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Multisystem Proteinopathy 1 (MSP1) disease is a rare genetic disorder caused by mutations in the Valosin-Containing Protein (VCP) gene with clinical features of inclusion body myopathy (IBM), frontotemporal dementia (FTD), and Pagets disease of bone (PDB). We performed bone scan imaging in twelve patients (6 females, 6 males) with confirmed VCP gene mutation six (50%) of which has myopathy alone, four (33%) with both PDB and myopathy, and two (15%) were presymptomatic carriers. We aim to characterize the PDB in diagnosed individuals, and potentially identify PDB in the myopathy and presymptomatic groups. Interestingly, two patients with previously undiagnosed PDB had positive diagnostic findings on the bone scan and subsequent radiograph imaging. Among the individuals with PDB, increased radiotracer uptake of the affected bones were of typical distribution as seen in conventional PDB and those reported in other MSP1 cohorts which are the thoracic spine and ribs (75%), pelvis (75%), shoulder (75%) and calvarium (15%). Overall, we show that technetium-99m bone scans done at regular intervals are a sensitive screening tool in patients with MSP1 associated VCP variants at risk for PDB. However, diagnostic confirmation should be coupled with clinical history, biochemical analysis, and skeletal radiographs to facilitate early treatment and prevention complications, acknowledging its limited specificity.

Interleukin-2 (IL-2) has been used in cancer treatment for over 30 years. However, due to its high toxicity, new mutant variants have been developed. These variants retain some of the biological properties of the original molecule but offer other therapeutic advantages. At the Center of Molecular Immunology, the IL-2 no-alpha mutein, an IL-2 agonist with lower toxicity than wtIL-2, has been designed, produced, and is currently being evaluated in a Phase I/II clinical trial. The mutein is produced in E. coli as an insoluble material that must be refolded in vitro to yield a fully active protein. Controlled oxidation steps are essential in the purification process of recombinant proteins produced in E. coli to ensure the proper formation of the disulfide bonds in the molecules. In this case, the new purification process includes a copper-catalyzed air oxidation step to induce disulfide bond establishment. The optimal conditions of pH, copper, protein and detergent concentration for this step were determined through screening. The produced protein demonstrated a conserved 3D structure, higher purity, and greater biological activity than the obtained by established process without the oxidation step. Four batches were produced and evaluated, demonstrating the consistency of the new process. • The controlled oxidation of the disulfide bond results in an enhanced efficiency of the reaction. • Oxidative refolding system recovers a higher protein specific activity and preserves protein structure. • Low-cost copper-catalyzed air oxidation process is suitable for high-purity protein production. [ABSTRACT FROM AUTHOR]

Aggregation of aberrant proteins is a common pathological hallmark in neurodegeneration such as polyglutamine (polyQ) and other repeat-expansion diseases. Here through overexpression of ataxin3 C-terminal polyQ expansion in Drosophila gut enterocytes, we generated an intestinal obstruction model of spinocerebellar ataxia type3 (SCA3) and reported a new role of nuclear-associated endosomes (NAEs)–the delivery of polyQ to the nucleoplasm. In this model, accompanied by the prominently increased RAB5-positive NAEs are abundant nucleoplasmic reticulum enriched with polyQ, abnormal nuclear envelope invagination, significantly reduced endoplasmic reticulum, indicating dysfunctional nucleocytoplasmic trafficking and impaired endomembrane organization. Consistently, Rab5 but not Rab7 RNAi further decreased polyQ-related NAEs, inhibited endomembrane disorganization, and alleviated disease model. Interestingly, autophagic proteins were enriched in polyQ-related NAEs and played non-canonical autophagic roles as genetic manipulation of autophagic molecules exhibited differential impacts on NAEs and SCA3 toxicity. Namely, the down-regulation of Atg1 or Atg12 mitigated while Atg5 RNAi aggravated the disease phenotypes both in Drosophila intestines and compound eyes. Our findings, therefore, provide new mechanistic insights and underscore the fundamental roles of endosome-centered nucleocytoplasmic trafficking and homeostatic endomembrane allocation in the pathogenesis of polyQ diseases. [ABSTRACT FROM AUTHOR]

Valosin-containing protein (VCP) gene mutations have been associated with a rare autosomal dominant, adult-onset progressive disease known as multisystem proteinopathy 1 (MSP1), or inclusion body myopathy (IBM), Paget's disease of bone (PDB), frontotemporal dementia (FTD), (IBMPFD), and amyotrophic lateral sclerosis (ALS). We report the clinical and genetic analysis findings in five patients, three from the same family, with novel VCP gene variants: NM_007126.5 c.1106T>C (p.I369T), c.478G>A (p.A160T), and c.760A>T (p.I254F), associated with cardinal MSP1 manifestations including myopathy, PDB, and FTD. Our report adds to the spectrum of heterozygous pathogenic variants found in the VCP gene and the high degree of clinical heterogeneity. This case series prompts increased awareness and early consideration of MSP1 in the differential diagnosis of myopathies and/or PDB, dementia, or ALS to improve the diagnosis and early management of clinical symptoms.

BackgroundValosin containing protein (VCP) is an important protein with many vital functions mostly related to the ubiquitin-proteasome system that provides protein quality control. VCP-associated inclusion body myopathy with Paget disease of bone and frontotemporal dementia, also termed VCP disease and multisystem proteinopathy (MSP 1), is an autosomal dominant disorder caused by monoallelic variants in the VCP gene on human chromosome 9. VCP has also been strongly involved in cancer, with over-activity of VCP found in several cancers such as prostate, pancreatic, endometrial, esophageal cancers and osteosarcoma. Since MSP1 is caused by gain of function variants in the VCP gene, we hypothesized our patients would show increased risk for developing malignancies. We describe cases of 3 rare malignancies and 4 common cancers from a retrospective dataset.ResultsUpon surveying 106 families with confirmed VCP variants, we found a higher rate of rare tumors including malignant peripheral nerve sheath tumor, anaplastic pleomorphic xanthoastrocytoma and thymoma. Some of these subjects developed cancer before displaying other classic VCP disease manifestations. We also present cases of common cancers; however, we did not find an increased rate compared to the general population. This could be related to the early mortality associated with this disease, since most patients die in their 50-60 s due to respiratory failure or cardiomyopathy which is earlier than the age at which most cancers appear.ConclusionThis is the first study that expands the phenotype of VCP disease to potentially include rare cancers and highlights the importance of further investigation of the role of VCP in cancer development. The results of this study in VCP disease patients suggest that patients may be at an increased risk for rare tumors. A larger study will determine if patients with VCP disease develop cancer at a higher rate than the general population. If that is the case, they should be followed up more frequently and screened for recurrence and metastasis of their cancer.

Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding. Real-time intrinsic fluorescence monitoring in protein refolding [ABSTRACT FROM AUTHOR]

The aim of this study was to develop heat-tolerant cellulase enzymes to improve the utilization rate of cellulosic feed and the economic benefits of livestock and poultry. The sequence information of an exonuclease CelS from Clostridium thermocellum was obtained from the protein information database UniProt, and the overexpression of CelS was achieved by signal peptide deletion and codon optimization, and the expression system of Escherichia, coli was used. Through the optimization of self-induced expression conditions, the proportion of soluble expression of CelS was effectively increased. The intracellular supernatant was separated and purified by nickel ion affinity chromatography and heat treatment to obtain soluble CelS, and the intracellular precipitate was washed, denatured and renatured to obtain inclusion body renatured CelS. The enzymatic characteristics showed that there was no significant difference in the enzyme activities of the two forms of CelS, the optimal reaction temperature was 70 贮, the optimal reaction pH was 5.5-6.0, and the activity of amorphous cellulose (PASC) was the highest, followed by crystalline cellulose (Avicel) and corn stover (CS). CelS and EG12B synergistically hydrolyzed CS with Thermotoga maritima, which increased the activity of hydrolase by 81.8% compared with hydrolase alone. The results showed that recombinant CelS exhibited superior heat tolerance and cellulose hydrolysis activity, which provided strong support for the further development of enzyme preparations, and was conducive to improving the utilization rate of cellulose feed. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) is a mosquito-borne infectious disease caused by the Japanese encephalitis virus (JEV). There is currently no effective treatment for JE, and all approved Japanese encephalitis vaccine products originated from the JEV genotype III (GIII). In recent years, JEV genotype I (GI) has gradually replaced GIII as the dominant genotype, and a new symptom of peripheral nerve injury (PNI) caused by JEV NX1889 strain has attracted wide attention, in which JEV envelope (E) protein may be involved in early peripheral nerve injury. Here, the envelope (E) gene of JEV GIb, NX1889 strain was cloned into pET-32a (+) plasmid, and then the E protein was successfully expressed as the high-yield inclusion body form in Escherichia coli (E. coli) BL21 (DE3). Subsequently, we successfully developed a simplified approach to generate soluble JEV E protein by solubilization, and a stepwise dialysis refolding approach with the introduction of a redox system. The refolding recombinant E protein was identified by anti-JEV-E polyclonal antibody and the serum anti-E titer of mice immunized with refolded E protein was higher than 1: 409,600, which proves it has highly immunoreactive. Moreover, the recombinant E protein could recognize antibodies in samples of multiple species and types, such as mice, rabbits, and humans. The approach described above is economical and does not require complex eukaryotic expression systems to produce immunoreactive JEV E protein, which may be of great significance for the diagnosis and pathogenesis of JEV GI strain.

This study aimed to evaluate the correlation between various outcome measures used to assess disease severity and progression in inclusion body myositis (IBM) clinical trials. A cross-sectional study, involving 51 IBM patients meeting the European Neuromuscular Center (ENMC) 2011 criteria for clinically defined or probable IBM, was completed at the University of California, Irvine. Clinical details, demographic data, and functional data including timed get up (TGU), manual muscle testing, hand grip, pinch dynamometry, as well as IBM functional rating scale (IBMFRS), modified Rankin score, forced vital capacity (FVC), and modified ocular bulbar facial respiratory scale (mOBFRS) were collected on all patients. Descriptive statistics and Pearsons r correlation were performed to analyze the data. Age of the patient did not correlate with any of the outcome measures. Greater severity of IBMFRS scores correlated with longer disease duration as well as greater severity for FVC, strength outcomes, TGU, modified Rankin, and mOBFRS. Additionally, TGU strongly correlated with muscle strength measurements, modified Rankin, and mOBFRS. mOBFRS moderately correlated with IBMFRS, muscle strength, FVC, TGU and modified Rankin score. We demonstrate moderate to strong correlations among the disease severity outcome measures in this study.

PURPOSE OF REVIEW: To discuss recent developments in our understanding of epidemiology, diagnostics, biomarkers, pathology, pathogenesis, outcome measures, and therapeutics in inclusion body myositis (IBM). RECENT FINDINGS: Recent epidemiology data confirms a relatively higher prevalence in the population aged above 50 years and the reduced life expectancy. Association with cancer and other systemic disorders is better defined. The role of magnetic resonance imaging (MRI) and ultrasound in diagnosis as well as in following disease progression has been elucidated. There are new blood and imaging biomarkers that show tremendous promise for diagnosis and as outcome measures in therapeutic trials. Improved understanding of the pathogenesis of the disease will lead to better therapeutic interventions, but also highlights the importance to have sensitive and responsive outcome measures that accurately quantitate change. SUMMARY: There are exciting new developments in our understanding of IBM which should lead to improved management and therapeutic options.

Inclusion Body Myopathy, Paget's Disease of Bone, with Frontotemporal Dementia is a progressive autosomal dominant disease that affects the ubiquitin-proteasome complex, that is caused by variants in the Valosin Containing Protein (VCP) gene. We report the first case of concurrent pathogenic variants in both MYBPC3 and VCP that led to earlier onset of congestive heart failure with features of dilated cardiomyopathy. Cardiomyopathy has previously been associated with VCP inclusion body myopathy mostly at an advanced stage of the disease. Due to acute onset of cardiomyopathy in a previous asymptomatic individual, a cardiomyopathy gene panel was obtained which revealed an additional c.177_187del variant of the MYBPC3 gene. We report a first case of concurrent pathogenic variants in both c.177_187del gene of MYBPC3 and p.R155C VCP that led to earlier onset and a more severe form of the cardiomyopathy.

A broad application spectrum ranging from clinical diagnostics to biosensors in a variety of sectors, makes the enzyme Lactate dehydrogenase (LDH) highly interesting for recombinant protein production. Expression of recombinant LDH is currently mainly carried out in uncontrolled shake-flask cultivations leading to protein that is mostly produced in its soluble form, however in rather low yields. Inclusion body (IB) processes have gathered a lot of attention due to several benefits like increased space-time yields and high purity of the target product. Thus, to investigate the suitability of this processing strategy for ldh L1 production, a fed-batch fermentation steering the production of IBs rather than soluble product formation was developed. It was shown that the space-time-yield of the fermentation could be increased almost 3-fold by increasing q s to 0.25 g g−1 h−1 which corresponds to 21% of q s , max , and keeping the temperature at 37°C after induction. Solubilization and refolding unit operations were developed to regain full bioactivity of the ldh L1. The systematic approach in screening for solubilization and refolding conditions revealed buffer compositions and processing strategies that ultimately resulted in 50% product recovery in the refolding step, revealing major optimization potential in the downstream processing chain. The recovered ldh L1 showed an optimal activity at pH 5.5 and 30∘C with a high catalytic activity and K M values of 0.46 mM and 0.18 mM for pyruvate and NADH, respectively. These features, show that the here produced LDH is a valuable source for various commercial applications, especially considering low pH-environments. • Steering an Escherichia coli fermentation towards inclusion body formation for increased production of Lactate dehydrogenase originating from Lactobacillus plantarum. • Systematic investigation of solubilization and refolding unit operations for increased product recovery. • Cross unit operation comparison of processing efficiency reveals optimization potential along processing chain. • Scalable production of ldh L1 for low pH applications. [ABSTRACT FROM AUTHOR]

