Malnutrition and maternal vaccination against typhoid toxin.

Children are particularly susceptible to typhoid fever caused by the bacterial pathogen Salmonella Typhi. Typhoid fever is prevalent in developing countries where diets can be less well-balanced. Here, using a murine model, we investigated the role of the macronutrient composition of the diet in maternal vaccination efficacies of two subunit vaccines targeting typhoid toxin: ToxoidVac and PltBVac. We found that maternal vaccinations protected all offspring against a lethal-dose typhoid toxin challenge in a balanced, normal diet (ND) condition, but the declined protection in a malnourished diet (MD) condition was observed in the PltBVac group. Despite the comparable antibody titers in both MD and ND mothers, MD offspring had a significantly lower level of typhoid toxin neutralizing antibodies than their ND counterparts. We observed a lower expression of the neonatal Fc receptor on the yolk sac of MD mothers than in ND mothers, agreeing with the observed lower antibody titers in MD offspring. Protein supplementation to MD diets, but not fat supplementation, increased FcRn expression and protected all MD offspring from the toxin challenge. Similarly, providing additional typhoid toxin-neutralizing antibodies to MD offspring was sufficient to protect all MD offspring from the toxin challenge. These results emphasize the significance of balanced/normal diets for a more effective maternal vaccination transfer to their offspring.

Enrichment of Activated Fibroblasts as a Potential Biomarker for a Non-Durable Response to Anti-Tumor Necrosis Factor Therapy in Patients with Crohn's Disease.

We investigated whether the response to anti-tumor necrosis factor (anti-TNF) treatment varied according to inflammatory tissue characteristics in Crohn's disease (CD). Bulk RNA sequencing (RNA-seq) data were obtained from inflamed and non-inflamed tissues from 170 patients with CD. The samples were clustered based on gene expression profiles using principal coordinate analysis (PCA). Cellular heterogeneity was inferred using CiberSortx, with bulk RNA-seq data. The PCA results displayed two clusters of CD-inflamed samples: one close to (Inflamed_1) and the other far away (Inflamed_2) from the non-inflamed samples. Inflamed_1 was rich in anti-TNF durable responders (DRs), and Inflamed_2 was enriched in non-durable responders (NDRs). The CiberSortx results showed that the cell fraction of activated fibroblasts was six times higher in Inflamed_2 than in Inflamed_1. Validation with public gene expression datasets (GSE16879) revealed that the activated fibroblasts were enriched in NDRs over Next, we used DRs by 1.9 times pre-treatment and 7.5 times after treatment. Fibroblast activation protein (FAP) was overexpressed in the Inflamed_2 and was also overexpressed in the NDRs in both the RISK and GSE16879 datasets. The activation of fibroblasts may play a role in resistance to anti-TNF therapy. Characterizing fibroblasts in inflamed tissues at diagnosis may help to identify patients who are likely to respond to anti-TNF therapy.

Identification of Bacterial Membrane Selectivity of Romo1-Derived Antimicrobial Peptide AMPR-22 via Molecular Dynamics.

The abuse or misuse of antibiotics has caused the emergence of extensively drug-resistant (XDR) bacteria, rendering most antibiotics ineffective and increasing the mortality rate of patients with bacteremia or sepsis. Antimicrobial peptides (AMPs) are proposed to overcome this problem; however, many AMPs have attenuated antimicrobial activities with hemolytic toxicity in blood. Recently, AMPR-11 and its optimized derivative, AMPR-22, were reported to be potential candidates for the treatment of sepsis with a broad spectrum of antimicrobial activity and low hemolytic toxicity. Here, we performed molecular dynamics (MD) simulations to clarify the mechanism of lower hemolytic toxicity and higher efficacy of AMPR-22 at an atomic level. We found four polar residues in AMPR-11 bound to a model mimicking the bacterial inner/outer membranes preferentially over eukaryotic plasma membrane. AMPR-22 whose polar residues were replaced by lysine showed a 2-fold enhanced binding affinity to the bacterial membrane by interacting with bacterial specific lipids (lipid A or cardiolipin) via hydrogen bonds. The MD simulations were confirmed experimentally in models that partially mimic bacteremia conditions in vitro and ex vivo. The present study demonstrates why AMPR-22 showed low hemolytic toxicity and this approach using an MD simulation would be helpful in the development of AMPs.

