Asymmetric Synthesis of Tetrahydrobenzodiazepines Using Transfer Hydrogenation by Chiral Phosphoric Acid.

Asymmetric synthesis of tetrahydrobenzodiazepines was achieved by transfer hydrogenation of dihydrobenzodiazepines with benzothiazoline having a hydrogen-bonding donor substituent by means of a newly synthesized chiral phosphoric acid. This method was applicable to various racemic dihydrobenzodiazepines to give the corresponding products in good yields with excellent diastereoselectivities and enantioselectivities taking advantage of the dynamic kinetic resolution. Furthermore, the effect of bulky substituent at 3,3'-position on the catalyst and hydrogen-bonding donor substituent on benzothiazoline was fully elucidated by the theoretical study.

Impact of Human Herpesvirus-6 Reactivation on Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation.

Human herpesvirus-6 (HHV-6) is known to reactivate after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and may be associated with development of acute graft-versus-host disease (GVHD) and nonrelapse mortality (NRM). However, the clinical significance of HHV-6 reactivation after allo-HSCT remains unclear. Therefore, we conducted a retrospective analysis to elucidate the impact of HHV-6 reactivation on transplantation outcomes. Of 236 patients who underwent allo-HSCT, 138 (58.5%) developed HHV-6 reactivation and 98 (41.5%) did not. Univariate analysis indicated that at 3 years, patients with HHV-6 reactivation had significantly higher NRM (27.7% versus 13.7%, P = .003) and worse overall survival (42.1% versus 59.0%, P = .008) than those without reactivation. In multivariate analysis, HHV-6 reactivation was associated with higher incidence of acute GVHD (hazard ratio [HR], 1.87; P = .01), cytomegalovirus reactivation (HR, 2.24; P < .001), and NRM (HR, 2.73; P = .007). Subgroup analysis stratified according to conditioning intensity indicated that a significant impact of HHV-6 reactivation on acute GVHD was observed only in patients who received myeloablative conditioning (MAC). These results indicate that HHV-6 reactivation was associated with development of acute GVHD, cytomegalovirus reactivation, and NRM. Furthermore, adverse impact of HHV-6 reactivation on transplantation outcomes was prominent in the setting of MAC.

European Treatment and Outcome Study score does not predict imatinib treatment response and outcome in chronic myeloid leukemia patients.

The Sokal and Hasford scores were developed in the chemotherapy and interferon era and are widely used as prognostic indicators in patients with chronic myeloid leukemia (CML). Recently, a new European Treatment and Outcome Study (EUTOS) scoring system was developed. We performed a multicenter retrospective study to validate the effectiveness of each of the three scoring systems. The study cohort included 145 patients diagnosed with CML in chronic phase who were treated with imatinib. In the EUTOS low- and high-risk groups, the cumulative incidence of complete cytogenetic response (CCyR) at 18 months was 86.9% and 87.5% (P = 0.797) and the 5-year overall survival rate was 92.6% and 93.3% (P = 0.871), respectively. The cumulative incidence of CCyR at 12 months, 5-year event-free survival and 5-year progression-free survival were not predicted using the EUTOS scoring system. However, there were significant differences in both the Sokal score and Hasford score risk groups. In our retrospective validation study, the EUTOS score did not predict the prognosis of patients with CML in chronic phase treated with imatinib.

Peripheral blood absolute lymphocyte/monocyte ratio as a useful prognostic factor in diffuse large B-cell lymphoma in the rituximab era.

OBJECTIVES: The tumor microenvironment, including tumor-infiltrating lymphocytes and myeloid-derived cells, is an important factor in the pathogenesis and clinical behavior of malignant lymphoma. However, the prognostic significance of peripheral lymphocytes and monocytes in lymphoma remains unclear. METHODS: We evaluated the prognostic impact of the absolute lymphocyte count (ALC), absolute monocyte count (AMC), and lymphocyte/monocyte ratio (LMR) in 359 diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). RESULTS: The median follow-up time of the surviving patients was 58 months. Low ALC and an elevated AMC were both associated with poor survival rates. Receiver operating characteristic curve analysis showed that LMR was the best predictor of survival, with 4.0 as the cutoff point. Patients with LMR ≤4.0 were more likely to have an aggressive tumor, and this was associated with poor treatment responses. Patients with LMR ≤4.0 at diagnosis had significantly poorer overall survival (OS) and progression-free survival (PFS) than those with LMR >4.0. Multivariate analysis, which included prognostic factors of the International Prognostic Index, showed LMR ≤4.0 to be an independent predictor for the OS (hazard ratio [HR], 2.507; 95% confidence interval [CI], 1.255-5.007; P = 0.009) and PFS (HR, 2.063; 95% CI, 1.249-3.408; P = 0.005). CONCLUSIONS: The LMR at diagnosis, as a simple index which reflects host systemic immunity, predicts clinical outcomes in DLBCL patients treated with R-CHOP.

