The Long-Term Effect of Weight Loss on the Prevention of Progression to Cirrhosis among Patients with Obesity and MASH-Related F3 Liver Fibrosis.

This multi-center retrospective study examined the effect of weight loss on the prevention of progression to cirrhosis in a sample exclusively composed of patients with obesity and MASH-related F3 liver fibrosis. Adult patients with obesity and biopsy-confirmed MASH-related F3 liver fibrosis (n = 101) from two liver transplant centers in the US were included in the study. A higher proportion of patients who did not progress to cirrhosis achieved >5% weight loss at follow-up (59% vs. 30%, p = 0.045). In multivariable analysis, patients with >5% weight loss at follow-up had a lower hazard of developing cirrhosis compared to patients with no weight loss or weight gain (HR: 0.29, 95%, CI: 0.08-0.96); whereas, diabetes (HR: 3.24, 95%, CI: 1.21-8.67) and higher LDL levels (HR: 1.02, 95%, CI: 1.01-1.04) were associated with higher hazards of progression to cirrhosis. Weight loss >5% has the potential to prevent disease progression to cirrhosis in patients with obesity and MASH-related F3 liver fibrosis. The realization of this benefit requires weight loss maintenance longer than one year. Larger prospective studies are needed to determine how weight loss impacts other patient-centered outcomes such as mortality, hepatic decompensation, and hepatocellular carcinoma in patients with obesity and MASH-related F3 liver fibrosis.

Hepatic lipid droplet-associated proteome changes distinguish dietary-induced fatty liver from glucose tolerance in male mice.

Fatty liver is characterized by the expansion of lipid droplets (LDs) and is associated with the development of many metabolic diseases. We assessed the morphology of hepatic LDs and performed quantitative proteomics in lean, glucose-tolerant mice compared with high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD- and HStD-fed mice had more and larger LDs than Chow-fed animals. We observed striking differences in liver LD proteomes of HFD- and HStD-fed mice compared with Chow-fed mice, with fewer differences between HFD and HStD. Taking advantage of our diet strategy, we identified a fatty liver LD proteome consisting of proteins common in HFD- and HStD-fed mice, as well as a proteome associated with glucose tolerance that included proteins shared in Chow and HStD but not HFD-fed mice. Notably, glucose intolerance was associated with changes in the ratio of adipose triglyceride lipase to perilipin 5 in the LD proteome, suggesting dysregulation of neutral lipid homeostasis in glucose-intolerant fatty liver. We conclude that our novel dietary approach uncouples ectopic lipid burden from insulin resistance-associated changes in the hepatic lipid droplet proteome.NEW & NOTEWORTHY This study identified a fatty liver lipid droplet proteome and one associated with glucose tolerance. Notably, glucose intolerance was linked with changes in the ratio of adipose triglyceride lipase to perilipin 5 that is indicative of dysregulation of neutral lipid homeostasis.

KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

Diphtheria case detection and carrier study of close contacts: A step towards diphtheria eradication.

INTRODUCTION: Diphtheria is an infectious disease caused by gram-positive bacilli C. diphtheriae involving nasal, pharyngeal, tonsillar, or laryngeal mucus membranes. The mortality rate is as high as 20%, with India contributing almost 78% of the world incidence. AIMS AND OBJECTIVES: We report a fatal case of nasopharyngeal diphtheria with carrier study in close contacts. MATERIALS AND METHODS: Seven years child presented with fever, throat pain, and earache for 3 days followed by neck swelling and noisy respiration. On examination, membrane was present in the throat, which was received for Albert and Gram staining and reported as positive for C. diphtheria like organisms followed by culture. The patient was treated with ADS and antibiotics, and intensively managed, but still succumbed to death. Follow-up was done for carriage of C. diphtheriae on the throat and nasopharyngeal swabs of siblings and close contacts. It was isolated in 3 of them. Samples were processed for Gram, Albert stain, and culture. Identification, antibiotic sensitivity, and toxigenicity were done. RESULTS AND DISCUSSION: Four samples, one from the patient and three from contacts showed the presence of gram-positive slender bacilli with cuneiform arrangement, less cellular infiltrate on the Gram stain, and the presence of few metachromatic granules in the Albert stain. C. diphtheriae was grown on Potassium Tellurite agar. Antibiogram of all isolates was similar with resistance to Erythromycin and sensitivity to Penicillin. Isolates were confirmed by PCR and ToxA gene was detected. Contacts were treated with Penicillin and repeat swabs were negative. CONCLUSION: Present health statistics and this study suggests, fight against diphtheria in India is far from being over. It still lurks in some remote areas. It is a need to remain vigilant, keep tracing, and treating contacts to curtail down the rate of infection. In view of the resurgence, Government has given directives to replace TT with Td in UIP. Still, a lot needs to be done.

