Rice small secreted peptide, OsRALF26, recognized by FERONIA-like receptor 1 induces immunity in rice and Arabidopsis.

Rapid alkalinization factors (RALFs), belonging to a family of small secreted peptides, have been considered as important signaling molecules in diverse biological processes, including immunity. Current studies on RALF-modulated immunity mainly focus on Arabidopsis, but little is reported in crop plants. The rice immune receptor XA21 confers immunity to the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo). Here, we pursued functional characterization of rice RALF26 (OsRALF26) up-regulated by Xoo during XA21-mediated immune response. When applied exogenously as a recombinant peptide, OsRALF26 induced a series of immune responses, including pathogenesis-related genes (PRs) induction, reactive oxygen species (ROS) production, and callose deposition in rice and/or Arabidopsis. Transgenic rice and Arabidopsis overexpressing OsRALF26 exhibited significantly enhanced resistance to Xoo and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), respectively. In yeast two-hybrid, pull-down assays, and co-immunoprecipitation analyses, rice FER-like receptor 1 (OsFLR1) was identified as a receptor of OsRALF26. Transient expression of OsFLR1 in Nicotiana benthamiana leaves displayed significantly increased ROS production and callose deposition after OsRALF26 treatment. Together, we propose that OsRALF26 induced by Xoo in an XA21-dependent manner is perceived by OsFLR1 and may play a novel role in the enforcement of XA21-mediated immunity.

Genetic ablation of synaptotagmin-9 alters tomosyn-1 function to increase insulin secretion from pancreatic ß-cells improving glucose clearance.

Stimulus-coupled insulin secretion from the pancreatic islet ß-cells involves the fusion of insulin granules to the plasma membrane (PM) via SNARE complex formation-a cellular process key for maintaining whole-body glucose homeostasis. Less is known about the role of endogenous inhibitors of SNARE complexes in insulin secretion. We show that an insulin granule protein synaptotagmin-9 (Syt9) deletion in mice increased glucose clearance and plasma insulin levels without affecting insulin action compared to the control mice. Upon glucose stimulation, increased biphasic and static insulin secretion were observed from ex vivo islets due to Syt9 loss. Syt9 colocalizes and binds with tomosyn-1 and the PM syntaxin-1A (Stx1A); Stx1A is required for forming SNARE complexes. Syt9 knockdown reduced tomosyn-1 protein abundance via proteasomal degradation and binding of tomosyn-1 to Stx1A. Furthermore, Stx1A-SNARE complex formation was increased, implicating Syt9-tomosyn-1-Stx1A complex is inhibitory in insulin secretion. Rescuing tomosyn-1 blocked the Syt9-knockdown-mediated increases in insulin secretion. This shows that the inhibitory effects of Syt9 on insulin secretion are mediated by tomosyn-1. We report a molecular mechanism by which ß-cells modulate their secretory capacity rendering insulin granules nonfusogenic by forming the Syt9-tomosyn-1-Stx1A complex. Altogether, Syt9 loss in ß-cells decreases tomosyn-1 protein abundance, increasing the formation of Stx1A-SNARE complexes, insulin secretion, and glucose clearance. These outcomes differ from the previously published work that identified Syt9 has either a positive or no effect of Syt9 on insulin secretion. Future work using ß-cell-specific deletion of Syt9 mice is key for establishing the role of Syt9 in insulin secretion.

Deciphering the virome of Chunkung (Cnidium officinale) showing dwarfism-like symptoms via a high-throughput sequencing analysis.