A Novel Peptide Derived from the Transmembrane Domain of Romo1 Is a Promising Candidate for Sepsis Treatment and Multidrug-Resistant Bacteria.

The emergence of multidrug-resistant (MDR) bacteria through the abuse and long-term use of antibiotics is a serious health problem worldwide. Therefore, novel antimicrobial agents that can cure an infection from MDR bacteria, especially gram-negative bacteria, are urgently needed. Antimicrobial peptides, part of the innate immunity system, have been studied to find bactericidal agents potent against MDR bacteria. However, they have many problems, such as restrained systemic activity and cytotoxicity. In a previous study, we suggested that the K58-R78 domain of Romo1, a mitochondrial protein encoded by the nucleus, was a promising treatment candidate for sepsis caused by MDR bacteria. Here, we performed sequence optimization to enhance the antimicrobial activity of this peptide and named it as AMPR-22 (antimicrobial peptide derived from Romo1). It showed broad-spectrum antimicrobial activity against 17 sepsis-causing bacteria, including MDR strains, by inducing membrane permeabilization. Moreover, treatment with AMPR-22 enabled a remarkable survival rate in mice injected with MDR bacteria in a murine model of sepsis. Based on these results, we suggest that AMPR-22 could be prescribed as a first-line therapy (prior to bacterial identification) for patients diagnosed with sepsis.

Hepatitis C Virus p7 Induces Membrane Permeabilization by Interacting with Phosphatidylserine.

Hepatitis C virus (HCV) p7 is known to be a nonselective cation channel for HCV maturation. Because the interaction of HCV proteins with host lipids in the endoplasmic reticulum membrane is crucial for the budding process, the identification of p7-lipid interactions could be important for understanding the HCV life cycle. Here, we report that p7 interacts with phosphatidylserine (PS) to induce membrane permeabilization. The interaction of p7 with PS was not inhibited by Gd3+ ions, which have been known to interact with negatively charged lipids, but channel activity and p7-induced mitochondrial depolarization were inhibited by Gd3+ ions. From the present results, we suggest that the p7-PS interaction plays an essential role in regulating its ion channel function and could be a potential molecular target for anti-HCV therapy.

A comparison of patients with anorexia nervosa and women who are constitutionally thin.

OBJECTIVE: Many of the clinical features of anorexia nervosa (AN) can be secondary problems associated with malnutrition, which mask the primary features of AN. This study aimed to investigate the intrinsic features of AN by comparing AN patients to women who are constitutionally thin (CT). METHOD: Twenty-six patients with AN and 53 CT women of similar body mass index (AN: 17.13 ± 1.73 kg/m2 , CT: 16.62 ± 0.61 kg/m2 ) participated in the study. We examined medical findings, psychiatric features, and disturbed body image between the two groups. RESULTS: Patients with AN had earlier menarche but less frequent menstrual periods compared to CT women. They had lower blood pressure, lower triiodothyronine, and fasting glucose levels than CT women. Patients with AN had more disturbed body images and restricted diet patterns and also had higher neuroticism compared to CT women. CONCLUSIONS: The patients with AN viewed their current body shape as fatter and had more restrained eating than the CT women. The results suggest that the disturbed body image may be a salient feature of AN differentiating from CT women, the underlying neural mechanism of which requires further study.

Mobile Self-Help Interventions as Augmentation Therapy for Patients with Anorexia Nervosa.

