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Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

Abstract Bone mass is maintained by the balance between osteoclast-induced bone resorption and osteoblast-triggered bone formation. In inflammatory arthritis such as rheumatoid arthritis (RA), however, increased osteoclast differentiation and activity skew this balance resulting in progressive bone loss. O-GlcNAcylation is a posttranslational modification with attachment of a single O-linked β-D-N-acetylglucosamine (O-GlcNAc) residue to serine or threonine residues of target proteins. Although O-GlcNAcylation is one of the most common protein modifications, its role in bone homeostasis has not been systematically investigated. We demonstrate that dynamic changes in O-GlcNAcylation are required for osteoclastogenesis. Increased O-GlcNAcylation promotes osteoclast differentiation during the early stages, whereas its downregulation is required for osteoclast maturation. At the molecular level, O-GlcNAcylation affects several pathways including oxidative phosphorylation and cell-cell fusion. TNFα fosters the dynamic regulation of O-GlcNAcylation to promote osteoclastogenesis in inflammatory arthritis. Targeted pharmaceutical or genetic inhibition of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) arrests osteoclast differentiation during early stages of differentiation and during later maturation, respectively, and ameliorates bone loss in experimental arthritis. Knockdown of NUP153, an O-GlcNAcylation target, has similar effects as OGT inhibition and inhibits osteoclastogenesis. These findings highlight an important role of O-GlcNAcylation in osteoclastogenesis and may offer the potential to therapeutically interfere with pathologic bone resorption.

Introduction. Pseudoaneurysms of superior mesenteric artery and its branches are extremely rare but life threatening when complications occur. However, the diagnosis is often missed or delayed because of its non-specific signs and symptoms. Treatments depend on patient’s characteristics. In contrast to conventional open surgery, endovascular therapy has been reported as an effective option with minimally invasion, low complication, short recovery time and good outcome, especially in high-risk patients. Case presentation. We describe a case of ruptured pseudoaneurysm of ileal artery, a branch of superior mesenteric artery, presenting with gastrointestinal bleeding signs while on anticoagulant therapy. Both upper and lower gastrointestinal endoscopy were inconclusive. The diagnosis was confirmed by angiography and the patient was successfully treated by coil embolization. Conclusions. Ruptured pseudoaneurysm of superior mesenteric artery and its branches should be suspected in patients with unexplained gastrointestinal hemorrhage. Endovascular intervention is a feasible approach, especially in patients with multiple comorbidities and high bleeding risk.

Price stability is the ultimate objective of the State Bank of Vietnam (SBV). In addition, in a small export-led economy, such as Vietnam, the study of exchange rate volatility is crucial for the country’s economy due to its impact on inflation via exchange rate pass-through (ERPT). This study investigated ERPT in Vietnam by employing both autoregressive distributed lag (ARDL) and nonlinear-ARDL (NARDL) approaches with data spanning from January 2009 to May 2020. Our main findings suggest that the exchange rate and money supply are the two most important factors influencing the consumer price index (CPI) and explain the change in the CPI structure using the threshold model. In addition, the transition to a central-rate mechanism successfully lowered the inflation rate in Vietnam. Furthermore, the NARDL model displays short- and long-run exchange rate asymmetries. Based on this evidence, several policy implications are provided to support price-level stability.

Servelle-Martorell syndrome, also known as angio-osteohypotrophic syndrome, is a congenital venous malformation that rarely involves the arterial system. There are many different, mainly conservative, treatment methods. We present the case of a 28-year-old woman with a history of left arm and back lesions, which appeared at birth and had enlarged over time. She had been diagnosed with a vascular malformation but had not yet received treatment. She presented to our hospital with numbness in her left arm and was diagnosed with Servelle-Martorell syndrome based on computed tomography (CT) angiography. This case emphasizes that CT angiography is a useful modality for evaluating the extent of the venous malformations in patients with Servelle-Martorell syndrome.

BackgroundRecent trials including DIRECT-MT, DEVT, and SKIP have found that direct mechanical thrombectomy (MT) is equally effective as the combination of MT and intravenous thrombolysis. However, the results of the other trials, namely MR-CLEAN NO-IV and the SWIFT-DIRECT trial have failed to confirm the non-inferiority of direct MT vs. the combination therapy.AimWe aimed to identify prognostic factors of direct MT for anterior circulation large vessel occlusion within 4.5 h.Materials and MethodsData from January 2018 to January 2022 were retrospectively collected and analyzed. Adult patients with confirmed anterior circulation large vessel occlusion within 4.5 h of onset with baseline NIHSS of ≥6 and baseline ASPECTS of ≥6 treated using direct MT within 6 h were recruited.ResultsA total of 140 patients were enrolled in the study with a median age of 65.5 years [interquartile range (IQR), 59–76.5], median baseline NIHSS of 13.5 (IQR, 11–16), and median baseline ASPECTS of 8 (IQR, 7–8). Direct MT was feasible in all patients (100%). Successful reperfusion (mTICI 2b-3) was achieved in 124/140 patients (88.6%) with a low rate of complications (8/140, 5.7%). Any type of intracranial hemorrhage (ICH) and symptomatic ICH occurred in 44/140 (31.4%) and 5/140 (3.6%), respectively. Overall, a good outcome (mRS 0–2) was achieved in 93/140 (66.4%), and the mortality rate was 9.3% (13/140 patients). Using multivariate analysis, lower age [odds ratio (OR), 0.96; 95% CI, 0.92–1.00; P = 0.05], low baseline NIHSS (OR, 0.82; 95% CI, 0.74–0.92; P = 0.00), and absence of ICH (OR, 0.29; 95% CI, 0.10–0.81; P = 0.02) were independently associated with favorable outcome. Independent predictors of mortality were baseline NIHSS (OR, 1.21; 95% CI, 1.01–1.46; P = 0.04), successful reperfusion (OR, 0.02; 95% CI, 0.00–0.58; P = 0.02), and ICH (OR, 0.12; 95% CI, 0.02–0.75; P = 0.02). Further analysis showed that the median mRS at 90 days was significantly better in the MCA occlusion group compared to the ICA plus M1 occlusion group [1 (IQR 0–3) vs. 2 (IQR 1–4); P = 0.05].ConclusionsOur findings suggest that direct thrombectomy may be an adequate clinical option for younger patients (≤70) experiencing proximal middle artery occlusion within 4.5 h and who have low baseline NIHSS (≤14).