BACKGROUND: Viruses have notable effects on agroecosystems, wherein they can adversely affect plant health and cause problems (e.g., increased biosecurity risks and economic losses). However, our knowledge of their diversity and interactions with specific host plants in ecosystems remains limited. To enhance our understanding of the roles that viruses play in agroecosystems, comprehensive analyses of the viromes of a wide range of plants are essential. High-throughput sequencing (HTS) techniques are useful for conducting impartial and unbiased investigations of plant viromes, ultimately forming a basis for generating further biological and ecological insights. This study was conducted to thoroughly characterize the viral community dynamics in individual plants. RESULTS: An HTS-based virome analysis in conjunction with proximity sampling and a tripartite network analysis were performed to investigate the viral diversity in chunkung (Cnidium officinale) plants. We identified 61 distinct chunkung plant-associated viruses (27 DNA and 34 RNA viruses) from 21 known genera and 6 unclassified genera in 14 known viral families. Notably, 12 persistent viruses (7 DNA and 5 RNA viruses) were exclusive to dwarfed chunkung plants. The detection of viruses from the families Partitiviridae, Picobirnaviridae, and Spinareoviridae only in the dwarfed plants suggested that they may contribute to the observed dwarfism. The co-infection of chunkung by multiple viruses is indicative of a dynamic and interactive viral ecosystem with significant sequence variability and evidence of recombination. CONCLUSIONS: We revealed the viral community involved in chunkung. Our findings suggest that chunkung serves as a significant reservoir for a variety of plant viruses. Moreover, the co-infection rate of individual plants was unexpectedly high. Future research will need to elucidate the mechanisms enabling several dozen viruses to co-exist in chunkung. Nevertheless, the important insights into the chunkung virome generated in this study may be relevant to developing effective plant viral disease management and control strategies.

Gene therapy for glycogen storage diseases.

Glycogen storage disorders (GSDs) are inherited disorders of metabolism resulting from the deficiency of individual enzymes involved in the synthesis, transport, and degradation of glycogen. This literature review summarizes the development of gene therapy for the GSDs. The abnormal accumulation of glycogen and deficiency of glucose production in GSDs lead to unique symptoms based upon the enzyme step and tissues involved, such as liver and kidney involvement associated with severe hypoglycemia during fasting and the risk of long-term complications including hepatic adenoma/carcinoma and end stage kidney disease in GSD Ia from glucose-6-phosphatase deficiency, and cardiac/skeletal/smooth muscle involvement associated with myopathy +/- cardiomyopathy and the risk for cardiorespiratory failure in Pompe disease. These symptoms are present to a variable degree in animal models for the GSDs, which have been utilized to evaluate new therapies including gene therapy and genome editing. Gene therapy for Pompe disease and GSD Ia has progressed to Phase I and Phase III clinical trials, respectively, and are evaluating the safety and bioactivity of adeno-associated virus vectors. Clinical research to understand the natural history and progression of the GSDs provides invaluable outcome measures that serve as endpoints to evaluate benefits in clinical trials. While promising, gene therapy and genome editing face challenges with regard to clinical implementation, including immune responses and toxicities that have been revealed during clinical trials of gene therapy that are underway. Gene therapy for the glycogen storage diseases is under development, addressing an unmet need for specific, stable therapy for these conditions.

Complete nucleotide sequence of chrysanthemum virus D, a polero-like virus.

A putative new polerovirus, named "chrysanthemum virus D" (ChVD), was detected in a Chrysanthemum morifolium plant in South Korea. The virus was identified by high-throughput sequencing and confirmed by reverse transcription polymerase chain reaction. The entire ChVD genome is composed of 5,963 nucleotides and contains seven open reading frames (ORF0-5 and ORF3a), which are arranged similarly to those of other poleroviruses. These ORFs encode the putative proteins P0-5 and P3a, respectively. Pairwise amino acid sequence comparisons showed that the ChVD P0-5 and P3a proteins have 30.45-75% sequence identity to the corresponding proteins of other members of the genus Polerovirus. Since one of the species demarcation criteria for the genus Polerovirus is > 10% difference in the amino acid sequence of any gene product, the sequence comparisons indicate that ChVD represents a new species in this genus. Phylogenetic analysis of the P1-P2 and P3 amino acid sequences further indicate that ChVD is a novel polerovirus.

Complete genome sequence of a new virus, a tentative member of the family Geminiviridae, infecting hedge bindweed (Calystegia sepium) in South Korea.

The family Convolvulaceae comprises approximately 50-60 genera with approximately 1600-1700 species, exhibiting a rich diversity of morphological characteristics and occupying a broad range of ecological habitats. High-throughput sequencing identified a tentative new virus in the family Geminiviridae infecting Calystegia sepium var. japonica in South Korea. The 2,706 nt long genome comprises six open reading frames (ORFs). The analysis of the nucleotide sequence of the genome and the putative amino acid sequences of ORFs indicated that the virus was closely related to the members of the family Geminiviridae. The genome organization of the virus was similar to that of members of the genus Topilevirus in terms of the number of ORFs and splicing signal. However, the virus, tentatively named bindweed mottle virus (BWMV), may not be assigned to current genera into the family Geminiviridae.