Development and optimization of inclusion body (IB) refolding processes is often based on empirical strategies. Thus, generation of in-depth process understanding is crucial to shift towards more knowledge-driven approaches. Within this contribution the goal was to establish a systematic approach for the reliable quantification of process dynamics in protein refolding processes. Established offline- analytical tools like HPLC-based methods or photometric assays were analyzed regarding their specifications and consequent suitability to quantify protein states. An in-silico study was conducted to define requirements for state quantification by analyzing the influences of error, discrete sampling intervals and the pace of the reaction on signal quality. Refolding and aggregation reaction rates were calculated by finite difference approximation with errors propagated from the corresponding folding state. Application of the defined principles on two real Lactate dehydrogenase fed-batch refolding processes resulted in two individual sampling strategies. At reaction rates of up to 0.58 g h−1 description of the ongoing dynamics could be done accurately with high sampling intervals during the first few hours of processing as shown by comparison to a noise free simulation. The proposed method has the potential to be transferred to other proteins and to facilitate the development of model-based monitoring & control strategies. [Display omitted] • Systematic selection of analytical tools for protein refolding states. • Analysis of influencing factors on signal quality of protein refolding and aggregation rates. • Quantitative analysis of folding dynamics in fed-batch refolding processes. • Sampling strategy for maximization of information via offline data generation. [ABSTRACT FROM AUTHOR]

Summary: Phase separation has emerged as a fundamental principle for organizing viral and cellular membraneless organelles. Although these subcellular compartments have been recognized for decades, their biogenesis and mechanisms of regulation are poorly understood.Here, we investigate the formation of membraneless inclusion bodies (IBs) induced during the infection of a plant rhabdovirus, tomato yellow mottle‐associated virus (TYMaV). We generated recombinant TYMaV encoding a fluorescently labeled IB constituent protein and employed live‐cell imaging to characterize the intracellular dynamics and maturation of viral IBs in infected Nicotiana benthamiana cells.We show that TYMaV IBs are phase‐separated biomolecular condensates and that viral nucleoprotein and phosphoprotein are minimally required for IB formation in vivo and in vitro. TYMaV IBs move along the microfilaments, likely through the anchoring of viral phosphoprotein to myosin XIs. Furthermore, pharmacological disruption of microfilaments or inhibition of myosin XI functions suppresses IB motility, resulting in arrested IB growth and inefficient virus replication.Our study establishes phase separation as a process driving the formation of liquid viral factories and emphasizes the role of the cytoskeletal system in regulating the dynamics of condensate maturation. [ABSTRACT FROM AUTHOR]

Therapeutic proteins treat many acute and chronic diseases that were until recently considered untreatable. However, their high development cost keeps them out of reach of most patients around the world. One plausible solution to lower-cost manufacturing is to adopt newer technologies like using Escherichia coli to express larger molecules, including full-length antibodies, generally relegated to Chinese Hamster Ovary (CHO) cells, adopt continuous manufacturing, and convert the manufacturing to cell-free synthesis. The advantages of using E. coli include a shorter production cycle, little risk of viral contamination, cell host stability, and a highly reproducible post-translational modification. [ABSTRACT FROM AUTHOR]

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Background and objectivesTo evaluate the therapeutic potential of targeting highly differentiated T cells in patients with inclusion body myositis (IBM) by establishing high-resolution mapping of killer cell lectin-like receptor subfamily G member 1 (KLRG1+) within the T and natural killer (NK) cell compartments.MethodsBlood was collected from 51 patients with IBM and 19 healthy age-matched donors. Peripheral blood mononuclear cells were interrogated by flow cytometry using a 12-marker antibody panel. The panel allowed the delineation of naive T cells (Tn), central memory T cells (Tcm), 4 stages of effector memory differentiation T cells (Tem 1-4), and effector memory re-expressing CD45RA T cells (TemRA), as well as total and subpopulations of NK cells based on the differential expression of CD16 and C56.ResultsWe found that a population of KLRG1+ Tem and TemRA were expanded in both the CD4+ and CD8+ T-cell subpopulations in patients with IBM. KLRG1 expression in CD8+ T cells increased with T-cell differentiation with the lowest levels of expression in Tn and highest in highly differentiated TemRA and CD56+CD8+ T cells. The frequency of KLRG1+ total NK cells and subpopulations did not differ between patients with IBM and healthy donors. IBM disease duration correlated with increased CD8+ T-cell differentiation.DiscussionOur findings reveal that the selective expansion of blood KLRG1+ T cells in patients with IBM is confined to the TemRA and Tem cellular compartments.

Therapeutic proteins treat many acute and chronic diseases that were until recently considered untreatable. However, their high development cost keeps them out of reach of most patients around the world. One plausible solution to lower-cost manufacturing is to adopt newer technologies like using Escherichia coli to express larger molecules, including full-length antibodies, generally relegated to Chinese Hamster Ovary (CHO) cells, adopt continuous manufacturing, and convert the manufacturing to cell-free synthesis. The advantages of using E. coli include a shorter production cycle, little risk of viral contamination, cell host stability, and a highly reproducible post-translational modification.

The present study aimed to express soluble recombinant class IIa bacteriocin in Escherichia coli. The plantaricin LPL-1 gene was cloned into an arabinose promoter, and the C-terminus was fused with the his-tag sequence for expression. The inhibitory activity of recombinant plantaricin LPL-1 against Listeria monocytogenes 54002 was used as the response variable. A novel cell factory, E. coil (ΔtrxB + Δgor + ahpCM), suitable for class IIa bacteriocin expression was established by CRISPR gene editing technology. It was derived from E. coil BW25331 by deleting the thioredoxin reductase (trxB) gene and glutathione reductase (gor) gene and mutating the gene encoding peroxidase (ahpC). The recombinant plantaricin LPL-1 was purified by affinity chromatography, and its physicochemical properties were analyzed. The purified plantaricin LPL-1 possessed wide pH stability (2–11), high thermal stability (60–100 ℃), and surfactant stability. The recombinant bacteriocin has wide potential for food industrial applications.

Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease in adults over age 50, yet it remains unclear whether the disease is primarily driven by T cell–mediated autoimmunity. IBM muscle biopsies display nuclear clearance and cytoplasmic aggregation of TDP-43 in muscle cells, a pathologic finding observed initially in neurodegenerative diseases, where nuclear loss of TDP-43 in neurons causes aberrant RNA splicing. Here, we show that loss of TDP-43–mediated splicing repression, as determined by inclusion of cryptic exons, occurs in skeletal muscle of subjects with IBM. Of 119 muscle biopsies tested, RT-PCR–mediated detection of cryptic exon inclusion was able to diagnose IBM with 84% sensitivity and 99% specificity. To determine the role of T cells in pathogenesis, we generated a xenograft model by transplanting human IBM muscle into the hindlimb of immunodeficient mice. Xenografts from subjects with IBM displayed robust regeneration of human myofibers and recapitulated both inflammatory and degenerative features of the disease. Myofibers in IBM xenografts showed invasion by human, oligoclonal CD8+ T cells and exhibited MHC-I up-regulation, rimmed vacuoles, mitochondrial pathology, p62-positive inclusions, and nuclear clearance and cytoplasmic aggregation of TDP-43, associated with cryptic exon inclusion. Reduction of human T cells within IBM xenografts by treating mice intraperitoneally with anti-CD3 (OKT3) suppressed MHC-I up-regulation. However, rimmed vacuoles and loss of TDP-43 function persisted. These data suggest that T cell depletion does not alter muscle degenerative pathology in IBM.