Chemical Shifts of Artificial Monomers Used to Construct Heterogeneous-Backbone Protein Mimetics in Random Coil and Folded States.

The construction of protein-sized synthetic chains that blend natural amino acids with artificial monomers to create so-called heterogeneous-backbones is a powerful approach to generate complex folds and functions from bio-inspired agents. A variety of techniques from structural biology commonly used to study natural proteins have been adapted to investigate folding in these entities. In NMR characterization of proteins, proton chemical shift is a straightforward to acquire, information-rich metric that bears directly on a variety of properties related to folding. Leveraging chemical shift to gain insight into folding requires a set of reference chemical shift values corresponding to each building block type (i.e., the 20 canonical amino acids in the case of natural proteins) in a random coil state and knowledge of systematic changes in chemical shift associated with particular folded conformations. Although well documented for natural proteins, these issues remain unexplored in the context of protein mimetics. Here, we report random coil chemical shift values for a library of artificial amino acid monomers frequently used to construct heterogeneous-backbone protein analogues as well as a spectroscopic signature associated with one monomer class, ß3-residues bearing proteinogenic side chains, adopting a helical folded conformation. Collectively, these results will facilitate the continued utilization of NMR for the study of structure and dynamics in protein-like artificial backbones.

Magnetically-assembled multifunctional magnetic-plasmonic SERS substrate for low-concentration analyte detection.

Multifunctional particles with combined magnetic and optical properties are promising materials for applications such as sensing and detection of analytes, and contrast agents for imaging techniques such as MRI, and photocatalysis. While the magnetic property allows for non-contact manipulation of the nanoparticles, optical properties can be harnessed for such sensing applications. We present the synthesis and large-scale assembly of inter-layered magnetic-plasmonic nanoparticles with graphene oxide (GO) spacer (Fe3O4@GO@Ag). The multifunctional composite particles were prepared using simple chemical methods and had an average size of 225 nm. The prepared samples were characterized using different techniques including powder XRD, FT-IR, Raman scattering, SEM, and TEM imaging. By using an external magnetic field, it is possible to form an assembly of these multifunctional particles on a large scale. Due to the chain-like formation in the presence of a magnetic field, such assemblies are good substrates for surface-enhanced Raman scattering (SERS). Here, we demonstrate the application of these magnetically-assembled particles for the detection of very low concentrations of analyte molecules (4-mercaptopyridine) using SERS. These multifunctional composite particles are good candidates for potential applications involving chemical detection, photocatalytic reactions, optoelectronic devices, and photothermal effects.

Hepatocyte mARC1 promotes fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ∼25% worldwide, with significant public health consequences yet few effective treatments. Human genetics can help elucidate novel biology and identify targets for new therapeutics. Genetic variants in mitochondrial amidoxime-reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality; however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods: Analyses including multi-trait colocalisation and Mendelian randomisation were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra Amylin NASH (GAN)-diet non-alcoholic steatohepatitis mouse model treated with hepatocyte-specific N-acetylgalactosamine (GalNAc)-siRNA to understand the in vivo impacts of MTARC1. Results: We showed that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids, and body composition, and these associations are attributable to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian randomisation, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion, and in vivo GalNAc-siRNA-mediated knockdown of mARC1 lowered hepatic but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions: Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD. Impact and implications: We report that genetically predicted increases in MTARC1 mRNA associate with poor liver health. Furthermore, knockdown of mARC1 reduces hepatic steatosis in primary human hepatocytes and a murine NASH model. Together, these findings further underscore the therapeutic potential of targeting hepatocyte MTARC1 for NAFLD.

A Comparison of Changes in the Mean Arterial Blood Pressure and Mean Uterine Artery Pulsatility Index from 11-14 to 19-24 + 6 Gestation Weeks in Low-Risk and High-Risk Asian Indian Pregnant Women.

