SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression.

Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using the leaky scanning model of translation initiation. In this approach termed "stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes" (SEMPER), adjacent ORFs are translated from a single mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant monoclonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through in vitro transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A record of this paper's transparent peer review process is included in the supplemental information.

The effect of intermittent fasting on preventing obesity-related early aging from a molecular and cellular perspective.

Obesity is a global health concern owing to its association with numerous degenerative diseases and the fact that it may lead to early aging. Various markers of aging, including telomere attrition, epigenetic alterations, altered protein homeostasis, mitochondrial dysfunction, cellular senescence, stem cell disorders, and intercellular communication, are influenced by obesity. Consequently, there is a critical need for safe and effective approaches to prevent obesity and mitigate the onset of premature aging. In recent years, intermittent fasting (IF), a dietary strategy that alternates between periods of fasting and feeding, has emerged as a promising dietary strategy that holds potential in counteracting the aging process associated with obesity. This article explores the molecular and cellular mechanisms through which IF affects obesity-related early aging. IF regulates various physiological processes and organ systems, including the liver, brain, muscles, intestines, blood, adipose tissues, endocrine system, and cardiovascular system. Moreover, IF modulates key signaling pathways such as AMP-activated protein kinase (AMPK), sirtuins, phosphatidylinositol 3-kinase (PI3K)/Akt, mammalian target of rapamycin (mTOR), and fork head box O (FOXO). By targeting these pathways, IF has the potential to attenuate aging phenotypes associated with obesity-related early aging. Overall, IF offers promising avenues for promoting healthier lifestyles and mitigating the premature aging process in individuals affected by obesity.

Sirolimus to treat chronic and steroid-resistant allograft rejection-related fibrosis in pediatric liver transplantation.

This study aimed to report our experience with the use of sirolimus in pediatric liver transplant patients with chronic rejection or steroid-resistant rejection with hepatic fibrosis, focusing on their histological evolution. All pediatric liver transplant recipients who received off-label treatment with sirolimus for chronic ductopenic rejection or cortico-resistant rejection between July 2003 and July 2022 were included in the study. All nine patients included in the study showed improvement in liver enzymes and cholestasis parameters as soon as 1-month after postsirolimus introduction. A decrease in fibrosis stage was observed in 7/9 (77.7%) patients at 36 months. All but one patient experienced an improvement in the Rejection Activity Index and ductopenia at 12 months. A single patient had to discontinue sirolimus treatment owing to nephrotic proteinuria. In conclusion, sirolimus may be a safe and effective treatment for chronic and steroid-resistant rejection and may improve allograft rejection-related fibrosis and ductal damage.

Fc engineering by monoclonal mammalian cell display for improved affinity and selectivity towards FcγRs.

Fc optimization can significantly enhance therapeutic efficacy of monoclonal antibodies. However, existing Fc engineering approaches are sub-optimal with noted limitations, such as inappropriate glycosylation, polyclonal libraries, and utilizing fragment but not full-length IgG display. Applying cell cycle arrested recombinase-mediated cassette exchange, this study constructed high-quality monoclonal Fc libraries in CHO cells, displayed full-length IgG on cell surface, and preformed ratiometric fluorescence activated cell sorting (FACS) with the antigen and individual FcγRs. Identified Fc variants were quantitatively evaluated by flow cytometry, ELISA, kinetic and steady-state binding affinity measurements, and cytotoxicity assays. An error-prone Fc library focusing on the hinge-CH2 region was constructed in CHO cells with a functional diversity of 7.5 × 106. Panels of novel Fc variants with enhanced affinity and selectivity for FcγRs were isolated. Particularly, clone 2a-10 (G236E/K288R/K290W/K320M) showed increased binding strength towards FcγRIIa-131R and 131H allotypes with kinetic dissociation constants (KD-K) of 140 nM and 220 nM, respectively, while reduced binding strength towards FcγRIIb compared to WT Fc; clone 2b-1 (K222I/V302E/L328F/K334E) had KD-K of 180 nM towards FcγRIIb; clone 3a-2 (P247L/K248E/K334I) exhibited KD-K of 190 nM and 100 nM towards FcγRIIIa-176F and 176 V allotypes, respectively, and improved potency of 2.0 ng/ml in ADCC assays. Key mutation hotspots were identified, including P247 for FcγRIIIa, K290 for FcγRIIa, and K334 for FcγRIIb bindings. Discovery of Fc variants with enhanced affinity and selectivity towards individual FcγR and the identification of novel mutation hotspots provide valuable insights for further Fc optimization and serve as a foundation for advancing antibody therapeutics development.