Valosin-containing protein (VCP) associated multisystem proteinopathy (MSP) is a rare inherited disorder that may result in multisystem involvement of varying phenotypes including inclusion body myopathy, Paget's disease of bone (PDB), frontotemporal dementia (FTD), parkinsonism, and amyotrophic lateral sclerosis (ALS), among others. An international multidisciplinary consortium of 40+ experts in neuromuscular disease, dementia, movement disorders, psychology, cardiology, pulmonology, physical therapy, occupational therapy, speech and language pathology, nutrition, genetics, integrative medicine, and endocrinology were convened by the patient advocacy organization, Cure VCP Disease, in December 2020 to develop a standard of care for this heterogeneous and under-diagnosed disease. To achieve this goal, working groups collaborated to generate expert consensus recommendations in 10 key areas: genetic diagnosis, myopathy, FTD, PDB, ALS, Charcot Marie Tooth disease (CMT), parkinsonism, cardiomyopathy, pulmonology, supportive therapies, nutrition and supplements, and mental health. In April 2021, facilitated discussion of each working group's conclusions with consensus building techniques enabled final agreement on the proposed standard of care for VCP patients. Timely referral to a specialty neuromuscular center is recommended to aid in efficient diagnosis of VCP MSP via single-gene testing in the case of a known familial VCP variant, or multi-gene panel sequencing in undifferentiated cases. Additionally, regular and ongoing multidisciplinary team follow up is essential for proactive screening and management of secondary complications. The goal of our consortium is to raise awareness of VCP MSP, expedite the time to accurate diagnosis, define gaps and inequities in patient care, initiate appropriate pharmacotherapies and supportive therapies for optimal management, and elevate the recommended best practices guidelines for multidisciplinary care internationally.

ABSTRACT Filoviruses depend on the nucleoprotein (NP) to accomplish multiple functions during the viral life cycle. NP is the most abundantly expressed viral protein in infected cells and the main component of the viral nucleocapsid. It can be structurally divided into amino- and carboxy- terminal domains (NTD and CTD). The NTD can homo-oligomerize to interact and protect the (−) ssRNA genome, forming long helical structures. The flexible CTD is responsible for the binding of other nucleocapsid proteins and is involved in the formation of inclusion bodies (IBs)—the cytoplasmic sites of nucleocapsid formation and genome replication. The CTD ends in a ~100-residue globular tail. Měnglà virus (MLAV) is the only member of the new Dianlovirus genus within the Filoviridae family. Their differential characteristics and the possibility of becoming a threat for human health justify the interest in better understanding of its structure and function. In this work, we present the structure of the globular tail of the MLAV NP CTD, showing an overall conformation closely related to that previously reported for the equivalent NP region in MARV. Moreover, analyses of the CTD-CTD interactions in the crystal asymmetric unit revealed a higher-order helicoidal structure. Mutational studies underscore the crucial role of a number of residues, located at the CTD-CTD contact interface, for IB formation. Site-directed mutagenesis of amino acids L653 and F687, involved in the formation of these helicoidal assemblies, abrogate the growth of IBs when the full-length MLAV NP was ectopically expressed in cells. Our findings confirm the role of NP CTD in IB formation, also for MLAV, and focus the attention on particular residues as starting point for further analysis. IMPORTANCE Filoviruses are the causative agents of severe and often fatal hemorrhagic disease in humans. Měnglà virus (MLAV) is a recently reported filovirus, isolated from fruit bats that is capable to replicate in human cells, representing a potential risk for human health. An in-depth structural and functional knowledge of MLAV proteins is an essential step for antiviral research on this virus that can also be extended to other emerging filoviruses. In this study, we determined the first crystal structures of the C-terminal domain (CTD) of the MLAV nucleoprotein (NP), showing important similarities to the equivalent domain in MARV. The structural data also show that the NP CTD has the ability to form large helical oligomers that may participate in the control of cytoplasmic inclusion body formation during viral replication.

ABSTRACT One of the current promising solutions to address problems of CO2 emissions is the development of biological carbon fixation strategies. This strategy leads to higher chemical biosynthetic productivity through improved biological CO2 fixation. In a previous study, we introduced the Calvin-Benson Bassham (CBB) genes of the photosynthetic bacterium Cereibacter sphaeroides into Escherichia coli to develop a strain capable of endogenous CO2 recycling by heterologous expression of the CBB genes. However, the heterologous expression of recombinant proteins in E. coli is often hampered by inclusion bodies (IB), which are protein aggregates. Various factors contribute to IB formation, including host cell metabolism, protein synthesis, transformation machinery, and target protein properties. In this study, we investigated the influence of environmental conditions, particularly culture temperature, on IB formation and CO2 recycling in the CBB strain. As a result, by reducing the culture temperature from 37°C to 30°C, a significant suppression of IB formation was achieved, resulting in a remarkable decrease in CO2 release by approximately 5.76 times. In addition, interestingly, an enhancement in the accumulation of pyruvate by approximately 2.3 times was observed at the same time. These results demonstrate the simultaneous improvements in CO2 recycling and the synthesis of organic acids achieved through temperature control. Based on the findings of this study, we believe that temperature control would be a promising approach to increasing chemical biosynthetic productivity as well as biological CO2 fixation activity in integrated autotrophic biorefinery strategies. IMPORTANCE In a previous study, we successfully engineered Escherichia coli capable of endogenous CO2 recycling through the heterologous expression of the Calvin-Benson Bassham genes. Establishing an efficient gene expression environment for recombinant strains is crucial, on par with the importance of metabolic engineering design. Therefore, the primary objective of this study was to further mitigate greenhouse gas emissions by investigating the effects of culture temperature on the formation of inclusion bodies (IB) and CO2 fixation activity in the engineered bacterial strain. The findings demonstrate that lowering the culture temperature effectively suppresses IB formation, enhances CO2 recycling, and concurrently increases the accumulation of organic acids. This temperature control approach, without adding or modifying compounds, is both convenient and efficient for enhancing CO2 recycling. As such, additional optimization of various environmental parameters holds promise for further enhancing the performance of recombinant strains efficiently.

Protein misfolding, aggregation, and accumulation cause neurodegenerative disorders. One such disorder, Huntington’s disease, is caused by an increased number of glutamine-encoding trinucleotide repeats CAG in the first exon of the huntingtin (HTT) gene. Mutant proteins of Htt exon 1 with polyglutamine expansion are prone to aggregation and form pathological inclusion bodies in neurons. Extensive studies have shown that misfolded proteins are cleared by the ubiquitin-proteasome system or autophagy to alleviate their cytotoxicity. Misfolded proteins can form small soluble aggregates or large insoluble inclusion bodies. Previous works have elucidated the role of autophagy in the clearance of misfolded protein aggregates, but autophagic clearance of inclusion bodies remains poorly characterised. Here we use mutant Htt exon 1 with 103 polyglutamine (Htt103QP) as a model substrate to study the autophagic clearance of inclusion bodies in budding yeast. We found that the core autophagy-related proteins were required for Htt103QP inclusion body autophagy. Moreover, our evidence indicates that the autophagy of Htt103QP inclusion bodies is selective. Interestingly, Cue5/Tollip, a known autophagy receptor for aggrephagy, is dispensable for this inclusion body autophagy. From the known selective autophagy receptors in budding yeast, we identified three that are essential for inclusion body autophagy. Amyloid beta peptide (Aβ42) is a major component of amyloid plaques found in Alzheimer’s disease brains. Interestingly, a similar selective autophagy pathway contributes to the clearance of Aβ42 inclusion bodies in budding yeast. Therefore, our results reveal a novel autophagic pathway specific for inclusion bodies associated with neurodegenerative diseases, which we have termed IBophagy.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In this study, for the first time in the literature, is investigated the effects of different chaperones on outer transmembrane membrane protein solubility by modeling the expression of the recombinant Omp25 outer membrane protein, which is one of the main antigens against brucellosis and known to form inclusion bodies in the cytoplasm. [ABSTRACT FROM AUTHOR]