Background: It has been suggested that patients with anorexia nervosa (AN) who are resistant to first-line treatment would benefit from second-level interventions targeting specific features, an adaptive form of intervention. Guided self-help programs administered via mobile technology have the dual focus of instigating behavior change and managing anxiety associated with eating disorders in the moment and in users' naturalistic environments. Introduction: We evaluated the feasibility, acceptability, and preliminary augmentative effects of mobile-based, guided self-help interventions (Recovery vodcasts) after initial unsuccessful first-line treatments for patients with AN. Materials and Methods: Patients with AN who were unsuccessful in their treatments were recruited to access Recovery vodcasts as augmentation treatment for 3 weeks in Korea. Acceptability and feasibility of the intervention were evaluated, and qualitative feedback was collected. Preliminary treatment effects of adding the Recovery vodcasts were assessed, including eating disorder pathology, anxiety and depression symptoms, and body mass index. Results: The Recovery vodcasts were acceptable for patients with AN. The patients' psychopathologies of eating disorders improved with augmentation of the vodcasts in their first-line treatments. In addition, there was a tendency toward improvement of affective symptoms. The participants' feedback suggested that the intervention could be improved. Conclusions: The study demonstrated that the Recovery vodcasts were well accepted by Korean patients with AN. Moreover, augmentation of the Recovery vodcasts could facilitate improvements in psychopathology of eating disorders, anxiety, and mood symptoms for patients with AN.

Determinants of binge eating disorder among normal weight and overweight female college students in Korea.

PURPOSE: The aim of the present study was to describe the clinical features of binge eating disorder (BED) in normal weight and overweight undergraduate Korean women. METHODS: 117 overweight (BMI ≥ 25 kg/m2) and 346 normal weight (18 kg/m2 ≤ BMI < 25 kg/m2) undergraduate Korean women completed questionnaires to assess for BED. Their emotional eating behaviors, binge eating-related behaviors, a spectrum of compulsive behaviors such as substance abuse and obsessive-compulsive disorder, and psychological profiles were evaluated through personal interviews and questionnaires. The features of those with BED were compared to those without BED in the overweight and normal weight groups. RESULTS: Both normal weight and overweight BED women had higher levels of functional impairment, eating disorder psychopathology including emotional and external eating behaviors, and neuroticism than their non-BED counterparts. In the normal weight group, BED women had more frequent alcohol consumption and obsessive-compulsive symptoms than non-BED women. In the overweight group, BED women had higher levels of depression and lower extraversion than non-BED women. CONCLUSIONS: BED is associated with global functional impairment and mental health problems. Thus, the association with high functional impairments and psychiatric comorbidities suggest that people with BED may benefit from treatment. LEVEL III: Evidence obtained from well-designed case-control analytic studies, from more than one center.

Feasibility and acceptability of a prevention program for eating disorders (Me, You and Us) adapted for young adolescents in Korea.

PURPOSE: The aim of this study was to assess the adaptability and acceptability of a prevention program. METHODS: A total of 169 Korean students (83 boys and 86 girls) with a mean age of 12.3 years from a 6th grade class at an elementary school participated in the study. Mental health social workers delivered Me, You and Us, a school-based body image intervention program originally developed in the UK, through a set of six sessions. The participants were assessed in terms of their body satisfaction and self-esteem before the program, after the program, and at 1-month follow-up. They were also surveyed about their satisfaction and acceptability levels after the program. RESULTS: At baseline, girls had lower body satisfaction and self-esteem than boys, and their body satisfaction and self-esteem improved after the program. The improved body satisfaction was maintained at the 1-month follow-up. The efficacy of the program on body satisfaction was positively correlated with the frequency of their baseline level of "fat talk." The program was more effective in girls with possible symptoms of an eating disorder at baseline. 93.7% of boys and 77.4% of girls responded that they enjoyed the program. CONCLUSIONS: The program Me, You and Us was well-accepted by early adolescents in Korea and it can play a role in increasing body satisfaction and self-esteem by reducing "fat talk" in 6th grade students. LEVEL OF EVIDENCE: Level III, cohort study with intervention.

Hepatitis C virus p7 mediates membrane-to-membrane adhesion.