Genome sequencing projects reveal new insights into the mammalian Gonadotropin-releasing Hormone II system.

The type II gonadotropin-releasing hormone (GnRH-II) was first discovered in chicken (Gallus gallus) brain and then shown to be present in many vertebrates. Indeed, its structure is conserved unchanged throughout vertebrate evolution from teleost fish through to mammals suggesting a crucial function. Yet the functional significance has been largely unexplored. Studies in comparative endocrinology show that the GnRH-II system is differentially functional in mammalian species. Intact GnRH-II neuropeptide and receptor genes (GnRH2 and GnRH receptor 2 GnRHR2) occur in marmoset monkeys (Callithrix jacchus), musk shrews (Suncus murinus) and pigs (Sus scrofa). However, one or other or both of these genes are inactivated in other species, where mutations or remnants affecting GnRH2 neuropeptide and/or type II GnRHR exons are retained in conserved genomic loci. New data from DNA sequencing projects facilitate extensive analysis of species-specific variation in these genes. Here, we describe GnRH2 and GnRHR2 genes spanning a collection of 21 taxonomic orders, encompassing around 140 species from Primates, Scandentia, Eulipotyphla, Rodentia, Lagomorpha, Artiodactyla, Carnivora, Perissodactyls, Pholidota, Chiroptera, Afrotheria, Xenarthra and Marsupialia. Intact coding exons for both GnRH2 and GnRHR2 occur in monkeys, tree shrews, shrews, moles, hedgehogs, several rodents (degu, kangaroo-rat, pocket mouse), pig, pecarry and warthog, camels and alpaca, bears, Weddell seal, hyena, elephant, aardvark and marsupials. Inactivating mutations affecting GnRH2 and GnRHR2, some located at conserved sites within exons, occur in species of primates, most rodents, lagomorphs, bovidae, cetaceans, felidae, canidae and other carnivora, pangolins, most bats, armadillo, brushtail and echidna. A functional GnRH-II system appears retained within several taxonomic families of mammals, but intact retention does not extend to whole taxonomic orders. Defining how endogenous GnRH-II neuropeptide operates in different mammals may afford functional insight into its actions in the brain, especially as, unlike the type I GnRH system, it is expressed in the mid brain and not the hypothalamus.

Complex Factors in Hydrocephalus Development in Tuberous Sclerosis Complex: A Case Report of Subependymal Giant Cell Astrocytoma.

Subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis complex (TSC) occurs in 5-20% of TSC patients, with a subset developing hydrocephalus. We present a case of a 14-year-old male diagnosed with TSC in the neonatal period who developed SEGA and subsequent hydrocephalus. Despite reducing the tumor size with the mammalian target of rapamycin (mTOR) inhibitors, ventricular enlargement persisted, indicating that obstructive hydrocephalus due to the foramen of Monro blockage was not the sole mechanism. Elevated cerebrospinal fluid (CSF) protein levels suggested additional factors like impaired CSF outflow. This case underscores the need for comprehensive treatment strategies and further research to better understand and manage hydrocephalus in TSC patients with SEGA.

Alpha-Gal Syndrome: An Underrated Serious Disease and a Potential Future Challenge.

Over the past decades, red meat allergy, also known as mammalian meat allergy, which manifests differently from classic food allergies, has been reported in different countries and regions, including China. The allergen of this disease is not a protein but an oligosaccharide: galactose-α-1,3-galactose, i.e., alpha-gal or α-gal. Therefore, this clinical syndrome is also called α-gal syndrome (AGS). It clinically manifests as delayed anaphylaxis, i.e., patients generally develop allergic symptoms 2-6 h after ingesting red meat. This clinical manifestation is believed to be related to sensitization to α-gal after tick bites. Sensitized individuals may also develop anaphylaxis after ingesting food and medicine or being exposed to medical equipment containing α-gal, such as cetuximab and gelatin. Here, the literature on AGS is reviewed for a better understanding of its pathogenesis, clinical diagnosis, and treatment.

Ambulatory Management of Bite Injuries to the Hand: A Safe and Cost-Effective Option.