VCP associated inclusion body myopathy, Paget's disease of bone, and Frontotemporal Dementia (IBMPFD, VCP disease, or multisystem proteinopathy type 1 (MSP1)) is an autosomal dominant disease caused by missense mutations in the VCP gene, which plays a crucial role in ubiquitin-proteasome dependent degradation of cytosolic proteins. Those diagnosed with the disorder often suffer from cardiovascular complications in the advanced stages. We conducted an observational cross-section study to investigate echocardiographic features of asymptomatic carriers and those affected by the disease to determine the differences and potential early features of the VCP-associated cardiomyopathy. The study cohort constituted of 32 patients with VCP mutations including 23 affected individuals diagnosed with myopathy +/- Paget disease of bone, and 9 asymptomatic carriers. Among the affected individuals, 95.7% had myopathy, 43.5% had Paget's disease of bone, and none had frontotemporal dementia, and the carriers were asymptomatic. Not surprisingly the carriers were younger (mean age 38.4 ± 3.8 years), than the affected cohort (mean age 50.6 ± 9.1 years; p

ObjectiveTo assess long-term (2 years) effects of bimagrumab in participants with sporadic inclusion body myositis (sIBM).MethodsParticipants (aged 36-85 years) who completed the core study (RESILIENT [Efficacy and Safety of Bimagrumab/BYM338 at 52 Weeks on Physical Function, Muscle Strength, Mobility in sIBM Patients]) were invited to join an extension study. Individuals continued on the same treatment as in the core study (10 mg/kg, 3 mg/kg, 1 mg/kg bimagrumab or matching placebo administered as IV infusions every 4 weeks). The co-primary outcome measures were 6-minute walk distance (6MWD) and safety.ResultsBetween November 2015 and February 2017, 211 participants entered double-blind placebo-controlled period of the extension study. Mean change in 6MWD from baseline was highly variable across treatment groups, but indicated progressive deterioration from weeks 24-104 in all treatment groups. Overall, 91.0% (n = 142) of participants in the pooled bimagrumab group and 89.1% (n = 49) in the placebo group had ≥1 treatment-emergent adverse event (AE). Falls were slightly higher in the bimagrumab 3 mg/kg group vs 10 mg/kg, 1 mg/kg, and placebo groups (69.2% [n = 36 of 52] vs 56.6% [n = 30 of 53], 58.8% [n = 30 of 51], and 61.8% [n = 34 of 55], respectively). The most frequently reported AEs in the pooled bimagrumab group were diarrhea 14.7% (n = 23), involuntary muscle contractions 9.6% (n = 15), and rash 5.1% (n = 8). Incidence of serious AEs was comparable between the pooled bimagrumab and the placebo group (18.6% [n = 29] vs 14.5% [n = 8], respectively).ConclusionExtended treatment with bimagrumab up to 2 years produced a good safety profile and was well-tolerated, but did not provide clinical benefits in terms of improvement in mobility. The extension study was terminated early due to core study not meeting its primary endpoint.Clinical trial registrationClinicaltrials.gov identifier NCT02573467.Classification of evidenceThis study provides Class IV evidence that for patients with sIBM, long-term treatment with bimagrumab was safe, well-tolerated, and did not provide meaningful functional benefit. The study is rated Class IV because of the open-label design of extension treatment period 2.

ObjectiveTo define the clinicopathologic features and diagnostic utility associated with anti-cytosolic 5'-nucleotidase 1A (NT5C1A) antibody seropositivity in idiopathic inflammatory myopathies (IIMs).MethodsAnti-NT5C1A antibody status was clinically tested between 2014 and 2019 in the Washington University neuromuscular clinical laboratory. Using clinicopathologic information available for 593 patients, we classified them as inclusion body myositis (IBM), dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy (IMNM), nonspecific myositis, or noninflammatory muscle diseases.ResultsOf 593 patients, anti-NT5C1A antibody was found in 159/249 (64%) IBM, 11/53 (21%) dermatomyositis, 7/27 (26%) antisynthetase syndrome, 9/76 (12%) IMNM, 20/84 (24%) nonspecific myositis, and 6/104 (6%) noninflammatory muscle diseases patients. Among patients with IBM, anti-NT5C1A antibody seropositive patients had more cytochrome oxidase-negative fibers compared with anti-NT5C1A antibody seronegative patients. Among 14 IBM patients initially negative for anti-NT5C1A antibody, three patients (21%) converted to positive. Anti-NT5C1A antibody seropositivity did not correlate with malignancy, interstitial lung disease, response to treatments in dermatomyositis, antisynthetase syndrome, and IMNM, or survival in IIMs.InterpretationAnti-NT5C1A antibody is associated with IBM. However, the seropositivity can also be seen in non-IBM IIMs and it does not correlate with any prognostic factors or survival.

The article discusses the case of a patient with sarcoid myopathy that can resemble inclusion body myositis (IBM). Topics discussed include the patient's presentation of progressive extremity weakness, gait instability, falls and swelling in shoulders and neck, the clinical overlap of sarcoid myopathy and sporadic IBM, and immunosuppression therapy in patients with sarcoid myopathy.

Knock-in homozygote VCPR155H/R155H mutant mice are a lethal model of valosin-containing protein (VCP)-associated inclusion body myopathy associated with Paget disease of bone, frontotemporal dementia and amyotrophic lateral sclerosis. Ceramide (d18:1/16:0) levels are elevated in skeletal muscle of the mutant mice, compared to wild-type controls. Moreover, exposure to a lipid-enriched diet reverses lethality, improves myopathy and normalizes ceramide levels in these mutant mice, suggesting that dysfunctions in lipid-derived signaling are critical to disease pathogenesis. Here, we investigated the potential role of ceramide in VCP disease using pharmacological agents that manipulate the ceramide levels in myoblast cultures from VCP mutant mice and VCP patients. Myoblasts from wild-type, VCPR155H/+ and VCPR155H/R155H mice, as well as patient-induced pluripotent stem cells (iPSCs), were treated with an inhibitor of ceramide degradation to increase ceramide via acid ceramidase (ARN082) for proof of principle. Three chemically distinct inhibitors of ceramide biosynthesis via serine palmitoyl-CoA transferase (L-cycloserine, myriocin or ARN14494) were used as a therapeutic strategy to reduce ceramide in myoblasts. Acid ceramidase inhibitor, ARN082, elevated cellular ceramide levels and concomitantly enhanced pathology. Conversely, inhibitors of ceramide biosynthesis L-cycloserine, myriocin and ARN14494 reduced ceramide production. The results point to ceramide-mediated signaling as a key contributor to pathogenesis in VCP disease and suggest that manipulating this pathway by blocking ceramide biosynthesis might exert beneficial effects in patients with this condition. The ceramide pathway appears to be critical in VCP pathogenesis, and small-molecule inhibitors of ceramide biosynthesis might provide therapeutic benefits in VCP and related neurodegenerative diseases.

