High Molecular Weight Research Articles

Extensional rheology of elastoviscous aqueous PEO/PEG or DMS Boger fluids and weakly elastic alternatives for investigating viscoelastic flows and instabilities

Boger fluids that exhibit a rate-independent shear viscosity (typically ∼ 1 Pa∙s = 1000x water viscosity) and elasticity measurable using torsional rheometers (modulus, relaxation time, or first normal stress difference) are considered as the realization of viscoelastic fluids described by the Oldroyd-B model. However, Boger fluids, conventionally formulated as dilute solutions of high molecular weight (Mw) polymers in relatively high viscosity solvents (or solutions of oligomers or lower Mw polymer), are unsuitable for emulating polymeric fluids used in coating formulations that are lower in viscosity, appear inelastic in torsional shear rheometry, and yet appear prone to viscoelastic instabilities. Therefore, Dontula, Macosko, and Scriven (DMS) (AIChE J, 1998) chose to create an alternative model of elastic fluids by dissolving ultrahigh molecular weight (UHMw) poly(ethylene oxide) (PEO) in an aqueous solution of its lower Mw analog, often referred to as poly(ethylene glycol) or PEG. Even though numerous studies of printing and coating flow instabilities use this aqueous PEO/PEG or DMS Boger fluids as model viscoelastic fluids, only a few explicitly measured elastic properties, especially using relaxation time, and hardly any characterized extensional rheology. In this contribution, we recreate the DMS Boger fluids to examine their elasticity and extensional rheology response using dripping-onto-substrate (DoS) rheometry that relies on an analysis of capillarity-driven pinching dynamics. Though the aqueous PEG solution is often treated as a viscous and Newtonian solvent, we discover that in DoS rheometry, both the PEG solution and the DMS Boger fluid display power law response followed by elastocapillary, in contradiction with the assumptions and the response expected of the Oldroyd-B model. Furthermore, the solution of entangled PEG chains influences specific viscosity, pinching dynamics, and measured extensional rheology response in striking contrast with Newtonian solvents of the same viscosity. Lastly, we describe the possibility of using dilute, aqueous solutions of UHMw PEO as model viscoelastic fluids for coating flows, for both elasticity and extensional viscosity can be determined using DoS rheometry. Due to their water-like viscosity, the aqueous PEO solutions appeal as model fluids that have elasticity, emulate Newtonian fluids in the early stages of pinching, and have a relaxation time tunable by changing polymer Mw or concentration.

Periodic membrane fractionation of freshwater organic matter reveals various reactivity patterns during chlorine/chloramine disinfection

Although drinking water disinfection has significantly reduced mortality from waterborne diseases, the formation of potentially harmful disinfection by-products (DBPs) remains a concern today. These DBPs emerge from the reaction of dissolved organic matter (DOM) with chlor(am)ine during disinfection. Identifying these DBP precursors from heterogeneous and complex DOM mixtures is challenging and to address this, a novel membrane fractionation protocol was developed to isolate and identify the organic DBP precursors from fresh water sources. A tight ultrafiltration and nanofiltration membrane were carefully selected to partition DOM into three molecular weight (MW) fractions: high (>20 kDa), medium (0.3–20 kDa) and low (<0.3 kDa) as defined by high performance-size exclusion chromatography- total organic carbon analysis. A mathematical tool was developed to optimize the fractionation protocol. Therefore, ultrafiltration was executed before nanofiltration and the concentration factor was maximized without inducing fouling to obtain the purest fractions. The mathematical tool was also set in place to predict the necessary diafiltration factor for each individual fractionation experiment based on the initial organic matter composition of the surface water allowing the fractionation protocol to be effective at all times. The protocol was applied to surface water samples collected six times across three seasons yielding a fraction enriched in high MW compounds (up to 50 %), a fraction having more than 80 % of medium MW compounds and a fraction only containing low MW compounds. Although the medium MW fraction showed the highest reactivity for the majority of the investigated DBPs, the low MW fraction showed high reactivity for iodinated trihalomethanes during chlorination and for haloacetonitriles during chloramination. The high MW fraction had the lowest reactivity towards DBPs, which can have important implications for a drinking water treatment since this fraction is generally the most effectively removed by e.g. coagulation, while the more important fractions for DBP formation such as the medium and low MW compounds remain in the water until the disinfection step.