Viroporin p7 of the hepatitis C virus (HCV) acts as an ion channel for pH equilibration to stabilize HCV particles; most studies of p7 have focused on this role. However, pH equilibration by p7 via its ion channel activity does not fully explain the importance of p7 in HCV particle production. Indeed, several researchers have suggested p7 to have an unidentified ion channel-independent function. Here, we show that p7 has a novel role as a lipid raft adhesion factor, which is independent of its ion channel activity. We found that p7 targets not only the liquid-disordered (Ld) phase, but also the negatively-charged liquid-ordered (Lo) phase that can be represented as a lipid raft. p7 clusters at the phase boundary of the neutral Ld phase and the negatively-charged Lo phase. Interestingly, p7 targeting the Lo phase facilitates membrane-to-membrane adhesion, and this activity is not inhibited by p7 ion channel inhibitors. Our results demonstrated that HCV p7 has dual roles as a viroporin and as a lipid raft adhesion factor. This ion channel-independent function of p7 might be an attractive target for development of anti-HCV compounds.

Single missense mutations in Vi capsule synthesis genes confer hypervirulence to Salmonella Typhi.

Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S. Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control Vi polymerization or acetylation is enough to result in different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper Vi capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo Vi capsule variants have primarily been identified in Africa, whereas the hyper Vi capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria.

Single missense mutations in Vi capsule synthesis genes confer hypervirulence to Salmonella Typhi.

Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S . Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control the length or acetylation of Vi is enough to create different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper-capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo-capsule variants have primarily been identified in Africa, whereas the hyper-capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria.

Antimicrobial properties of tomato juice and peptides against typhoidal Salmonella.

Tomatoes are readily available and affordable vegetables that offer a range of health benefits due to their bioactive molecules, such as antioxidants and antimicrobials. In contrast to the widely recognized antioxidant properties of tomatoes, their antimicrobial properties remain largely unexplored. Here, we present our findings on the antimicrobial properties of tomato juice and peptides, namely, tomato-derived antimicrobial peptides (tdAMPs), in relation to their effectiveness against typhoidal Salmonella. Our research has revealed that tomato juice demonstrates significant antimicrobial properties against Salmonella Typhi, a pathogen that specifically affects humans and is responsible for causing typhoid fever. By employing computational analysis of the tomato genome sequence, conducting molecular dynamics simulation, and performing functional analyses, we have successfully identified two tdAMPs, namely, tdAMP-1 and tdAMP-2. These tdAMPs have demonstrated potent antimicrobial properties by effectively disrupting bacterial membranes. The efficacy of tdAMP-2 is shown to be more effective than tdAMP-1. The efficacy of tdAMP-1 and tdAMP-2 has been demonstrated against drug-resistant S. Typhi, as well as hyper-capsular S. Typhi variants that possess hypervirulent characteristics, which are presently circulating in countries with endemicity. Tomato juice, along with the two tdAMPs, has demonstrated effectiveness against uropathogenic Escherichia coli as well. This underscores their potential as viable agents in combating certain Gram-negative pathogens. This study provides valuable insights into the development of effective and sustainable public health strategies that utilize tomato and its derivatives as lifestyle interventions.IMPORTANCEIn this study, we investigate the antimicrobial properties of tomato juice, the most widely consumed affordable vegetables, as well as tomato-derived antimicrobial peptides, in relation to their effectiveness against foodborne pathogens with an emphasis on Salmonella Typhi, a deadly human-specific pathogen.

Overexpression of Romo1 promotes production of reactive oxygen species and invasiveness of hepatic tumor cells.

BACKGROUND & AIMS: Chronic oxidative stress from reactive oxygen species (ROS) produced by the mitochondria promotes hepatocarcinogenesis and tumor progression. However, the exact mechanism by which mitochondrial ROS contributes to tumor cell invasion is not known. We investigated the role of ROS modulator 1 (Romo1) in hepatocellular carcinoma (HCC) development and tumor cell invasiveness. METHODS: We performed real-time, semi-quantitative, reverse transcriptase polymerase chain reaction; invasion and luciferase assays; and immunofluorescence and immunohistochemical analyses. The formation of pulmonary metastatic nodules after tumor cell injection was tested in severe combined immunodeficient mice. We analyzed Romo1 expression in HCC cell lines and tissues (n = 95). RESULTS: Expression of Romo1 was increased in HCC cells, compared with normal human lung fibroblast cells. Exogenous expression of Romo1 in HCC cells increased their invasive activity, compared with control cells. Knockdown of Romo1 in Hep3B and Huh-7 HCC cells reduced their invasive activity in response to stimulation with 12-O-tetradecanoylphorbol-13-acetate. Levels of Romo1 were increased compared with normal liver tissues in 63 of 95 HCC samples from patients. In HCC samples from patients, there was an inverse correlation between Romo1 overexpression and patient survival times. Increased levels of Romo1 also correlated with vascular invasion by the tumors, reduced differentiation, and larger tumor size. CONCLUSIONS: Romo1 is a biomarker of HCC progression that might be used in diagnosis. Reagents that inhibit activity of Romo1 and suppress mitochondrial ROS production, rather than eliminate up-regulated intracellular ROS, might be developed as cancer therapies.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