Complete genome sequence of a new putative member of the genus Pelarspovirus that infects Quercus aliena Blume in South Korea.

The complete nucleotide sequence of a newly discovered virus infecting Quercus aliena Blume, tentatively named "quercus leafroll virus" (QLRV), was determined through high-throughput and Sanger sequencing. The sequence comprises 3,940 nucleotides, has five open reading frames, and has a typical pelarspovirus genome organization, with neither 3' polyadenylation nor a 5' cap. The proteins encoded by QLRV share 17.9 to 44.2% amino acid sequence identity with known pelarspovirus proteins. The highest amino acid sequence identity values for the RNA-dependent RNA polymerase (RdRp) and coat protein were 67.5% and 55.2%, respectively, which are below the current thresholds for pelarspovirus species demarcation. On the basis of these results, we propose classifying QLRV as a new member of the genus Pelarspovirus, family Tombusviridae.

Long-term trajectories of densely reported depressive symptoms during an extended period of the COVID-19 pandemic in Switzerland: Social worries matter.

Previous mental health trajectory studies were mostly limited to the months before access to vaccination. They are not informing on whether public mental health has adapted to the pandemic. The aim of this analysis was to 1) investigate trajectories of monthly reported depressive symptoms from July 2020 to December 2021 in Switzerland, 2) compare average growth trajectories across regions with different stringency phases, and 3) explore the relative impact of self-reported worries related to health, economic and social domains as well as socio-economic indicators on growth trajectories. As part of the population-based Corona Immunitas program of regional, but harmonized, adult cohorts studying the pandemic course and impact, participants repeatedly reported online to the DASS-21 instrument on depressive symptomatology. Trajectories of depressive symptoms were estimated using a latent growth model, specified as a generalised linear mixed model. The time effect was modelled parametrically through a polynomial allowing to estimate trajectories for participants' missing time points. In all regions level and shape of the trajectories mirrored those of the KOF Stringency-Plus Index, which quantifies regional Covid-19 policy stringency. The higher level of average depression in trajectories of those expressing specific worries was most noticeable for the social domain. Younger age, female gender, and low household income went along with higher mean depression score trajectories throughout follow-up. Interventions to promote long-term resilience are an important part of pandemic preparedness, given the observed lack of an adaptation in mental health response to the pandemic even after the availability of vaccines in this high-income context.

Rice NLR protein XinN1, induced by a pattern recognition receptor XA21, confers enhanced resistance to bacterial blight.

KEY MESSAGE: Rice CC-type NLR XinN1, specifically induced by a PRR XA21, activates defense pathways against Xoo. Plants have evolved two layers of immune systems regulated by two different types of immune receptors, cell surface located pattern recognition receptors (PRRs) and intracellular nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs). Plant PRRs recognize conserved molecular patterns from diverse pathogens, resulting in pattern-triggered immunity (PTI), whereas NLRs are activated by effectors secreted by pathogens into plant cells, inducing effector-triggered immunity (ETI). Rice PRR, XA21, recognizes a tyrosine-sulfated RaxX peptide (required for activation of XA21-mediated immunity X) as a molecular pattern secreted by Xanthomonas oryzae pv. oryzae (Xoo). Here, we identified a rice NLR gene, XinN1, that is specifically induced during the XA21-mediated immune response against Xoo. Transgenic rice plants overexpressing XinN1 displayed increased resistance to infection by Xoo with reduced lesion length and bacterial growth. Overexpression of autoactive mutant of XinN1 (XinN1D543V) also displayed increased resistance to Xoo, accompanied with severe growth retardation and cell death. In rice protoplast system, overexpression of XinN1 or XinN1D543V significantly elevated reactive oxygen species (ROS) production and cytosolic-free calcium (Ca2+) accumulations. In addition, XinN1 overexpression additionally elevated the ROS burst caused by the interaction between XA21 and RaxX-sY and induced the transcription of PTI signaling components, including somatic embryogenesis receptor kinases (OsSERKs) and receptor-like cytoplasmic kinases (OsRLCKs). Our results suggest that XinN1 induced by the PRR XA21 activates defense pathways and provides enhanced resistance to Xoo in rice.