[T315I positive promyelocytic crisis of chronic myeloid leukemia].

Promyelocytic crisis (PMC) of chronic myelogenous leukemia (CML) is relatively rare. We report a patient who progressed to PMC with a T315I mutation during the initial treatment with dasatinib for CML. He obtained hematological remission after combination therapy with all-trans retinoic acid and chemotherapy for PMC, and PML-RARA was not detected by FISH analysis. Arsenic trioxide (ATO) and imatinib therapy induced a second complete cytogenetic response, and PML-RARA mRNA detected by real-time quantitative RT-PCR dropped below the detection limit. Finally, allogeneic stem cell transplantation was performed. This case suggests that combination therapy with imatinib and ATO achieves favorable outcomes for PMC.

MIB-1 labeling index as a prognostic factor for patients with follicular lymphoma treated with rituximab plus CHOP therapy.

The MIB-1 labeling index, which is based on Ki67 immunostaining, is widely used to evaluate the proliferation of tumor cells in lymphoma. However, its clinical significance has not been fully assessed. We retrospectively evaluated the prognostic impact of the MIB-1 labeling index at the time of diagnosis, in 98 patients with follicular lymphoma (FL) grade 1-3b who were treated uniformly with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) therapy. The 5-year progression-free survival (PFS) for an MIB-1 labeling index of ≥10% (n = 60) and <10% (n = 38) was 35% and 61%, respectively (P = 0.015). The 5-year overall survival (OS) for an MIB-1 labeling index of ≥10% and <10% was 77% and 92%, respectively (P = 0.025). Pathological grading was not correlated with PFS or OS. In multivariate analysis, an MIB-1 labeling index of ≥10% was independently associated with poor PFS and OS. In conclusion, an MIB-1 labeling index of 10% is a useful cut-off level for predicting the prognosis of patients with FL.

SHI family transcription factors regulate an interspecific barrier.

Pre-zygotic interspecies incompatibility in angiosperms is an important mechanism to prevent unfavourable hybrids between species. Here we report our identification of STIGMATIC PRIVACY 2 (SPRI2), a transcription factor that has a zinc-finger domain and regulates interspecies barriers in Arabidopsis thaliana, via genome-wide association study. Knockout analysis of SPRI2/SRS7 and its paralogue SPRI2-like/SRS5 demonstrated their necessity in rejecting male pollen from other species within female pistils. Additionally, they govern mRNA transcription of xylan O-acetyltransferases (TBL45 and TBL40) related to cell wall modification, alongside SPRI1, a pivotal transmembrane protein for interspecific pollen rejection. SPRI2/SRS7 is localized as condensed structures in the nucleus formed via liquid-liquid phase separation (LLPS), and a prion-like sequence in its amino-terminal region was found to be responsible for the formation of the condensates. The LLPS-regulated SPRI2/SRS7 discovered in this study may contribute to the establishment of interspecific reproductive barriers through the transcriptional regulation of cell wall modification genes and SPRI1.

Establishment of a monoclonal anti-pan HLA class I antibody suitable for immunostaining of formalin-fixed tissue: unusually high frequency of down-regulation in breast cancer tissues.

A novel monoclonal anti-pan human leukocyte antigen (HLA) class I heavy chain antibody, EMR8-5, was established. It could detect HLA-A, -B, and -C antigens in formalin-fixed paraffin embedded tissues. By immunohistochemical staining using the EMR8-5 antibody, various cancer tissues from 246 cases were examined for HLA class I expression. It was found that HLA class I expression was decreased in 20% to 42% of the cases of lung cancer, hepatocellular carcinoma, colon cancer, renal cell carcinoma, and urothelial carcinoma. In contrast, 85% of breast cancer cases had loss of or decreased HLA class I expression. Of the 35 breast cancer cases that had decreased HLA class I heavy chain expression, 33 (94%) also had decreased beta2-microglobulin expression detected by immunohistochemical staining. It was suggested that HLA class I down-regulation might be a common characteristic of breast cancer mostly caused by the down-regulation of beta2-microglobulin expression.

Co-existing Neuropeptide FF and Gonadotropin-Releasing Hormone 3 Coordinately Modulate Male Sexual Behavior.