RNA-Seq-Based Molecular Classification Analyses in Colorectal Cancer and Synchronous Adenoma.

Colorectal cancers (CRC) are classified into consensus molecular subtypes (CMS) based on gene expression profiles. The revised classification system iCMS was proposed by considering intrinsic epithelial status, microsatellite instability (MSI), and fibrosis. This study aimed to provide molecular evidence for the adenoma-carcinoma sequence concept by examining CRC and synchronous adenomas using iCMS. Epithelial CMS cell proportion was estimated using CiberSortx, an in silico cell fractionation method that included CMS cell types among the reference cell types. A random forest (RF) model estimated the posterior probabilities of CMS classes, which were compared with the CiberSortx results. Gene expression profiles of the published iCMS signature panel were retrieved from our dataset and subjected to heatmap clustering for classification. Bulk RNA sequencing data were collected from 29 adenocarcinomas and 11 adenoma samples. CiberSortx showed all CRC contained either CMS2 or CMS3 as the major epithelial cancer cell type. The RF model classified approximately half of the CRC as CMS4, whereas CMS4 was hardly detected by CiberSortx. Because they were enriched with myofibroblasts as per the CiberSortx classification, we tentatively designated them as iCMS2-F/iCMS3-F. iCMS coupled with the application of an in silico cell fractionation method can provide the molecular dissection of CRC and adenoma.

Glycan-mediated molecular interactions in bacterial pathogenesis.

Glycans are expressed on the surface of nearly all host and bacterial cells. Not surprisingly, glycan-mediated molecular interactions play a vital role in bacterial pathogenesis and host responses against pathogens. Glycan-mediated host-pathogen interactions can benefit the pathogen, host, or both. Here, we discuss (i) bacterial glycans that play a critical role in bacterial colonization and/or immune evasion, (ii) host glycans that are utilized by bacteria for pathogenesis, and (iii) bacterial and host glycans involved in immune responses against pathogens. We further discuss (iv) opportunities and challenges for transforming these research findings into more effective antibacterial strategies, and (v) technological advances in glycoscience that have helped to accelerate progress in research. These studies collectively offer valuable insights into new perspectives on antibacterial strategies that may effectively tackle the drug-resistant pathogens that are rapidly spreading globally.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

RASSF1A suppresses the activated K-Ras-induced oxidative DNA damage.

The mutant K-Ras elevates intracellular reactive oxygen species (ROS) levels and leads to oxidative DNA damage, resulting in malignant cell transformation. Ras association domain family 1 isoform A (RASSF1A) is known to play a role as a Ras effector. However, the suppressive effect of RASSF1A on K-RasV12-induced ROS increase and DNA damage has not been identified. Here, we show that RASSF1A blocks K-RasV12-triggered ROS production. RASSF1A expression also inhibits oxidative DNA damage and chromosomal damage. From the results obtained in this study, we suggest that RASSF1A regulates the cellular ROS levels enhanced by the Ras signaling pathway, and that it may function as a tumor suppressor by suppressing DNA damage caused by activated Ras.

Complete Genome Sequence of Salmonella enterica Serovar Typhi Strain ISP2825.

Salmonella enterica serovar Typhi ISP2825, isolated in 1983 from a Chilean patient, is one of the major S. Typhi strains used for research, along with strains Ty2, CT18, and H58. The complete genome sequence of ISP2825, consisting of a 4,774,014-bp circular chromosome, will help us understand typhoid pathogenesis and evolution.