Successful AAV8 readministration: Suppression of capsid-specific neutralizing antibodies by a combination treatment of bortezomib and CD20 mAb in a mouse model of Pompe disease.

BACKGROUND: A major challenge to adeno-associated virus (AAV)-mediated gene therapy is the presence of anti-AAV capsid neutralizing antibodies (NAbs), which can block viral vector transduction even at very low titers. In the present study, we examined the ability of a combination immunosuppression (IS) treatment with bortezomib and a mouse-specific CD20 monoclonal antibody to suppress anti-AAV NAbs and enable readministration of AAV vectors of the same capsid in mice. METHODS: An AAV8 vector (AAV8-CB-hGAA) that ubiquitously expresses human α-glucosidase was used for initial gene therapy and a second AAV8 vector (AAV8-LSP-hSEAP) that contains a liver-specific promoter to express human secreted embryonic alkaline phosphatase (hSEAP) was used for AAV readministration. Plasma samples were used for determination of anti-AAV8 NAb titers. Cells isolated from whole blood, spleen, and bone marrow were analyzed for B-cell depletion by flow cytometry. The efficiency of AAV readministration was determined by the secretion of hSEAP in blood. RESULTS: In näive mice, an 8-week IS treatment along with AAV8-CB-hGAA injection effectively depleted CD19+ B220+ B cells from blood, spleen, and bone marrow and prevented the formation of anti-AAV8 NAbs. Following administration of AAV8-LSP-hSEAP, increasing levels of hSEAP were detected in blood for up to 6 weeks, indicating successful AAV readministration. In mice pre-immunized with AAV8-CB-hGAA, comparison of IS treatment for 8, 12, 16, and 20 weeks revealed that the 16-week IS treatment demonstrated the highest plasma hSEAP level following AAV8-LSP-hSEAP readministration. CONCLUSIONS: Our data suggest that this combination treatment is an effective IS approach that will allow retreatment of patients with AAV-mediated gene therapy. A combination IS treatment with bortezomib and a mouse-specific CD20 monoclonal antibody effectively suppressed anti-AAV NAbs in naïve mice and in mice with pre-existing antibodies, allowing successful readministration of the same AAV capsid vector.

Complete genome sequence of Stellaria aquatica virus B, a novel polerovirus that infects Stellaria aquatica.

The complete genome sequence of Stellaria aquatica virus B (StAVB), a new member of the genus Polerovirus that infects Stellaria aquatica, was determined using high-throughput RNA sequencing with confirmation by Sanger sequencing. The complete StAVB genome (GenBank accession no. OP389993) is 5,900 nucleotide (nt) long with seven open reading frames (ORF0-5 and ORF3a) that encode putative proteins (P0-P5 and P3a) in a similar configuration to that of other typical poleroviruses. Pairwise sequence comparisons with other poleroviruses showed 38-50% nt sequence identity in the complete genome and 13-24%, 36-45%, 7-68%, and 6-50% amino acid sequence identity in (aa), for the P0, P1-2, P3, and P4 protein, respectively. These data, together with the results of phylogenetic analysis, indicate that StAVB should be classified as a new member of the genus Polerovirus, family Solemoviridae.

Molecular characterization of a novel umbra-like virus from Thuja orientalis (arborvitae) in South Korea.

A novel umbra-like virus was identified in arborvitae in South Korea using RNA sequencing (RNA-seq). The virus identified was tentatively named "arborvitae umbra-like virus" (AULV) and contained a 4,300-nucleotide genome organized into four non-structural open reading frames (ORFs). Cloning and Sanger sequencing were used to confirm the viral contig sequence and determine the size of the genome. Genome analysis indicated that ORF2 encodes an RNA-dependent RNA polymerase that is probably expressed through ribosomal frameshifting. ORF3 encodes a putative long-distance movement protein, while the functions of ORFs 1 and 4 are unknown. The virus lacks a coat protein gene. The genome of AULV shares 27.3%-48.4% nucleotide sequence identity with closely related umbraviruses. Phylogenetic analysis based on the complete genome sequences and amino acid sequences of the RNA-dependent RNA polymerase revealed that AULV forms a monophyletic lineage with Guiyang paspalum paspaloides tombus-like virus (GPpTV1). We suggest that AULV is a novel umbra-like virus belonging to the family Tombusviridae.