Introduction Animal or human hand bites are a common presentation to the emergency department. If hand bites are not treated adequately, they can give rise to significant local and systemic complications, potentially leading to functional deficits that impact patients' lives. Traditionally, hand bites require hospital admission for the administration of intravenous antibiotics and, in some cases, surgical intervention. A combination of the increasing incidence, hospital admission rates, and in-patient bed pressures prompted a change in our bite management protocol and a move toward ambulatory management of bite injuries. We found this new protocol to be safe, efficient, and cost-effective with a scope for wider implementation. Aim The primary outcome is to assess the feasibility of safely managing hand bites on an outpatient basis, by reviewing the local data before and after the change in practice. The secondary outcome is to compare the financial implications of treating hand bites with an outpatient approach. Material and methods All first-presentation adult consultations referred to Trauma and Orthopaedics from the emergency department over a three-month snapshot period were reviewed in 2017. This was repeated after the implementation of the updated handbite guidelines in 2023. Initial admission documentation as well as operation notes and clinic follow-up letters were each reviewed retrospectively.  Results  In 2017, 36 patients were identified over three months. The average time to surgery was 1.19 days with an average inpatient stay of 2.36 days. There were two re-operations and follow-up of two cases of osteomyelitis.  In 2023, 63 patients were identified over three months. The average time to surgery was 1.03 days with an average inpatient stay of 0.56 days. Thirty-seven surgeries were performed for 33 patients with 32% (20/63) of patients admitted directly from the emergency department. There were no documented cases of osteomyelitis on follow-up. The cost per patient episode decreased by 40% from 2017 to 2023, without accounting for inflation. Conclusions With the implementation of the new departmental guidelines, there has been a reduced average inpatient stay and reduced time to surgery without an increase in documented osteomyelitis. There is also a significant decrease in the average patient cost. This data suggests that without compromising patient safety it is possible to cost-effectively manage hand bites without the need for long inpatient stays.  However, it is imperative that there is close patient follow-up as well as prompt time to surgery to ensure patient safety. Our findings suggest a need for further research to strengthen the evidence supporting our conclusions.

Detection and characterization of H5N1 HPAIV in environmental samples from a dairy farm.

The recent expansion of HPAIV H5N1 infections in terrestrial mammals in the Americas, most recently including the outbreak in dairy cattle, emphasizes the critical need for better epidemiological monitoring of zoonotic diseases. In this work, we detected, isolated, and characterized the HPAIV H5N1 from environmental swab samples collected from a dairy farm in the state of Kansas, USA. Genomic sequencing of these samples uncovered two distinctive substitutions in the PB2 (E249G) and NS1 (R21Q) genes which are rare and absent in recent 2024 isolates of H5N1 circulating in the mammalian and avian species. Additionally, approximately 1.7% of the sequence reads indicated a PB2 (E627K) substitution, commonly associated with virus adaptation to mammalian hosts. Phylogenetic analyses of the PB2 and NS genes demonstrated more genetic identity between this environmental isolate and the 2024 human isolate (A/Texas/37/2024) of H5N1. Conversely, HA and NA gene analyses revealed a closer relationship between our isolate and those found in other dairy cattle with almost 100% identity, sharing a common phylogenetic subtree. These findings underscore the rapid evolutionary progression of HPAIV H5N1 among dairy cattle and reinforces the need for more epidemiological monitoring which can be done using environmental sampling.

Genome-Wide Profiling of Cis-regulatory Elements in Mammalian Skin.

The modulation of cis-regulatory elements (e.g., enhancers and promoters) is a major mechanism by which gene expression can be controlled in a temporal and spatially restricted manner. However, methods for both identifying these elements and inferring their activity are limited and often require a substantial investment of time, money, and resources. Here, using mammalian skin as a model, we demonstrate a streamlined protocol by which these hurdles can be overcome using a novel chromatin profiling technique (CUT&RUN) to map histone modifications genome-wide. This protocol can be used to map the location and activity of putative cis-regulatory elements, providing mechanistic insight into how differential gene expression is controlled in mammalian tissues.

Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells.

Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four-residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit can disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are powerful tools for expanding the CAR T cell engineering toolbox.

Back to the future: Forgotten protocols for optimizing the isolation of arthropod haemocytes.

Consideration is given to previous and more recent protocols for harvesting arthropod haemocytes from Galleria, Drosophila, mosquitoes, Limulus and crustaceans. The optimal harvesting of these cells is essential for meaningful studies of invertebrate immunity in vitro. The results of such experiments, however, have often been flawed due to a lack of understanding of the fragile nature of arthropod haemocytes on exposure to bacterial lipopolysaccharides, resulting in the aggregation and loss of cell types during haemolymph clotting. This article emphasizes that although there are similarities between mammalian neutrophils and arthropod haemocytes, the protocols required for the successful harvesting of these cells vary significantly. The various stages for the successful harvesting of arthropod haemocytes are described in detail and should provide invaluable advice to those requiring both high cell viability and recovery of the different cell types for subsequent experimentation.

Energy Content, Moisture Content, and Energy Assimilation Efficiency by Birds and Mammals of Oil-Containing Seeds and Implications for Seed Treatment Risk Assessments for Birds and Mammals.