Missense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson's disease. Here, we characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal-lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. We also establish multiple links between SVIP and VCP-dependent disease in our Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, we identify a human SVIP mutation and confirm the pathogenicity of this mutation in our Drosophila model. We propose a model for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles.

Paraoxonase 2 (PON2) is considered as a potential anti-biofilm agent due to the highest lactonase activity among the PON family members implicating quorum quenching in gram-negative bacteria. However, PON2 is expressed mostly in insoluble fractions in the bacterial expression host which limits its application as an anti-biofilm agent. Therefore, obtaining the native human PON2 (HuPON2) protein in soluble form, better protein yield, stability, and enzymatic activities is essential. In this study, procedures for obtaining a high yield of the native form of HuPON2 in soluble and active forms were optimized. Guanidinium hydrochloride solubilized the HuPON2 protein, however, it is lethal for several bacteria, and thus a major problem for studying the various downstream application of the protein. Therefore, another refolding process for native HuPON2 was optimized. Owing to the promiscuous nature of HuPON2, we hypothesized that it could inhibit the biofilm formation in Mycobacterium smegmatis also. Interestingly, we observed a significant inhibition of the biofilm formation by HuPON2_Rf. However, the primary target of HuPON2 and the probable mechanism behind the quorum quenching in M. smegmatis need to be further explored, which would help widen the scope of HuPON2 as a potential anti-biofilm agent beyond the gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Objective: Investigation of the frequency and progression of ventilatory muscle dysfunction in patients with inclusion body myositis, the most common myopathy after age of 50 yrs. Prior research is limited to case series and cross-section studies. Design: This is a retrospective review of pulmonary function tests, respiratory symptoms, and muscle strength testing. Results: Of the 54 patients reviewed (mean age: 65 ± 9 yrs and disease duration: 7 ± 7 yrs), the majority (n = 32, 59%) had restrictive forced vital capacity deficits at initial visit. Patients with reduced forced vital capacity showed higher prevalence of respiratory symptoms; but age, body mass index, and limb strength were similar when compared with patients without restrictive forced vital capacity. Mean rate of forced vital capacity decline of 0.108 l/yr in inclusion body myositis patients. Lower baseline limb strength correlated with longer disease duration and future forced vital capacity decline (eg, weaker patients experienced faster decline). Conclusions: Based on forced vital capacity, there is a high frequency of ventilatory pump muscle weakness in inclusion body myositis, which is associated with a higher burden of respiratory symptoms. Baseline strength may indicate risk of respiratory decline and need for vigilant screening. Importantly, ventilatory and limb muscle decline may not progress in a corresponding manner, highlighting the importance of pulmonary function surveillance. [ABSTRACT FROM AUTHOR]

The production of recombinant proteins is one of the most significant achievements of biotechnology in the last century. These proteins are produced in the eukaryotic or prokaryotic heterologous hosts. By increasing the omics data especially related to different heterologous hosts as well as the presence of new amenable genetic engineering tools, we can artificially engineer heterologous hosts to produce recombinant proteins in sufficient quantities. Numerous recombinant proteins have been produced and applied in various industries, and the global recombinant proteins market size is expected to be cast to reach USD 2.4 billion by 2027. Therefore, identifying the weakness and strengths of heterologous hosts is critical to optimize the large-scale biosynthesis of recombinant proteins. E. coli is one of the popular hosts to produce recombinant proteins. Scientists reported some bottlenecks in this host, and due to the increasing demand for the production of recombinant proteins, there is an urgent need to improve this host. In this review, we first provide general information about the E. coli host and compare it with other hosts. In the next step, we describe the factors involved in the expression of the recombinant proteins in E. coli. Successful expression of recombinant proteins in E. coli requires a complete elucidation of these factors. Here, the characteristics of each factor will be fully described, and this information can help to improve the heterologous expression of recombinant proteins in E. coli. [ABSTRACT FROM AUTHOR]

The wide applications of human epidermal growth factor (hEGF) as an efficient mitogen agent in the clinical, health, and cosmetic industries has made it an interesting research target. But its low abundance in natural resources has limited its utilization. Therefore, the high-level expression of recombinant hEGF (rhEGF) has received attention in recent years. However, expression of rhEGF in the Escherichia coli intracellular environment often results in the formation of inclusion bodies (IBs). In this study, to increase the expression level and solubility, the SUMO tag was fused to the mature hEGF gene and expressed in E. coli BL21 (DE3). Subsequently, the freeze-thawing method plus 2 M urea solubilized IBs. Further, the SUMO-rhEGF protein was purified by Ni-NTA affinity chromatography, and the biological activity of the purified protein was investigated on the NIH3T3 cell line by MTT assay. The results showed SUMO tag succeeded in increasing the fusion protein expression level, but ~80% of them were in the form of IBs. Further, it was found the freeze-thawing method could significantly solubilize and refold about 70% of the IBs. MTT assays also indicated the different concentrations of rhEGF had mitogenic activity in a dose-dependent manner and significantly increases the viability percentage of treated cells after 48 h. The results of this study showed that the freeze-thawing method has improved the expression yield of rhEGF, and due to its cost-effectiveness and high efficiency, it can be a promising strategy to produce rhEGF for pharmaceutical and industrial development. [ABSTRACT FROM AUTHOR]

ABSTRACT Methods for efficient insoluble protein production require further exploration. PagP, an Escherichia coli outer membrane protein with high β-sheet content, could function as an efficient fusion partner for inclusion body-targeted expression of recombinant peptides. The primary structure of a given polypeptide determines to a large extent its propensity to aggregate. Herein, aggregation “hot spots” (HSs) in PagP were analyzed using the web-based software AGGRESCAN, leading to identification of a C-terminal region harboring numerous HSs. Moreover, a proline-rich region was found in the β-strands. Substitution of these prolines by residues with high β-sheet propensity and hydrophobicity significantly improved its ability to form aggregates. Consequently, the absolute yields of recombinant antimicrobial peptides Magainin II, Metchnikowin, and Andropin were increased significantly when expressed in fusion with this refined version of PagP. We describe separation of recombinant target proteins expressed in inclusion bodies fused with the tag. An artificial NHT linker peptide with three motifs was implemented for separation and purification of authentic recombinant antimicrobial peptides. IMPORTANCE Fusion tag-induced formation of inclusion bodies provides a powerful means to express unstructured or toxic proteins. For a given fusion tag, how to enhance the formation of inclusion bodies remains to be explored. Our study illustrated that the aggregation HSs in a fusion tag played important roles in mediating its insoluble expression. Efficient production of inclusion bodies could also be implemented by refining its primary structure to form a more stable β-sheet with higher hydrophobicity. This study provides a promising method for improvement of the insoluble expression of recombinant proteins.