A single-center prospective study regarding time to return to activities of daily living after craniotomy for brain tumors.

BACKGROUND: There are few studies on the time to return to activities of daily living (ADL) after craniotomy in patients with brain tumors. This study aimed to investigate the duration before returning to ADLs after craniotomy for brain tumors and present data that can provide information and guidelines on the appropriate time needed. METHODS: Patients (n = 183 of 234) who underwent craniotomy for brain tumors between April 2021 and July 2021 capable of self-care upon discharge were enrolled, and data of 158 were collected. The start time of 85 ADL items was prospectively investigated for 4 months postoperatively, using the self-recording sheet. RESULTS: Over 89% and 87% of the patients performed basic ADL items within a month and instrumental ADL items within 2 months (medians: within 18 days), except for a few. Regarding work, 50% of the patients returned within 4 months. Washing hair with a wound was performed at 18 days of median value, after 4 months of dyeing/perming hair, 6 days of drinking coffee/tea, after 4 months of air travel, and 40 days of complementary and alternative medicine. In patients with infratentorial tumors or surgical problems, return times were much later for various items. CONCLUSIONS: It is possible to provide practical information and guidelines on the duration to return to ADL after craniotomy in brain tumor patients. These study findings also reduce uncertainty about recovery and daily life and help patients return to their daily life at the appropriate time, thereby maintaining function and daily well-being after surgery.

Depression trajectories during the COVID-19 pandemic in the high-quality health care setting of Switzerland: the COVCO-Basel cohort.

OBJECTIVES: During the pandemic, Switzerland avoided stringent lockdowns and provided funds to stabilize the economy. To assess whether and in what subgroups the pandemic impacted on depressive symptoms in this specific Swiss context, we derived depression trajectories over an extended pandemic period in a Swiss cohort and related them to individuals' sociodemographic characteristics. STUDY DESIGN: This was a population-based cohort study. METHODS: The population-based COVCO-Basel cohort in North-Western Switzerland invited 112,848 adult residents of whom 12,724 participated at baseline. Between July 2020 and December 2021, 6396 participants answered to additional 18 monthly online questionnaires. Depression symptoms were repeatedly measured by the DASS-21 scale. Group-based Trajectory Models methods were applied to identify clusters of similar depression trajectories. Trajectory clusters were characterized descriptively and with a Multinomial response model. RESULTS: Three distinct trajectories were identified. The 'Highly affected' trajectory (13%) had a larger presence of younger and female participants with lower average income, higher levels of past depression, and living alone. A majority of individuals in the 'Unaffected' trajectory (52%) were of medium or high average income, older average age, without previous depression symptoms, and not living alone. The 'Moderately affected' trajectory (35%) had a composition intermediate between the two opposite 'extreme' trajectories. CONCLUSIONS: This study is among few studies investigating depression trajectories up to the time when COVID-19 vaccination was readily available to the entire population. During these 18 months of the pandemic, depressive symptoms increased in a substantial percentage of participants. Economic support, high-quality health care system, and moderate containment measures did not sufficiently protect all population subgroups from adverse, potentially long-term psychological pandemic impacts.

First report of Kalanchoe Latent Virus naturally infecting common bean (Phaseolus vulgaris L.) in South Korea.

