Progressive encephalomyelitis with rigidity and myoclonus (PERM) is a rare and severe phenotype within stiff-person spectrum disorders (SPSD), frequently associated with glycine receptor antibodies (GlyR-Ab). Because typical rigidity and stimulus-sensitive spasms may be absent at disease onset, early diagnosis can be challenging. We report a 52-year-old man who presented with facial pruritus, facial stiffness, limited tongue protrusion, dysphagia, and left lower-limb pain. Early manifestations included diplopia, restricted extraocular movements, dysarthria, restricted mouth opening, involuntary jaw closure resembling trismus, and involuntary tongue biting. Nonspecific oropharyngeal and maxillofacial abnormalities on imaging initially raised concern for infectious multiple cranial neuropathy, tetanus, or orofacial infection; however, antimicrobial therapy and symptomatic treatment with diazepam and pregabalin produced no sustained improvement. The subsequent emergence of neck, truncal, and lower-limb stiffness, gait impairment, perioral hypersensitivity, and stimulus-induced facial muscle contractions prompted reconsideration of SPSD. Needle electromyography (EMG) demonstrated continuous synchronous motor unit activity in agonist and antagonist muscles at rest. Serum antibody testing was positive for glycine receptor 1 immunoglobulin G (GlyR1-IgG), with a reported semiquantitative value of 1:100, whereas serum glutamic acid decarboxylase 65 (GAD65) antibody was negative, supporting the diagnosis of GlyR-Ab-positive PERM. The patient showed marked initial improvement following combined immunotherapy and symptomatic treatment. During regular follow-up, the patient's symptoms progressively improved. At the latest follow-up, diplopia, facial sensory abnormalities, restricted mouth opening, and limb rigidity had resolved. Repeat serum testing at a different laboratory showed persistent GlyR1-IgG positivity, with a reported endpoint titer of 1:100; the two numerical results were not directly comparable because different titration protocols were used. The patient was advised to continue oral mycophenolate mofetil 500 mg twice daily as maintenance therapy. This case illustrates the diagnostic challenges of GlyR-Ab-positive PERM when facial pruritus and cranial-bulbar symptoms precede generalized stiffness. Recognition of evolving stimulus-induced muscle contractions, together with characteristic EMG findings and antibody testing, can help distinguish this presentation from infectious cranial neuropathy, tetanus, and local orofacial disease.

α-hydroxy ketones are important fine chemical building blocks. To improve in vivo cascade catalysis integration for preparing α-hydroxy ketones from racemic epoxides, we constructed a recombinant Escherichia coli/PeCbn strain based on a previous two-strain system for preparing 4'-chloro-2-hydroxyacetophenone (1c) from rac-4-chlorostyrene oxide (1a). It co-expressed the epoxide hydrolase RpEH[L360V], the (2,3)-butanediol dehydrogenase BsBDHA[I49L/V266L/G292A], and the NADH oxidase EfNOx via dual-plasmid co-transformation, integrating epoxide hydrolysis, alcohol oxidation, and NAD[+] regeneration into one host to simplify catalyst preparation. A silicone tube micro-membrane oxygen supply was introduced to enhance transmembrane oxygen transfer and stabilize EfNOx-mediated coenzyme regeneration. The results showed that micro-membrane oxygenation prevented foaming and cell flotation caused by direct aeration, enabling oxygen diffusion through polydimethylsiloxane (PDMS) walls. SDS-PAGE confirmed soluble expression of all target enzymes, yielding 22.9% 1c initially. Orthogonal optimization fixed induction conditions at OD600=0.3, 20 ℃, 120 mL liquid in a 1 000-mL flask, and 0.062 5 mmol/L IPTG. Further single-factor and orthogonal optimization set catalytic conditions at 80 mg dry cell weight (DCW)/mL cells, 15 mmol/L NAD[+], and 5.0 kPa inlet gauge pressure. In a 50 mL scale-up system, the titer of 1c produced from rac-1a reached 46.7 mmol/L at the time point of 24 h, with a peak average space-time yield of 6.05 mmol/(L·h) at 3 h. This single-strain, multi-enzyme co-expression system provides an experimental basis for green biomanufacturing and oxygen-dependent biocatalysis intensification.