The XpressCF+® cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50–100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+® reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant E. coli expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms.

Abstract Background Stored potato (Solanum tuberosum L.) tubers are sensitive to wet conditions that can cause rotting in long-term storage. To study the effect of water on the tuber surface during storage, microarray analysis, RNA-Seq profiling, qRT-PCR and phytohormone measurements were performed to study gene expression and hormone content in wet tubers incubated at two temperatures: 4 °C and 15 °C. The growth of the plants was also observed in a greenhouse after the incubation of tubers in wet conditions. Results Wet conditions induced a low-oxygen response, suggesting reduced oxygen availability in wet tubers at both temperatures when compared to that in the corresponding dry samples. Wet conditions induced genes coding for heat shock proteins, as well as proteins involved in fermentative energy production and defense against reactive oxygen species (ROS), which are transcripts that have been previously associated with low-oxygen stress in hypoxic or anoxic conditions. Wet treatment also induced senescence-related gene expression and genes involved in cell wall loosening, but downregulated genes encoding protease inhibitors and proteins involved in chloroplast functions and in the biosynthesis of secondary metabolites. Many genes involved in the production of phytohormones and signaling were also affected by wet conditions, suggesting altered regulation of growth by wet conditions. Hormone measurements after incubation showed increased salicylic acid (SA), abscisic acid (ABA) and auxin (IAA) concentrations as well as reduced production of jasmonate 12-oxo-phytodienoic acid (OPDA) in wet tubers. After incubation in wet conditions, the tubers produced fewer stems and more roots compared to controls incubated in dry conditions. Conclusions In wet conditions, tubers invest in ROS protection and defense against the abiotic stress caused by reduced oxygen due to excessive water. Changes in ABA, SA and IAA that are antagonistic to jasmonates affect growth and defenses, causing induction of root growth and rendering tubers susceptible to necrotrophic pathogens. Water on the tuber surface may function as a signal for growth, similar to germination of seeds.

This paper aims at discussing the theoretical basis as well as the pedagogical potential of using games in teaching mathematics at high schools. A study on the literature, namely international publications pertaining to this topic, enables us to confirm the role of Mathematical Games and Games for Teaching Mathematics - hereinafter referred to as Educational Games for Teaching Mathematics (EGTMs). We have also collected, tested and annotated 26 games in order to initiate a supporting library of EGTMs for interested colleagues.

Abstract Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

Using small colloidal clusters with dumbbell, triplet, tetrahedral, octahedral geometry as elementary building blocks, we investigate the templated-assisted cluster-cluster aggregation into hierarchical clusters, namely superclusters, by means of Monte Carlo simulations. We find that the colloidal superclusters made of dumbbells and triplets produces a unique structural motif that is similar to that of clusters of single colloids, whereas the superclusters composed of tetrahedral and octahedral clusters possess a more complex structure. However, despite their complexity, such structures can be predicted based on a decomposition using triplets as a core and the number of initial “building block” clusters. In particular, a majority of the observable superclusters are members of a particular category of nonconvex deltahedra (Cundy deltahedra). These findings could be useful in preparation of complex colloidal molecules.

The minimization principle of the second moment of the mass distribution ( M 2 ) is responsible for the unique structure of three-dimensional clusters by using emulsion droplet evaporation. Herein we study the structure of two-dimensional clusters of colloidal particles bound at the interface of liquid droplets in the plane. We found that, differently from the three-dimensional system, the two-dimensional clusters have multiple degenerate configurations (isomers). An interesting feature of such two-dimensional clusters is that they have the same packings as those belonging to a class of geometric figures known as polyiamonds. In particular, except for the six-particle cluster, many higher order clusters of polyiamond have not been reported previously. Using a simple geometrical approach, based on the number of ways to generate a packing, we calculated the occupation probabilities of distinct isomeric clusters. The level of agreement with the results of metropolis Monte Carlo simulations was good for clusters containing up to nine particles, suggesting that our two-dimensional cluster structures are not a result of the minimization of the second moment. In addition, the structure of these clusters is somewhat insensitive to the range and depth of the interparticle potential, in good agreement with the results in the literature.

Transforming growth factor [beta] (TGF-[beta]) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-[beta] remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-[beta] in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-[beta]. The activation of latent TGF-[beta] requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin [[alpha].sub.V] (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-[beta], rebalanced TGF-[beta] signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-[beta] in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach., Introduction Fibrosis is defined as the excessive deposition of extracellular matrix in the affected tissues. Activated myofibroblasts are the principal source of extracellular matrix production in physiologic tissue repair as [...]