The common bean (Phaseolus vulgaris; family: Fabaceae) is an economically and nutritionally important food crop worldwide (Ganesan et al. 2017). In 2021, several plants collected from different provinces in South Korea had symptoms of viral infections (e.g., mild yellow-greenish speckling, stunting, crinkling, and deformed leaves). To identify the causal pathogens, total RNA was isolated from pooled leaf tissues from all samples (n = 29) for paired-end high-throughput sequencing (HTS). The cDNA library was constructed after eliminating ribosomal RNA using the TruSeq RNA Sample Prep Kit and then sequenced using the Illumina NovaSeq 6000 platform (Macrogen, Korea). The 297,868,156 paired-end clean reads (150 nt) were de novo assembled using Trinity with default parameters. BLASTx was used for the contig analysis, which revealed the pooled samples were infected with several plant viruses (e.g., turnip mosaic virus, zucchini yellow mosaic virus, cucumber mosaic virus, lily mottle virus). Notably, the assembled contigs included a single viral contig (8,472 nt) comprising the nearly complete KLV genome (HTS mean coverage: 39.46%). Kalanchoe latent virus (KLV; genus: Carlavirus; family: Betaflexiviridae) has been detected in Kalanchoë blossfeldiana (Hearon 1982), Chenopodium quinoa (Dinesen et al. 2009), and Graptopetalum paraguayense (Sorrentino et al. 2017). The sequence was most similar (96.28% nucleotide identity; 99% query coverage) to KLV isolate DSMZ PV-0290 (GenBank: OP525283) from Denmark. The contig sequence was validated via reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from the 29 individually stored samples and nine primer sets specific for the KLV contig. All nine contig-specific overlapping fragments were amplified from only a P. vulgaris plant with mild yellowing mosaic symptoms collected on July 6, 2021, in Jeongseon County, South Korea. Additionally, 5' and 3' rapid amplification of cDNA ends (RACE)-specific primers were designed for the KLV contig sequence to determine the terminal ends of the genome of the South Korean KLV isolate using the 5'/3' RACE System (Invitrogen, Carlsbad, CA, USA). All of the amplified and overlapping fragments were cloned into the RBC T&A Cloning Vector (RBC Bioscience, Taipei, Taiwan) and sequenced using the Sanger method. The obtained full-length genomic sequence of the KLV isolate (KLV-SK22) was 8,517 nt long and was deposited in GenBank OQ718816. According to the BLASTn analysis, KLV-SK22 was highly similar (96.30% sequence identity; 100% query coverage) to the DSMZ PV-0290 isolate. Phylogenetic trees constructed on the basis of coat protein and RNA-dependent RNA polymerase amino acid sequences revealed that KLV-SK22 is closely related to the DSMZ PV-0290 and PV-0290B isolates from Denmark, respectively. At the genome and gene levels, the individual sequence identities between the carlaviruses and other KLV isolates were 96.29% to 100% (Adams et al. 2004). Additionally, an RT-PCR analysis using detection primers specific for KLV-SK22 did not detect KLV in 15 samples (P. vulgaris = 3, Glycine max = 8, Pueraria montana = 2, Trifolium repens = 1, and Vigna angularis = 1) randomly collected from different regions in South Korea. Based on these results, KLV infection may not be widespread at this time in South Korea. To the best of our knowledge, this is the first report of KLV in P. vulgaris in South Korea or elsewhere. Our findings will aid future research on the epidemiology and long-term management of KLV-related diseases.

Promise and Challenges of T Cell Immunotherapy for Osteosarcoma.

The cure rate for metastatic or relapsed osteosarcoma has not substantially improved over the past decades despite the exploitation of multimodal treatment approaches, allowing long-term survival in less than 30% of cases. Patients with osteosarcoma often develop resistance to chemotherapeutic agents, where personalized targeted therapies should offer new hope. T cell immunotherapy as a complementary or alternative treatment modality is advancing rapidly in general, but its potential against osteosarcoma remains largely unexplored. Strategies incorporating immune checkpoint inhibitors (ICIs), chimeric antigen receptor (CAR) modified T cells, and T cell engaging bispecific antibodies (BsAbs) are being explored to tackle relapsed or refractory osteosarcoma. However, osteosarcoma is an inherently heterogeneous tumor, both at the intra- and inter-tumor level, with no identical driver mutations. It has a pro-tumoral microenvironment, where bone cells, stromal cells, neovasculature, suppressive immune cells, and a mineralized extracellular matrix (ECM) combine to derail T cell infiltration and its anti-tumor function. To realize the potential of T cell immunotherapy in osteosarcoma, an integrated approach targeting this complex ecosystem needs smart planning and execution. Herein, we review the current status of T cell immunotherapies for osteosarcoma, summarize the challenges encountered, and explore combination strategies to overcome these hurdles, with the ultimate goal of curing osteosarcoma with less acute and long-term side effects.