We present a tool to help identify and develop shared goals for collaboration on technologies, standards, and regulatory understanding that will be needed to enable the biopharmaceutical factories the industry will need in the next five years. That tool is a publicly available design and economic analysis of a state-of-the-art commercial scale continuous biomanufacturing facility. Using current technology, equipment, quality and regulatory expectations, this design is described as a reference point against which new technology can be compared, and to illustrate the cost advantages of integrated continuous biomanufacturing. The facility can produce anywhere between 225kg to 3,600kg of mAb per year, at a capital cost of approximately $210 million. This compares to a traditional 6x15kL SS facility, which can produce about 170% more but has a capital cost more than 5 times higher. A 20-year net present cost analysis showed that the continuous facility with bioreactor volumetric productivity of 5 g/L bioreactor/day was favorable to the traditional 15kL six-pack at a titer of 10 g/L by half a billion to over a billion dollars, even at production rates up to 14,000 kg/yr.

Insect cytochrome P450 monooxygenases (CYPs) exert essential functions in xenobiotic detoxification and growth and development. Nevertheless, the physiological roles of the majority of CYP genes remain poorly characterized in the small hive beetle (SHB), Aethina tumida. Screening of CYP genes from the ovarian transcriptome of SHBs identified AtCYP6K1 as an ovary-enriched gene. RT-qPCR analysis revealed that ovarian AtCYP6K1 expression increased with female adult age, reaching the highest level at day 7 post-eclosion. RNA interference (RNAi)-mediated silencing of AtCYP6K1 caused defective ovarian development and markedly reduced female fecundity, egg hatchability, and juvenile hormone III (JHIII) titers. Furthermore, knockdown of AtCYP6K1 significantly downregulated the expression of key genes associated with vitellogenin synthesis (Vg1, Vg2, VgR) and the juvenile hormone signaling pathway (Met, Kr-h1). Notably, in vitro overexpression assays confirmed that AtCYP6K1 catalyzes JH III synthesis from methyl farnesoate (MF) with activity similar to CYP15A1. These findings establish that AtCYP6K1 plays an essential role in female reproduction by directly participating in JH III biosynthesis, which in turn regulates vitellogenin synthesis and ovarian maturation. This study advances our understanding of endogenous physiological functions of insect CYP6 proteins and the reproductive regulatory mechanism of SHB, and offers insights for developing future population management strategies against this pest.

This chapter describes the experimental methodology used to establish a rat model of oral food allergy. The final protocol has been optimized for the Wistar strain. The model mimics chronic oral allergen re-exposure and allows the study of effector-phase allergic responses. It encompasses all experimental stages, including preliminary procedures, intramuscular sensitization, the use of pro-type 2 immune response adjuvants, allergen re-sensitization, and the final oral challenge, using ovalbumin (OVA). Key outcomes include clinical parameters such as sign scoring, diarrhea classification, and body temperature changes during oral anaphylaxis. In addition, immunological and inflammatory markers such as OVA-specific IgE titer, mast cell protease II, or even histamine can be assessed.

Zebrafish (Danio rerio) has emerged as a robust vertebrate model for studying immune and allergic disorders, including food allergies. The alpha-Gal syndrome (AGS) is a tick-borne allergy characterized by an IgE-mediated delayed hypersensitivity to the oligosaccharide galactose-α-1,3-galactose (α-Gal), which is present in tick saliva and mammalian-derived foods and drugs. This chapter describes the experimental procedures used to establish and analyze the validated zebrafish model for AGS. Adult zebrafish were intramuscularly injected with Ixodes ricinus tick saliva and maintained on a mammalian meat-based diet to induce allergic responses. Clinical manifestations were monitored and correlated with serum anti-α-Gal IgM titers measured by ELISA, while the intestinal expression of allergic and immune biomarkers (ctss2.1, prkdc, il1b, il4, tnfα, tlr2, c3a, and ifn1) was analyzed by RT-qPCR. This animal model successfully reproduces key features of AGS such as delayed allergic reactions (hemorrhagic-type), altered behavior, and increased mortality, accompanied by elevated anti-α-Gal IgM antibody levels and modulation of immune gene expression. Importantly, zebrafish also enables the testing of specific salivary biomolecules from ticks or other arthropods to evaluate their allergenic potential. Overall, the zebrafish model provides a reproducible and cost-effective in vivo platform to elucidate molecular mechanisms underlying AGS and arthropod-induced allergies.