Animals properly perform sexual behaviors by using multiple sensory cues. However, neural mechanisms integrating multiple sensory cues and regulating motivation for sexual behaviors remain unclear. Here, we focused on peptidergic neurons, terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons, which receive inputs from various sensory systems and co-express neuropeptide FF (NPFF) in addition to GnRH. Our behavioral analyses using knockout medaka of GnRH (gnrh3) and/or NPFF (npff) demonstrated that some sexual behavioral repertoires were delayed, not disrupted, in gnrh3 and npff single knockout males, while the double knockout appeared to alleviate the significant defects that were observed in single knockouts. We also found anatomical evidence to show that both neuropeptides modulate the sexual behavior-controlling brain areas. Furthermore, we demonstrated that NPFF activates neurons in the preoptic area via indirect pathway, which is considered to induce the increase in motivation for male sexual behaviors. Considering these results, we propose a novel mechanism by which co-existing peptides of the TN-GnRH neurons, NPFF, and GnRH3 coordinately modulate certain neuronal circuit for the control of behavioral motivation. Our results may go a long way toward understanding the functional significance of peptidergic neuromodulation in response to sensory information from the external environments.

Effect of surface-mannose modification on aerosolized liposomal delivery to alveolar macrophages.

PURPOSE: The effect of surface-mannose modification on aerosolized liposomal delivery to alveolar macrophages (AMs) was evaluated in vitro and in vivo. METHOD: 4-Aminophenyl-α-D-mannopyranoside (Man) was used for surface-mannose modification, and mannosylated liposomes with various mannosylation rates (particle size: 1000 nm) were prepared. RESULTS: In the in vitro uptake experiments, the uptake of mannosylated liposomes by AMs was increased with the increase in the mannosylation rate over the range 2.4-9.1 mol% Man and became constant at over 9.1%. Thus, the most efficient mannosylation rate was 9.1 mol% Man. Furthermore, free mannose inhibited the uptake of mannosylated liposomes by AMs. This indicates that the uptake mechanism of mannosylated liposomes by AMs is mannose receptor-mediated endocytosis. In the in vivo animal experiments, the mannosylated liposomes (mannosylation rate, 9.1 mol% Man) were more efficiently delivered to AMs after pulmonary aerosolization to rats than nonmodified liposomes and did not harm lung tissues. CONCLUSION: These results indicate that surface-mannose modification is useful for efficient aerosolized liposomal delivery to AMs.

Isolation and characterization of Streptococcus thermophilus possessing prtS gene from raw milk in Japan.

Streptococcus thermophilus is widely used for producing fermented dairy products such as yogurt and cheese. Some S. thermophilus strains possessing the cell-wall protease PrtS show high proteolytic activity and fast acidification properties, which are very useful in industrial starters. However, few S. thermophilus strains possessing the prtS gene have been isolated from the environment. To clarify whether or not S. thermophilus strains possessing the prtS gene are present in Japan, we isolated S. thermophilus from raw milk collected in Japan from 2011 to 2017 and investigated the strains for the presence of prtS by PCR. A total of 172 S. thermophilus strains were isolated, and 59 strains were confirmed to possess prtS. We measured fermentation times of 59 prtS-positive strains in skim milk broth and found that 53 strains showed fast acidification properties, finishing fermentation within 10 hr. However, the remaining 6 prtS-positive strains showed slow acidification properties, and they had several amino acid mutations in PrtS compared with fast acidifying S. thermophilus LMD-9 and 4F44. These results demonstrate that S. thermophilus strains possessing prtS are prevalent in Japan and that some prtS-positive strains could lose their fast acidifying properties through mutations in PrtS.

Preschoolers' nonsymbolic arithmetic with large sets: is addition more accurate than subtraction?

Adult and developing humans share with other animals analog magnitude representations of number that support nonsymbolic arithmetic with large sets. This experiment tested the hypothesis that such representations may be more accurate for addition than for subtraction in children as young as 3(1/2)years of age. In these tasks, the experimenter hid two equal sets of cookies, visibly added to or subtracted from the sets, and then asked 3(1/2)-year-olds which set had more cookies. Initial set size was either large (7 or 9) or very large (18 or 30), and the final sets differed by either a high proportion (ratio of 1:2) or a low proportion (difference of 1 cookie). Children's addition performance exceeded chance, as well as their subtraction performance, across set sizes and proportions, whereas subtraction performance did not exceed chance. Arithmetic performance was also independent of counting ability. Addition performance was remarkably accurate when ratios between outcomes were close to 1, in contrast to previous findings. Interpretations for the asymmetry between addition and subtraction are discussed with respect to the nature of representations for nonsymbolic arithmetic with large sets.