Nonsegmented negative-strand RNA viruses, including measles virus (MeV), a member of the Paramyxoviridae family, are assumed to replicate in cytoplasmic inclusion bodies. These cytoplasmic viral factories are not membrane bound, and they serve to concentrate the viral RNA replication machinery. Although inclusion bodies are a prominent feature in MeV-infected cells, their biogenesis and regulation are not well understood. Here, we show that infection with MeV triggers inclusion body formation via liquid-liquid phase separation (LLPS), a process underlying the formation of membraneless organelles. We find that the viral nucleoprotein (N) and phosphoprotein (P) are sufficient to trigger MeV phase separation, with the C-terminal domains of the viral N and P proteins playing a critical role in the phase transition. We provide evidence suggesting that the phosphorylation of P and dynein-mediated transport facilitate the growth of these organelles, implying that they may have key regulatory roles in the biophysical assembly process. In addition, our findings support the notion that these inclusions change from liquid to gel-like structures as a function of time after infection, leaving open the intriguing possibility that the dynamics of these organelles can be tuned during infection to optimally suit the changing needs during the viral replication cycle. Our study provides novel insight into the process of formation of viral inclusion factories, and taken together with earlier studies, suggests that Mononegavirales have broadly evolved to utilize LLPS as a common strategy to assemble cytoplasmic replication factories in infected cells.IMPORTANCE Measles virus remains a pathogen of significant global concern. Despite an effective vaccine, outbreaks continue to occur, and globally ∼100,000 measles-related deaths are seen annually. Understanding the molecular basis of virus-host interactions that impact the efficiency of virus replication is essential for the further development of prophylactic and therapeutic strategies. Measles virus replication occurs in the cytoplasm in association with discrete bodies, though little is known of the nature of the inclusion body structures. We recently established that the cellular protein WD repeat-containing protein 5 (WDR5) enhances MeV growth and is enriched in cytoplasmic viral inclusion bodies that include viral proteins responsible for RNA replication. Here, we show that MeV N and P proteins are sufficient to trigger the formation of WDR5-containing inclusion bodies, that these structures display properties characteristic of phase-separated liquid organelles, and that P phosphorylation together with the host dynein motor affect the efficiency of the liquid-liquid phase separation process.

Lk2 and Lk3 were thermostable recombinant lipase and highly expressed in Escherichia coli BL21 (DE3). However, Lk2 and Lk3 accumulated as an inclusion body. To further characterize both recombinant lipases, the soluble enzyme must be obtained first. This study aimed to optimize the disruption of the cell membrane in order to obtain soluble and active lipases. The effects of temperature lysis, pH, and SDS concentration on lipolytic activity Lk2 and Lk3 were investigated using a three-factor Box-Behnken design response surface methods. The optimum condition for the temperature variables at 50°C, pH 8, and 0.34% SDS which gave a lipolytic activity of 0.9 U for Lk2. Meanwhile, Lk3 lipolytic activity of 0.9 U obtained at the temperature of 50°C, pH 8, and 0.1% SDS. This result showed efficient one-step membrane disruption methods using thermolysis with addition of a low concentration of detergent at pH 8. The methods used were effective and applicable in the production of active and soluble thermostable recombinant lipase.

Transforming growth factor-beta 3 (TGF-β3) is an important cytokine involved in various biological processes. TGF-β3 is used as a scar-reducing antifibrotic agent for acute and chronic wounds and fibrosing disorders. TGF-β3, a valuable therapeutic protein, is produced recombinantly in different expression systems. TGF-β3, produced in the Escherichia coli (E. coli) expression system, widely used due to its various advantages in recombinant production, is commercially available. However, the main problem encountered in protein expression in E. coli cells is the formation of an inclusion body. Various approaches have been developed to solve this problem. The use of a fusion tag is one of the most powerful strategies used to obtain protein in the soluble active form in the E. coli expression system. Superfolder GFP (sfGFP) is one of the fusion tags used to increase the solubility of the fusion partner in E. coli. In this study, TGF-β3 with sfGFP fusion tag (sfGFP-TGFβ3) was successfully produced in soluble form in E. coli BL21 (DE3) in high yield and purity for the first time. Purified protein was identified by western blot and SDS-PAGE. 20 mg of protein with 98% purity was obtained from 1 L of bacterial culture. It was determined that the obtained high purity protein did not have a cytotoxic effect on BJ normal human skin fibroblast cells. The impact of sfGFP-TGFβ3 fusion protein on wound healing was evaluated with in vitro scratch wound healing assay. The results showed that the sfGFP-TGFβ3 fusion protein produced in soluble form in the E. coli expression system has the potential to support the wound healing process. [ABSTRACT FROM AUTHOR]

Background and Aims: Cytomegalovirus (CMV) infection is a common occurrence in liver transplantation (LT) even in an era of preventive strategies. However, the diagnosis of CMV colitis remains challenging. This study aimed to focus on the clinical significance of endoscopic biopsy-proven CMV colitis in patients following living donor liver transplantation (LDLT). Methods: From January 2007 to December 2021, a total of 55 CMV colitis cases were retrospectively enrolled and divided into a non-LDLT group in 53 and an LDLT group in 2 cases. Clinical demographics, diagnostic measurement, histopathology, and anti-viral therapy were investigated. Results: There were 1630 cases undergoing LDLT in the period 2007–2021, with only 2 recipients being confirmed to have CMV colitis in 2021 (2/114, 1-year incidence: 1.75%). Comparisons between the 53 non-LDLT cases and 2 LDLT cases are as follows: Serum anti-CMV immunoglobulin M (IgM) was shown to be positive (n = 3, 5.5% vs. n = 0, p = 1.0) and negative (n = 20, 37.7% vs. n = 2, 100%, p = 0.16); anti-CMV immunoglobulin G (IgG) was positive (n = 19, 35.8% vs. n = 2, 100%, p = 0.14) and none were negative; CMV DNAemia was shown to be detectable (n = 14, 26.4% vs. n = 1, 50%, p = 0.47) and undetectable (n = 14, 26.4% vs. n = 1, 50%, p = 0.47). Among the two recipients with CMV colitis, one had CMV DNAemia and the other had no CMV DNAemia upon the development of symptoms; negative anti-CMV-IgM and positive anti-CMV-IgG were observed both pre-transplant and post-transplant; finally, CMV colitis was documented based on the presence of inclusion bodies and positive immunohistochemistry (IHC) staining in histology. Conclusion: Patients with immunocompromised status, in particular organ transplantation, may have positive serum anti-CMV IgM/IgG antibodies both before and after transplantation. This study emphasized the fact that endoscopic biopsy with IHC staining may be a more powerful tool for making an accurate diagnosis of CMV colitis in the setting of living donor liver transplantation. [ABSTRACT FROM AUTHOR]

Autophagy is emerging as a critical player in host defense against diverse infections, in addition to its conserved function to maintain cellular homeostasis. Strikingly, some pathogens have evolved strategies to evade, subvert or exploit different steps of the autophagy pathway for their lifecycles. Here, we present a new viral mechanism of manipulating autophagy for its own benefit with severe fever with thrombocytopenia syndrome bunyavirus (SFTSV, an emerging high‐pathogenic virus) as a model. SFTSV infection triggers autophagy, leading to complete autophagic flux. Mechanistically, we show that the nonstructural protein of SFTSV (NSs) interacts with mTOR, the pivotal regulator of autophagy, by targeting its kinase domain and captures mTOR into viral inclusion bodies (IBs) induced by NSs itself. Furthermore, NSsimpairs mTOR‐mediated phosphorylation of unc‐51‐like kinase 1 (ULK1) at Ser757, disrupting the inhibitory effect of mTOR on ULK1 activity and thus contributing to autophagy induction. Pharmacologic treatment and Beclin‐1 knockout experimental results establish that, in turn, autophagy enhances SFTSV infection and propagation. Moreover, the minigenome reporter system reveals that SFTSV ribonucleoprotein (the transcription and replication machinery) activity can be bolstered by autophagy. Additionally, we found that the NSs proteins of SFTSV‐related bunyaviruses have a conserved function of targeting mTOR. Taken together, we unravel a viral strategy of inducing pro‐viral autophagy by interacting with mTOR, sequestering mTOR into IBs and hence provoking the downstream ULK1 pathway, which presents a new paradigm for viral manipulation of autophagy and may help inform future development of specific antiviral therapies against SFTSV and related pathogens. [ABSTRACT FROM AUTHOR]