Aim The aim of this study was to determine the changes in the mean arterial blood pressure (MAP) and mean uterine artery (UtA) pulsatility index (PI) from 11-14 to 19-24 + 6 gestation weeks in Asian Indian pregnant women. Methods Clinical and demographic details, MAP, and mean UtA PI measures were ascertained for pregnant women at 11 to 14 gestation weeks and 19-24 + 6 gestation weeks. Women were categorized as a high-or-low risk for preterm preeclampsia using the Fetal Medicine Foundation algorithm and 1 in 150 cutoff. High-risk pregnant women were recommended low-dose aspirin 150 mg daily at bedtime. Changes in MAP and mean UtA PI were compared for gestational age intervals and high-and-low risk women using nonparametric tests. Results The study analyzed the results of 1,163 pregnant women. Both MAP (mean difference: 5.14, p < 0.001) and mean UtA PI (mean difference: 0.14, p < 0.001) remained significantly higher at the second-trimester assessment in high-risk pregnant women compared to low-risk pregnant women. Seventy-seven (35.16%) of the 219 pregnant women with abnormal mean UtA PI in the first trimester had an abnormal mean UtA PI in the second-trimester assessment. One hundred (10.59%) of the 944 pregnant women with normal mean UtA PI in the first trimester had an abnormal mean UtA PI in the 19-24 + 6 weeks assessment. Seventy-seven pregnant women (6.62% of 1,163 women, 95% confidence interval: 5.33, 8.20) had an abnormal mean UtA PI at both gestation age intervals. High-risk pregnant women taking low-dose aspirin daily showed a larger reduction in mean UtA PI compared to high-risk pregnant women that did not report the use of low-dose aspirin (0.89 vs. 0.62, p <0.001) Conclusion MAP and mean UtA PI decreased significantly from the first to the second trimester of pregnancy. Sequential assessment of the MAP and mean UtA PI in the first and second trimesters of pregnancy will be useful for fetal radiologists in India to identify a subgroup of women with abnormal mean UtA PI at both trimesters that may need more intense surveillance and follow-up till childbirth.

MLX plays a key role in lipid and glucose metabolism in humans: Evidence from in vitro and in vivo studies.

BACKGROUND AND AIM: Enhanced hepatic de novo lipogenesis (DNL) has been proposed as an underlying mechanism for the development of NAFLD and insulin resistance. Max-like protein factor X (MLX) acts as a heterodimer binding partner for glucose sensing transcription factors and inhibition of MLX or downstream targets has been shown to alleviate intrahepatic triglyceride (IHTG) accumulation in mice. However, its effect on insulin sensitivity remains unclear. As human data is lacking, the aim of the present work was to investigate the role of MLX in regulating lipid and glucose metabolism in primary human hepatocytes (PHH) and in healthy participants with and without MLX polymorphisms. METHODS: PHH were transfected with non-targeting or MLX siRNA to assess the effect of MLX knockdown on lipid and glucose metabolism, insulin signalling and the hepatocellular transcriptome. A targeted association analysis on imputed genotype data for MLX on healthy individuals was undertaken to assess associations between specific MLX SNPs (rs665268, rs632758 and rs1474040), plasma biochemistry, IHTG content, DNL and gluconeogenesis. RESULTS: MLX knockdown in PHH altered lipid metabolism (decreased DNL (p < 0.05), increased fatty acid oxidation and ketogenesis (p < 0.05), and reduced lipid accumulation (p < 0.001)). Additionally, MLX knockdown increased glycolysis, lactate secretion and glucose production (p < 0.001) and insulin-stimulated pAKT levels (p < 0.01) as assessed by transcriptomic, steady-state and dynamic measurements. Consistent with the in vitro data, individuals with the rs1474040-A and rs632758-C variants had lower fasting plasma insulin (p < 0.05 and p < 0.01, respectively) and TG (p < 0.05 and p < 0.01, respectively). Although there was no difference in IHTG or gluconeogenesis, individuals with rs632758 SNP had notably lower hepatic DNL (p < 0.01). CONCLUSION: We have demonstrated using human in vitro and in vivo models that MLX inhibition favored lipid catabolism over anabolism and increased glucose production, despite increased glycolysis and phosphorylation of Akt, suggesting a metabolic mechanism that involves futile cycling.

Diagnostic Effectiveness of Third-Trimester Fetal Doppler Studies in Pregnancy to Predict Late-and-Term Stillbirth and Neonatal Mortality in the Samrakshan Program in India.