Conventional microbial biosynthetic pathways for isoprenoid often require intricate metabolic balancing and multigene regulation, which are not only time-consuming but also prone to interference from host cellular metabolism and feedback inhibition by toxic intermediate metabolites or end-products. In this study, we developed a novel cell-free multienzyme catalytic system for the synthesis of lycopene, a representative isoprenoid. By integrating the isopentenol utilization pathway with cell-free metabolic engineering strategies, the system eliminates the need for complex in vivo genetic modifications and avoids inherent cellular metabolic interference. The in vitro reaction employs seven enzymes directly, enabling rapid and efficient lycopene production without relying on costly substrates isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The optimized cell-free reaction mixture consisted of isoprenol and prenol (3:1 molar ratio, 15mM), seven key enzymes (each at a concentration of 0.6mg/mL), 8mM Mg[2+], 1mM NAD, 1mM ATP, 1mM CoA, and 0.05% (w/v) Tween 20 in 30mM Bis-Tris buffer (pH 6.5). After incubation at 30 °C for 20hours, the lycopene titer reached 128.04mg/L. Further in vivo validation achieved a final lycopene titer of 270.0mg/L. This novel approach offers a promising alternative strategy to overcome common challenges associated with the construction and regulation of complex natural isoprenoid biosynthetic pathways.

Carfentanil, an ultrapotent synthetic opioid, poses a severe public health threat because of its rapid brain penetration and potent activation of μ-opioid receptors. Conventional anti-drug vaccines are limited by the poor hapten immunogenicity and dominant anti-carrier immunity. Here, we report a molecularly encoded synthetic carfentanil vaccine (MECVPLA-PEI) integrating a carfentanil hapten, TLR7/8 agonist, and T-helper epitope within a polymeric scaffold to coordinate B and T cell activation. MECVPLA-PEI exhibited markedly enhanced lymph node targeting, with 21.2-fold higher accumulation at 24 h and retention over 7 days, leading to a 1.5-fold increase in plasma cell differentiation and enhanced dendritic cell activation. Compared with an OVA-carrier protein-based vaccine, MECVPLA-PEI induced 22-fold higher antibody titers with a very low level of anti-carrier antibodies and attenuated carfentanil-induced antinociceptive and reward-related behaviors. Importantly, MECVPLA-PEI exhibited a favorable safety profile. This work establishes a precise synthetic vaccine strategy for anti-carfentanil therapy.

Lipid nanoparticles (LNPs) administered parenterally often show poor localization to multiple tissue types, including the pancreas and gastrointestinal (GI) tract. Furthermore, patients typically prefer orally administered drugs to those that are given parenterally. We therefore investigated whether GI LNP administration to buccal, gastric, small intestinal, colonic, and rectal submucosal tissues could simultaneously circumvent parenteral administration while enhancing LNP delivery to previously inaccessible locations. We found that, in addition to local targeting, gastric LNP administration achieved an order of magnitude greater pancreas delivery in rodents than intravenous administration. Gastric dosing of glucagon-like peptide-1 mRNA also improved blood glucose regulation in mice compared with phosphate-buffered saline controls. Additionally, we found that buccal LNP administration targeted local and lymphatic tissues. Furthermore, dosing mice with ovalbumin mRNA resulted in equivalent immunoglobulin G blood titers to those of intramuscular delivery. These results suggest that GI administration can reprogram LNP biodistribution, expanding therapeutic opportunities for both local and systemic nucleic acid delivery.

Viral infections pose a persistent challenge to global public health and cause millions of infections and substantial mortality each year. There is therefore an urgent need for effective antiviral strategies to limit the transmission of viruses such as influenza A virus and SARS-CoV-2. In this work, we report a copper imidazole-carboxylate coordinated nanozyme (Cu-ImCar) with high esterase-mimicking activity and potent antiviral performance. Mechanistic studies reveal that Cu-ImCar nanozyme catalyzes the hydrolysis of lipid components in the viral envelope, leading to structural disruption and rapid loss of viral infectivity. In addition, the nanozyme demonstrates excellent catalytic stability and tolerance to extreme conditions. When functionalized onto fabrics, Cu-ImCar nanozyme efficiently inactivated influenza virus and SARS-CoV-2 pseudovirus over a wide temperature range (-20 to 37 °C). Notably, at a moderately elevated temperature of 50 °C, it reduces viral titers to undetectable levels within 5 min, even at the low loading level (6.25 μg cm-2). These findings establish Cu-ImCar as a robust antiviral nanozyme with broad environmental adaptability and highlight the potential of esterase-mimicking catalysis as an effective strategy for viral inactivation and transmission control.