The RUS (ROOT UVB SENSITIVE 1) proteins characterized by their unique DUF647 domain are widely distributed in eukaryotes. Their functional roles are largely unknown except for the possible involvement of Arabidopsis RUS1 and RUS2 in early seedling development. To investigate the biochemical roles of the RUS proteins, full length and truncated Arabidopsis RUS1 were seamlessly fused with GFP and cloned into prokaryotic expression vector pQE-100 which allows proteins expressed with an N-terminal 6×His tag. Expression of the full length RUS1-GFP could not be detected after adding the inducer IPTG, while a truncated RUS1-GFP was expressed at high levels and formed inclusion bodies in Escherichia coli. The inclusion bodies were dissolved in a denaturing buffer, and then the truncated RUS1-GFP fusion protein in the supernatant was bound to a Ni-NTA slurry. The bound proteins were eluted after the non-specific binding proteins were washed away. The purified truncated proteins were detected as a single clear band of the expected size in SDS-PAGE, and were further confirmed by the Western blot test. Our results suggest that the impossible expression of the full length RUS1 protein in E. coli can be expressed in truncated form, and inclusion bodies can be effectively solubilized.

Inclusion body myopathy (IBM) associated with Paget disease of the bone and frontotemporal dementia or IBMPFD is an autosomal dominant degenerative disorder caused by mutations in the valosin-containing protein (VCP) gene. We aim to establish a detailed clinical phenotype of VCP disease amongst 35 (28 affected individuals, 7 presymptomatic gene carriers) individuals versus 14 unaffected first-degree relatives in 14 families to establish useful biomarkers for IBMPFD and identify the most meaningful tests for monitoring disease progression in future clinical trials. Comprehensive studies included the Inclusion Body Myositis Functional Rating Scale (IBMFRS) and fatigue severity scale questionairres, strength measurements using the Manual Muscle Test with Medical Research Council (MRC) scales, hand-held dynamometry using the microFET and Biodex dynamometers, 6 minute walk test (6MWT), and pulmonary function studies. Strong correlation was observed between the IBMFRS and measurements of muscle strength with dynamometry and the other functional tests, indicating that it may be utilized in long-term follow-up assessments due to its relative simplicity. This cross-section study represents the most comprehensive evaluation of individuals with VCP disease to date and provides a useful guide for evaluating and possible monitoring of muscle weakness and pulmonary function progression in this unique cohort of individuals.

Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5-deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication.IMPORTANCE Measles virus is a human pathogen that remains a global concern, with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here, we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus.

Many negative-sense RNA viruses, including measles virus (MeV), are thought to carry out much of their viral replication in cytoplasmic membraneless foci known as inclusion bodies (IBs). The mechanisms by which IBs facilitate efficient viral replication remain largely unknown but may involve an intricate network of regulation at the host-virus interface. Viruses are able to modulate such interactions by a variety of strategies including adaptation of their genomes and "hijacking" of host proteins. The latter possibility broadens the molecular reservoir available for a virus to enhance its replication and/or antagonize host antiviral responses. Here, we show that the cellular 5'-3' exoribonuclease, XRN1, is a host protein hijacked by MeV. We found that upon MeV infection, XRN1 is translocated to cytoplasmic IBs where it acts in a proviral manner by preventing the accumulation of double-stranded RNA (dsRNA) within the IBs. This leads to the suppression of the dsRNA-induced innate immune responses mediated via the protein kinase R (PKR)-integrated stress response (ISR) pathway. [ABSTRACT FROM AUTHOR]

Many negative-sense RNA viruses, including the highly pathogenic Ebola virus (EBOV), use cytoplasmic inclusion bodies (IBs) for viral RNA synthesis. However, it remains unclear how viral mRNAs are exported from these IBs for subsequent translation. We recently demonstrated that the nuclear RNA export factor 1 (NXF1) is involved in a late step in viral protein expression, i.e., downstream of viral mRNA transcription, and proposed it to be involved in this mRNA export process. We now provide further evidence for this function by showing that NXF1 is not required for translation of viral mRNAs, thus pinpointing its function to a step between mRNA transcription and translation. We further show that RNA binding of both NXF1 and EBOV NP is necessary for export of NXF1 from IBs, supporting a model in which NP hands viral mRNA over to NXF1 for export. Mapping of NP-NXF1 interactions allowed refinement of this model, revealing two separate interaction sites, one of them directly involving the RNA binding cleft of NP, even though these interactions are RNA-independent. Immuno-fluorescence analyses demonstrated that individual NXF1 domains are sufficient for its recruitment into IBs, and complementation assays helped to define NXF1 domains important for its function in the EBOV life cycle. Finally, we show that NXF1 is also required for protein expression of other viruses that replicate in cytoplasmic IBs, including Lloviu and Junín virus. These data suggest a role for NXF1 in viral mRNA export from IBs for various viruses, making it a potential target for broadly active antivirals. [ABSTRACT FROM AUTHOR]

Introduction: Overexpression of the EGFR is associated with carcinogenesis, and it is observed in more than 70% of head and neck cancers. The expression of an immunotoxin against EGFR designed as an alternative to full antibody led to the production of aggregated protein in the form of inclusion bodies. This study aimed to investigate the 8M urea and 6M guanidine hydrochloride approaches for obtaining the immunotoxin as the soluble and effective form with correct folding. Material & Methods: The BL21 (DE3) cells containing the pET28a-huimmunotoxin construct were induced by 1 mM IPTG at 37°C for 24 h, and the amount of expression was checked by SDS-PAGE. This immunotoxin was in the form of inclusion bodies and was solubilized individually in 8 M urea and 6 M guanidine hydrochloride and then purified by Ni-NTA affinity chromatography, which was observed as a single band in SDS-PAGE analysis. To correctly refold the obtained immunotoxin, the purified samples were poured into a dialysis bag, and denaturing agents were removed in a multi-step process called stepwise dialysis. The reactivity assessment of the purified and refold immunotoxin was assessed by ELISA technique using A431 cell lysate. Findings: The immunotoxin (17 mg/ml) was expressed using the bacteria cells in the form of inclusion bodies. The refolded humanized immunotoxin had a high reactivity with A431 cells, indicating the suitable folding of the purified immunotoxin. The 50% binding activity rates of humanized immunotoxin obtained from urea and guanidine hydrochloride approaches were 0.8 and 1.7 µg/ml, respectively. Discussion & Conclusion: The results of this study revealed that the urea approach was very effective in solubilizing and proper refolding of immunotoxins that were expressed in bacteria cells as inclusion bodies