Aim To determine the diagnostic effectiveness of third-trimester fetal Doppler studies in pregnancy for stillbirths and neonatal mortality in the Samrakshan program of the Indian Radiological and Imaging Association (IRIA). Methods The mean uterine artery (UtA) pulsatility index (PI) > 95th percentile, umbilical artery PI > 95th percentile, middle cerebral artery (MCA) PI < 5th percentile, and/or cerebroplacental ratio (CPR) < 5th percentile in the third trimester fetal Doppler study was considered as abnormal. The results of the fetal Doppler study closest to childbirth were considered for analysis. Late stillbirth (SB) was defined as a fetal loss between 28 and 36 gestation weeks and the term SB was defined as a fetal loss at ≥ 37 gestation weeks. Neonatal death was defined as the demise of a live-born baby within the first 28 days of life. Parameters of diagnostic effectiveness such as sensitivity, specificity, positive and negative predictive values and likelihood ratios, diagnostic odds ratio, and the area under receiver operator characteristic (AUROC) curve were assessed. Results Screening of 1,326 pregnant women in the third trimester of pregnancy between September 2019 and February 2022, identified 308 (23.23%) abnormal Doppler studies, 11 (0.83%) SB, and 11 (0.84%) neonatal deaths. An abnormal Doppler study was significantly associated with late stillbirths (OR 37.2, 95% CI: 2.05, 674) but not with term SB (OR: 3.38, 95% CI: 0.76, 15) or neonatal deaths (OR 1.39, 95% CI: 0.40, 4.87). Mean UtA PI, umbilical artery PI, MCA PI, and CPR were significantly associated with late SB and not term SB. The AUROC of Doppler measures was excellent for late SB but did not show discriminatory ability for term SB or neonatal deaths. Conclusion Integration of fetal Doppler with routine third-trimester antenatal scans can help identify pregnant women at high risk for late SB. The effectiveness of fetal Doppler to identify pregnant women at high risk for term SB and neonatal deaths needs further study on a larger sample size.

Effectiveness of the First Trimester Samrakshan Protocol for the Identification of Pregnant Women at High Risk for Preterm Pre-eclampsia.

Aim To determine the effectiveness of the first trimester Samrakshan protocol for the identification of pregnant women at high risk for preterm pre-eclampsia (PE). Methods Samrakshan uses a protocol that integrates routine first-trimester ultrasound assessment at 11 to 14 gestation weeks with the measurement of mean arterial blood pressure and mean uterine artery pulsatility index assessment to determine a customized risk for preterm PE and fetal growth restriction. Based on the risk assessment, pregnant women are classified as high or low risk. Results The protocol had a high specificity (90.4%, 95% CI: 89.4%, 91.2%) and negative predictive value (98.1%, 95% CI: 97.6%, 99%) for preterm PE. The odds ratio and positive likelihood ratio for preterm PE were 16.7 (95% CI: 12.3, 22.6) and 6.64 (95% CI: 5.77, 7.63), respectively. Conclusions The positive likelihood ratio and odds ratios indicate that pregnant women identified as high risk for preterm PE using the first-trimester protocol of Samrakshan are significantly more likely to develop preterm PE than low-risk women.

Prioritizing Fetal Structural Abnormalities Over Risk for Pre-Eclampsia and Fetal Growth Restriction in the 20-24 Gestation Week Assessment in India: Missing the Woods for the Trees?

Aim To compare the magnitude of fetuses with congenital anomalies, pregnant women identified at high risk for preterm pre-eclampsia (PE) or with preterm PE, and with early fetal growth restriction (FGR) or high risk for FGR at the second trimester assessment at 20 to 24 weeks of gestation. Methods A standardized trimester-specific protocol that included clinical and demographic details, fetal biometry, estimated fetal weight (EFW), fetal abdominal circumference (FAC), mean arterial blood pressure and fetal Doppler studies was used to identify high risk for preterm PE and FGR. The Targeted Imaging for Fetal Anomalies (TIFFA) scan was used to identify congenital anomalies. In addition, 95% confidence intervals of the point estimates were derived, and the p -value was estimated to assess the statistical significance of the difference in proportions. Results Analysis of the data of 4,572 pregnant women screened between 20 and 24 gestation weeks showed a significantly lower prevalence ( p < 0.001) of congenital abnormalities (3.81%) compared to women diagnosed with early PE (2.71%) or with a high risk for PE (4.00%) and women (6.80%) with early FGR or at higher risk for fetal growth restriction with both EFW and FAC < 10th percentile. Conclusion The data on prevalence from Samrakshan show that the second-trimester assessment of pregnant women in India must expand its scope from the TIFFA scan to also focus on screening to identify women at high risk for preterm PE and FGR.