Characteristics and Absorption Rate of Whey Protein Hydrolysates Prepared Using Flavourzyme after Treatment with Alcalase and Protamex.

The purpose of this study was to evaluate the physicochemical properties of whey protein hydrolysate and determine changes in absorption rate due to enzymatic hydrolysis. The molecular weight distribution analysis of whey protein concentrate (WPC) and low-molecule whey protein hydrolysate (LMWPH) using the Superdex G-75 column revealed that LMWPH is composed of peptides smaller than those in WPC. Fourier-transform infrared spectroscopy indicated differences in peak positions between WPC and LMWPH, suggesting hydrolysis-mediated changes in secondary structures. Moreover, LMWPH exhibited higher thermal stability and faster intestinal permeation than WPC. Additionally, oral LMWPH administration increased serum protein content at 20 min, whereas WPC gradually increased serum protein content after 40 min. Although the total amount of WPC and LMWPH absorption was similar, LMWPH absorption rate was higher. Collectively, LMWPH, a hydrolysate of WPC, has distinct physicochemical properties and enhanced absorptive characteristics. Taken together, LMWPH is composed of low-molecular-weight peptides with low antigenicity and has improved absorption compared to WPC. Therefore, LMWPH can be used as a protein source with high bioavailability in the development of functional materials.

HDAC inhibitor and proteasome inhibitor induce cleavage and exosome-mediated secretion of HSP90 in mouse pluripotent stem cells.

Heat shock protein 90 (HSP90), one of the molecular chaperones, stabilizes several proteins necessary to maintain pluripotency of embryonic stem (ES) cells. Recently, we reported that HDAC inhibitors and proteasome inhibitors down-regulate HSP90 activity through HSP90 cleavage induced by reactive oxygen species (ROS) generation and caspase 10 activation in various cancer cells. In this study, we investigated HSP90 cleavage in mouse ES cells. HDAC inhibitors and proteasome inhibitors induced HSP90 cleavage in the mouse ES cell line R1, and the cleaved HSP90 was barely found in the cells and instead secreted out of the cells through the exosome. The HSP90 cleavage was associated with ROS generation and caspase 10 activation. In addition, HDAC inhibitor and proteasome inhibitor induced Fas expression, and the inhibition of caspase 8, a downstream molecule of Fas, blocked HSP90 cleavage. Therefore, HDAC inhibitor- and proteasome inhibitor-mediated HSP90 cleavage was induced by ROS generation and Fas expression. We observed similar results in mouse induced pluripotent stem (iPS) cells. Taken together, HSP90 cleavage was induced in mouse pluripotent cells similarly to cancer cells but differently regulated through Fas expression and exosomal secretion. These findings will be helpful in elucidating the regulation of HSP90 upon stress in pluripotent stem cells.

Transcriptional responses of human intestinal epithelial HT-29 cells to spore-displayed p40 derived from Lacticaseibacillus rhamnosus GG.

BACKGROUNDS: The aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells. RESULTS: p40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action. CONCLUSIONS: This study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40.

Ablation of Sam68 in adult mice increases thermogenesis and energy expenditure.

Genetic deletion of Src associated in mitosis of 68kDa (Sam68), a pleiotropic adaptor protein prevents high-fat diet-induced weight gain and insulin resistance. To clarify the role of Sam68 in energy metabolism in the adult stage, we generated an inducible Sam68 knockout mice. Knockout of Sam68 was induced at the age of 7-10 weeks, and then we examined the metabolic profiles of the mice. Sam68 knockout mice gained less body weight over time and at 34 or 36 weeks old, had smaller fat mass without changes in food intake and absorption efficiency. Deletion of Sam68 in mice elevated thermogenesis, increased energy expenditure, and attenuated core-temperature drop during acute cold exposure. Furthermore, we examined younger Sam68 knockout mice at 11 weeks old before their body weights deviate, and confirmed increased energy expenditure and thermogenic gene program. Thus, Sam68 is essential for the control of adipose thermogenesis and energy homeostasis in the adult.