Energy content, moisture content, and energy assimilation efficiency are essential parameters in the food intake rate (FIR) and exposure calculations for bird and mammal risk assessments. The updated European Food Safety Authority guidance document on risk assessment for birds and mammals summarizes these parameters for different food items. For seed treatments, values for cereal seeds are proposed as surrogates for other crops. Oil-containing seeds are expected to have a higher energy content than cereal seeds. This would result in lower FIR and, thus, exposure from consuming such seeds. To be able to calculate reliable exposure values for risk-assessment purposes, we conducted a systematic literature review to collect information on these three parameters for oil-containing seeds (sunflower, oilseed rape, soybean, peanut, sesame, safflower, linseed [flax], white mustard, and castor bean). The search yielded 401 papers, of which 151 contained values for at least one of the parameters of the crops in focus. The overall average energy content value of oil-containing seeds was 24.25 kJ/g (n = 124, SD = 3.00), whereas that for moisture content was 6.57% (n = 296, SD = 1.15). Energy assimilation values were only available for peanut, oilseed rape, soy, linseed, and sunflower for a limited number of bird and mammal species. Mean energy assimilation efficiency for mammals was 82.69% (n = 4, SD = 1.55), whereas values for birds were 57.54% (n = 2, SD = 6.77) for Galliformes and 79.25% (n = 2, SD = 1.82) for Passeriformes. The values presented can be used to calculate appropriate FIR values for future bird and mammal risk assessments. Environ Toxicol Chem 2024;00:1-6. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Neurosurgical Management of Patients with Alpha-Gal Syndrome.

Alpha-gal syndrome (AGS) is an immunoglobulin E-mediated hypersensitivity to galatcose-alpha-1,3-galactose (alpha-gal), a carbohydrate compound present in nonprimate mammalian products. Initial exposure to alpha-gal most often occurs through a tick bite, most commonly the lone star tick in the United States. Repeated exposure to alpha-gal may elicit severe allergic reactions, including anaphylaxis. The allergy restricts dietary intake and may significantly impact perioperative risk, as many medications, anesthetics, and intraoperative surgical products utilize bovine or porcine-derived agents, including those containing magnesium stearate, glycerol, and gelatin. Here, we review the perineurosurgical care of two individuals with AGS and highlight pertinent clinical practices and perioperative management of these patients.

All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

A perspective-driven and technical evaluation of machine learning in bioreactor scale-up: A case-study for potential model developments.

Bioreactor scale-up and scale-down have always been a topical issue for the biopharmaceutical industry and despite considerable effort, the identification of a fail-safe strategy for bioprocess development across scales remains a challenge. With the ubiquitous growth of digital transformation technologies, new scaling methods based on computer models may enable more effective scaling. This study aimed to evaluate the potential application of machine learning (ML) algorithms for bioreactor scale-up, with a specific focus on the prediction of scaling parameters. Factors critical to the development of such models were identified and data for bioreactor scale-up studies involving CHO cell-generated mAb products collated from the literature and public sources for the development of unsupervised and supervised ML models. Comparison of bioreactor performance across scales identified similarities between the different processes and primary differences between small- and large-scale bioreactors. A series of three case studies were developed to assess the relationship between cell growth and scale-sensitive bioreactor features. An embedding layer improved the capability of artificial neural network models to predict cell growth at a large-scale, as this approach captured similarities between the processes. Further models constructed to predict scaling parameters demonstrated how ML models may be applied to assist the scaling process. The development of data sets that include more characterization data with greater variability under different gassing and agitation regimes will also assist the future development of ML tools for bioreactor scaling.

Extraction and physicochemical characterization of native and broiler chicken feet gelatin.

BACKGROUND: Poultry processing generates a large amount of industrial waste, which is rich in collagen content. This waste can be utilized for the extraction of valuable components such as gelatin, which can be used as an alternative to mammalian gelatin (porcine and bovine). RESULTS: Gelatins were analyzed for their yield, proximate analysis, pH, color, viscosity, bloom strength, and texture profile analysis. The yield of broiler chicken feet gelatin (BCFG) was slightly higher (7.93%) as compared to native chicken feet gelatin (NCFG) (7.06%). The protein content was 85.92% and 82.53% for BCFG and NCFG. Both gelatin had moisture content in the standard range (< 15) as given by Gelatin Manufacturers of Europe (GME). Both gelatins showed higher bloom strength (326 g for NCFG and 203 g for BCFG) at 6.67% gelatin concentration, classified as high bloom. Fourier-transform infrared (FTIR) analysis showed amide I, amide A, amide B at 1636 cm-1, 3302 cm-1, 2945 cm-1 for NCFG and 1738 cm-1, 3292 cm-1, 2920 cm-1 for BCFG. At 6.67% gelatin concentration, hardness and cohesiveness values were also higher than commercial gelatin previously studied. The pH values for NCFG were 5.43 and BCFG was 5.31. Both NCFG and BCFG viscosities (4.43 and 3.85 cP) were in the optimum range of commercial gelatins (2-7 cP). CONCLUSION: Hence, the present study concluded that both NCFG and BCFG have a huge potential to replace commercial mammalian gelatins (porcine and bovine) in the food industries. However further studies should be done to optimize the extraction process. © 2024 Society of Chemical Industry.

Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.

The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.

Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination.

The cytotoxic necrotizing factor (CNF) family of AB-type bacterial protein toxins catalyze two types of modification on their Rho GTPase substrates: deamidation and transglutamination. It has been established that E. coli CNF1 and its close homolog proteins catalyze primarily deamidation and Bordetella dermonecrotic toxin (DNT) catalyzes primarily transglutamination. The rapidly expanding microbial genome sequencing data have revealed that there are at least 13 full-length variants of CNF1 homologs. CNFx from E. coli strain GN02091 is the most distant from all other members of the CNF family with 50%-55% sequence identity at the protein level and 0.45-0.52 nucleotide substitutions per site at the DNA level. CNFx modifies RhoA, Rac1, and Cdc42, and like CNF1, activates downstream SRE-dependent mitogenic signaling pathways in human HEK293T cells, but at a 1,000-fold higher EC50 value. Unlike other previously characterized CNF toxins, CNFx modifies Rho proteins primarily through transglutamination, as evidenced by gel-shift assay and confirmed by MALDI mass spectral analysis, when coexpressed with Rho-protein substrates in E. coli BL21 cells or through direct treatment of HEK293T cells. A comparison of CNF1 and CNFx sequences identified two critical active-site residues corresponding to positions 832 and 862 in CNF1. Reciprocal site-specific mutations at these residues in each toxin revealed hierarchical rules that define the preference for deamidase versus a transglutaminase activity in CNFs. An additional unique Cys residue at the C-terminus of CNFx was also discovered to be critical for retarding cargo delivery.IMPORTANCECytotoxic necrotizing factor (CNF) toxins not only play important virulence roles in pathogenic E. coli and other bacterial pathogens, but CNF-like genes have also been found in an expanding number of genomes from clinical isolates. Harnessing the power of evolutionary relationships among the CNF toxins enabled the deciphering of the hierarchical active-site determinants that define whether they modify their Rho GTPase substrates through deamidation or transglutamination. With our finding that a distant CNF variant (CNFx) unlike other known CNFs predominantly transglutaminates its Rho GTPase substrates, the paradigm of "CNFs deamidate and DNTs transglutaminate" could finally be attributed to two critical amino acid residues within the active site other than the previously identified catalytic Cys-His dyad residues. The significance of our approach and research findings is that they can be applied to deciphering enzyme reaction determinants and substrate specificities for other bacterial proteins in the development of precision therapeutic strategies.

Targeting of CYP2E1 by miRNAs in alcohol-induced intestine injury.

Although binge alcohol-induced gut leakage has been studied extensively in the context of reactive oxygen species-mediated signaling, it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol (EtOH)-inducible cytochrome P450-2E1 (CYP2E1), a key enzyme in EtOH metabolism, promotes alcohol-induced hepatic steatosis and inflammatory liver disease, at least in part by mediating changes in intestinal permeability. For instance, gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that EtOH promotes CYP2E1 induction and activation, the mechanisms that regulate CYP2E1 expression in the context of intestinal damage remain poorly defined. Specific miRNAs, including miR-132, miR-212, miR-378, and miR-552, have been shown to repress the expression of CYP2E1, suggesting that these miRNAs contribute to EtOH-induced intestinal injury. Here, we have shown that CYP2E1 expression is regulated post-transcriptionally through miRNA-mediated degradation, as follows: (1) the RNA-binding protein AU-binding factor 1 (AUF1) binds mature miRNAs, including CYP2E1-targeting miRNAs, and this binding modulates the degradation of corresponding target mRNAs upon EtOH treatment; (2) the serine/threonine kinase mammalian Ste20-like kinase 1 (MST1) mediates oxidative stress-induced phosphorylation of AUF1. Those findings suggest that reactive oxygen species-mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation. Thus, our study demonstrates the critical functions of AUF1 phosphorylation by MST1 in the decay of miRNAs targeting CYP2E1, the stabilization of CYP2E1 mRNA in the presence of EtOH, and the relationship of this pathway to subsequent intestinal injury.