Preterm Birth Rates after Initiating the Third-Trimester Screening Protocol of Samrakshan in India: Initial Results.

Aim To determine the trends in preterm birth (PTB) rates in Samrakshan after initiating a third-trimester screening protocol. Methods The Samrakshan program of IRIA focused on clinical stage-based management of fetal growth restriction (FGR) in the third trimester integrating fetal Doppler studies with routine trimester-specific antenatal scans. Mean uterine artery pulsatility index (PI), umbilical artery PI, middle cerebral artery PI, and cerebroplacental ratio were assessed for all third-trimester pregnant women in the program. Results From 2019 to 2022, 249 (18.33%, 95% CI: 16.34, 20.54) women had PTB with 221 (16.67%, 95% CI: 14.73, 18.75) PTBs between 34 and 37 gestation weeks and 22 (1.66%, 95% CI: 1.10, 2.50) PTBs at gestation < 34 weeks. The overall preterm birth rates showed a significant (chi-square p < 0.001) declining trend each year from 23.18% ( n = 121) in 2019-2020 to 16.81% ( n = 99) in 2020-2021 and 10.75% ( n = 23) in 2021-2022. Conclusion The declining trend of PTB rates in the Samrakshan program shows that the reduction of PTB is an added benefit of the integration of fetal Doppler studies in the third trimester of pregnancy.

Cyanovirin-N binds to select SARS-CoV-2 spike oligosaccharides outside of the receptor binding domain and blocks infection by SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped positive stranded RNA virus which has caused the recent deadly pandemic called COVID-19. The SARS-CoV-2 virion is coated with a heavily glycosylated Spike glycoprotein which is responsible for attachment and entry into target cells. One, as yet unexploited strategy for preventing SARS-CoV-2 infections, is the targeting of the glycans on Spike. Lectins are carbohydrate-binding proteins produced by plants, algae, and cyanobacteria. Some lectins can neutralize enveloped viruses displaying external glycoproteins, offering an alternative therapeutic approach for the prevention of infection with virulent ß-coronaviruses, such as SARS-CoV-2. Here we show that the cyanobacterial lectin cyanovirin-N (CV-N) can selectively target SARS-CoV-2 Spike oligosaccharides and inhibit SARS-CoV-2 infection in vitro and in vivo. CV-N neutralizes Delta and Omicron variants in vitro better than earlier circulating viral variants. CV-N binds selectively to Spike with a Kd as low as 15 nM and a stoichiometry of 2 CV-N: 1 Spike but does not bind to the receptor binding domain (RBD). Further mapping of CV-N binding sites on Spike shows that select high-mannose oligosaccharides in the S1 domain of Spike are targeted by CV-N. CV-N also reduced viral loads in the nares and lungs in vivo to protect hamsters against a lethal viral challenge. In summary, we present an anti-coronavirus agent that works by an unexploited mechanism and prevents infection by a broad range of SARS-CoV-2 strains.

BioSentinel: A Biofluidic Nanosatellite Monitoring Microbial Growth and Activity in Deep Space.

Small satellite technologies, particularly CubeSats, are enabling breakthrough research in space. Over the past 15 years, NASA Ames Research Center has developed and flown half a dozen biological CubeSats in low Earth orbit (LEO) to conduct space biology and astrobiology research investigating the effects of the space environment on microbiological organisms. These studies of the impacts of radiation and reduced gravity on cellular processes include dose-dependent interactions with antimicrobial drugs, measurements of gene expression and signaling, and assessment of radiation damage. BioSentinel, the newest addition to this series, will be the first deep space biological CubeSat, its heliocentric orbit extending far beyond the radiation-shielded environment of low Earth orbit. BioSentinel's 4U biosensing payload, the first living biology space experiment ever conducted beyond the Earth-Moon system, will use a microbial bioassay to assess repair of radiation-induced DNA damage in eukaryotic cells over a duration of 6-12 months. Part of a special collection of articles focused on BioSentinel and its science mission, this article describes the design, development, and testing of the biosensing payload's microfluidics and optical systems, highlighting improvements relative to previous CubeSat life-support and bioanalytical measurement technologies.