Tick-borne encephalitis (TBE) is an escalating global health threat, increasingly exacerbated by climate change, and underscores the need for innovative vaccine strategies. Conventional subunit vaccines are limited by suboptimal spatiotemporal dissociation between antigens and adjuvants, as well as insufficient specific targeting, resulting in compromised immunogenicity. Herein, we report a biomimetic nanovaccine platform engineered to overcome these limitations through the synergistic co-delivery of antigen and adjuvant. This platform is constructed by co-displaying the TBE virus envelope protein domain III (fused to a dendritic cell-targeting peptide, Dcpep) together with the molecular adjuvant MAB2560 on the surface of bacterium-like particles (BLPs). The resulting co-display vaccine, EDc2&MAB2560-GEM BLPs, outperforms a simple mixture of its components, inducing significantly higher titers of TBEV-specific IgG antibodies, robust T-cell and B-cell immune responses, and durable immunity in mice. Mechanistic studies reveal that these enhanced immune responses arise from the platform's ability to precisely co-deliver antigen and adjuvant to dendritic cells, thereby triggering a potent cascading immunostimulatory effect. This work highlights the critical role of spatiotemporal coupling within targeted nanocarriers and establishes a versatile strategy for the development of next-generation subunit vaccines with enhanced potency and safety.

Francisella tularensis, the causative agent of tularemia, is a facultative intracellular pathogen categorized as a tier I bioterrorism agent. The current live vaccine strain (LVS) vaccine poses safety and virulence concerns, advocating the need for a safer alternative. In this study, we demonstrated a combination of antigens FopA, Tul4, SucB, and DnaK conjugated to biopolymer particles (BPs) as a multivalent subunit vaccine that not only induces LVS and SchuS4-specific antibody titers but also confers protective immunity with differential cytokine profiles upon LVS challenge. This multivalent vaccine conferred 80% survival in mice, with antibodies of opsonizing ability and rapid recovery from weight loss. The production of these biopolymer conjugate vaccines was achieved in one step inside engineered Escherichia coli and is not only cost-effective but also leads to thermostable formulations, eliminating the need for cold-chain transport and storage. E. coli fermentation-based production avoids the need for high-containment facilities. Collectively, the FopA-BP-Tul4, SucB-BP, and Dnak-BP combined formulations hold the promise for the development of a robust subunit vaccine for the prevention of tularemia.

The increasing demands of synthetic biology have shifted the central bottleneck from DNA assembly to DNA writing. As engineered systems grow in complexity, progress increasingly depends on the ability to write, test, and rewrite regulatory DNA to support fast design-build-test-learn cycles and precise control of gene expression. In this regard, enzymatic DNA synthesis offers a promising route toward next-generation DNA writing technologies for on-demand benchtop implementation. In particular, terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, provides a unique mechanism for generating sequence diversity without predefined templates. Here, we establish a programmable TdT-based enzymatic platform for the in-house synthesis of random-sequence oligonucleotide libraries with tunable base composition. Systematic kinetic characterization of TdT revealed a substrate-dependent bias, which we mitigated through optimization of divalent cofactors and dNTP input ratios, enabling user-defined nucleotide distributions. Using this platform, we expanded and screened composition-controlled libraries of promoters, 5'-UTRs, and transcription terminators in Escherichia coli, identifying functional bioparts spanning wide dynamic ranges of transcriptional and translational activity. Integration of TdT-synthesized regulatory libraries into a streamlined enzymatic workflow enabled rapid cloning and screening within hours. As a proof of concept, targeted promoter diversification of a lycopene biosynthetic operon yielded a 124.7-fold range in production and identified variants achieving up to 1.43-fold higher titers than those of the parental strain. These results establish TdT-mediated enzymatic DNA synthesis as a controllable DNA writing modality for rapid regulatory-part engineering in synthetic biology and metabolic engineering.

Cyanobacteria act as efficient "solar-powered" cell factories, converting CO2 and light into valuable products while thriving in seawater or wastewater. Cyanobacteria are attractive hosts for terpenoid bioproduction because they natively produce terpene precursors. Our review found that while cyanobacteria have been engineered to produce 26 different terpenoids, only 1 of the 63 terpene bioproduction studies achieved titers in the g/L range. To identify the path forward towards maximizing cyanobacterial terpene titers and productivity, this review has taken a twofold approach. First, it synthesizes progress and bottlenecks across three interconnected dimensions: synthetic biology, systems biology, and bioprocess engineering. Second, it addresses a limitation of the field by harmonizing all cyanobacterial terpenoid bioproduction titers into consistent units for the first time, enabling like-for-like comparisons. Through this, three factors were found consistent across the top five highest titers: terpene precursor overexpression, fermentation optimization (using nutrient-rich media and/or a bioreactor), and the production of a volatile terpenoid. On the other hand, while systems biology-informed metabolic engineering can significantly increase cellular health and productivity, it is the least utilized strategy. Taken together, this review highlights the importance of multifaceted approaches to improving cyanobacterial terpene production.