IgE binding epitope mapping with TL1A tagged peptides.

Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis.

Expression, purification, characterization, and patient IgE reactivity of new macadamia nut iso-allergen.

Structural and functional information about food allergens is essential for understanding the allergenicity of food proteins. All allergens belong to a small number of protein families. Various allergens from different families have been successfully produced recombinantly in E. coli for their characterization and applications in allergy diagnosis and treatment. However, recombinant hexameric 11S seed storage protein has not been reported, although numerous 11S legumins are known to be food allergens, including the recently identified macadamia nut allergen Mac i 2. Here we report the production of a macadamia nut legumin by expressing it in E. coli with a substrate site of HRV 3C protease and cleaving the purified protein with HRV 3C protease. The protease divided the protein into two chains and left a native terminus for the C-terminal chain, resulting in a recombinant hexameric 11S allergen for the first time after the residues upstream to the cleavage site flipped out of the way of the trimer-trimer interaction. The 11S allergens are known to have multiple isoforms in many species. The present study removed an obstacle in obtaining homogeneous allergens needed for studying allergens and mitigating allergenicity. Immunoreactivity of the protein with serum IgE confirmed it to be a new isoform of Mac i 2.

Determining the temporal, dose, and composition effects of nutritional substrates in an in vitro model of intrahepatocellular triglyceride accumulation.

Pathological accumulation of intrahepatic triglyceride underpins the early stages of nonalcoholic fatty liver disease (NAFLD) and can progress to fibrosis, cirrhosis, and cancer of the liver. Studies in humans suggest that consumption of a diet enriched in saturated compared to unsaturated fatty acids (FAs), is more detrimental to liver fat accumulation and metabolism. However, the reasons for the divergence remain unclear and physiologically-relevant cellular models are required. Therefore, the aims of this study were to investigate the effect of modifying media composition, concentration, and treatment frequency of sugars, FAs and insulin on intrahepatocellular triglyceride content and intracellular glucose, FA and circadian function. Huh7 cells were treated with 2% human serum and a combination of sugars and FAs (low fat low sugar [LFLS], high fat low sugar [HFLS], or high fat high sugar [HFHS]) enriched in either unsaturated (OPLA) or saturated (POLA) FAs for 2, 4, or 7 days with a daily or alternating treatment regime. Stable isotope tracers were utilized to investigate basal and/or insulin-responsive changes in hepatocyte metabolism in response to different treatment regimes. Cell viability, media biochemistry, intracellular metabolism, and circadian biology were quantified. The FA composition of the media (OPLA vs. POLA) did not influence cell viability or intracellular triglyceride content in hepatocytes. In contrast, POLA-treated cells had lower FA oxidation and media acetate, and with higher FA concentrations, displayed lower intracellular glycogen content and diminished insulin stimulation of glycogenesis, compared to OPLA-treated cells. The addition of HFHS also had profound effects on circadian oscillation and gene expression. Cells treated daily with HFHS for at least 4 days resulted in a cellular model displaying characteristics of early stage NAFLD seen in humans. Repeated treatment for longer durations (≥7 days) may provide opportunities to investigate lipid and glucose metabolism in more severe stages of NAFLD.

EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression.

Triple-negative breast cancers (TNBCs) are frequently poorly differentiated with high propensity for metastasis. Enhancer of zeste homolog 2 (EZH2) is the lysine methyltransferase of polycomb repressive complex 2 that mediates transcriptional repression in normal cells and in cancer through H3K27me3. However, H3K27me3-independent non-canonical functions of EZH2 are incompletely understood. We reported that EZH2 phosphorylation at T367 by p38α induces TNBC metastasis in an H3K27me3-independent manner. Here, we show that cytosolic EZH2 methylates p38α at lysine 139 and 165 leading to enhanced p38α stability and that p38 methylation and activation require T367 phosphorylation of EZH2. Dual inhibition of EZH2 methyltransferase and p38 kinase activities downregulates pEZH2-T367, H3K27me3, and p-p38 pathways in vivo and reduces TNBC growth and metastasis. These data uncover a cooperation between EZH2 canonical and non-canonical mechanisms and suggest that inhibition of these pathways may be a potential therapeutic strategy.