Validation of a global method for the simultaneous analysis of polar and non-polar pesticides by online extraction and LC-MS/MS

BackgroundMulti-residue methods for pesticide analysis in food are available for many compounds, but polar pesticides are not generally included due to their specific properties, which include high polarity and low molecular weight. Single residue methods are therefore needed for sample preparation, while chromatographic separation often requires derivatization, ion paring, or dedicated methods suitable for polar compounds, mostly ion chromatography and hydrophilic interaction liquid chromatography (HILIC). These challenges affect the important pesticide glyphosate and the related compounds aminomethylphosphonic acid (AMPA) and glufosinate. There are only a few methods including these compounds in large-scale analysis, mostly complex methods based on multidimensional chromatography. ResultsA new method, for the global online extraction and analysis of pesticides in beer was developed and validated. The method exploited an online trapping device, with reversed-phase (RP) and anion exchange properties, that can trap small molecules from liquid samples. The ion exchange mechanism was used to retain the very polar pesticides glyphosate, AMPA, and glufosinate. The hydrophobic properties of the trapping column were also exploited to trap pesticides suitable for multi-residue investigations. The chromatographic separation was optimized by comparison of HILIC and RP C30, which could separate pesticides, including the polar ones, with modulation by the trapping column after proper selection of the mobile phase composition and basic modifier. The validation for beer provided recoveries in the range 71–112 %, with <15 % RSD, and LOD and LOQ values of 0.02–1 and 0.3–3 μg L−1, respectively. The result was competitive with previous methods on polar pesticide analysis in beer. SignificanceThe method was validated for 15 pesticides, over the log Kow range from −4.4 to 4.5, using a methodology with single and fast chromatographic separation under conditions compatible with multi-residue analysis by RP-LC-MS/MS. In the case of beer, for which the method was validated, the sample preparation was also performed online, after simple degassing, and sample dilution.

Rapid Production of Native and Mirror-Image Tumor Necrosis Factor-α Enabled by Automated Flow Peptide Synthesis Technology.

Tumor necrosis factor-α (TNF-α) plays a central role in immune response regulation. Because elevated TNF-α production is correlated with a range of diseases, inhibiting the interaction of this protein with its native receptors has been thoroughly explored as a therapeutic avenue. Despite advancements in the development of TNF-α inhibitors, concerns remain regarding immunogenicity and loss of activity in vivo. To facilitate the discovery of stable and less immunogenic therapeutic modalities, we applied a single-shot automated fast-flow peptide synthesis (AFPS) strategy to produce full-length TNF-α, resulting in a complex reaction mixture. Leveraging the ability of AFPS to generate long peptides with high purity, we combined this technology with native chemical ligation (NCL). An NCL reaction using two fragments readily produced by AFPS afforded synthetic L- and D-TNF-α in milligram quantities (up to 5.5 mg, ∼28% yield). Following the oxidative folding of synthetic TNF-α using established conditions, higher molecular weight species were generated. Through high-throughput screening of refolding conditions, functional synthetic L- and mirror-image D-TNF-α were obtained, exhibiting characteristics analogous to those of the recombinant TNF-α. Overall, this approach can serve as a general protocol for accessing proteins that are intractable by modern protein synthesis methods, therefore, streamlining the development of novel therapeutics.

Anodic Commodity Polymer Recycling: The Merger of Iron-Electrocatalysis with Scalable Hydrogen Evolution Reaction.

Plastics are omnipresent in our everyday life, and accumulation of post-consumer plastic waste in our environment represents a major societal challenge. Hence, methods for plastic waste recycling are in high demand for a future circular economy. Specifically, the degradation of post-consumer polymers towards value-added small molecules constitutes a sustainable strategy for a carbon circular economy. Despite of recent advances, chemical polymer degradation continues to be largely limited to chemical redox agents or low energy efficiency in photochemical processes. We herein report a powerful iron-catalyzed degradation of high molecular weight polystyrenes through electrochemistry to efficiently deliver monomeric benzoyl products. The robustness of the ferraelectrocatalysis was mirrored by the degradation of various real-life post-consumer plastics, also on gram scale. The cathodic half reaction was largely represented by the hydrogen evolution reaction (HER). The scalable electro-polymer degradation could be solely fueled by solar energy through a commercially available solar panel, indicating an outstanding potential for a decentralized green hydrogen economy.

Multimaterial Digital-Light Processing of Metal-Organic Framework (MOF) Composites: A Versatile Tool for the Rapid Microfabrication of MOF-Based Devices.

Patterning Metal-Organic Frameworks (MOFs) is essential for their use in sensing, electronics, photonics, and encryption technologies. However, current lithography methods are limited in their ability to pattern more than two MOFs, hindering the potential for creating advanced multifunctional surfaces. Additionally, balancing design flexibility, simplicity, and cost often results in compromises. This study addresses these challenges by combining Digital-Light Processing (DLP) with a capillary-assisted stop-flow system to enable multimaterial MOF patterning. It demonstrates the desktop fabrication of multiplexed arbitrary micropatterns across cm-scale areas while preserving the MOF's pore accessibility. The ink, consisting of a MOF crystal suspension in a low volatile solvent, a mixture of high molecular weight oligomers, and a photoinitiator, is confined by capillarity in the DLP projection area and quickly exchanged using syringe pumps. The versatility of this method is demonstrated by the direct printing of a ZIF-8-based luminescent oxygen sensor, a 5-component dynamic information concealment method, and a PCN-224-based colorimetric sensor for amines, covering disparate pore and analyte sizes. The multi-MOF capabilities, simplicity, and accessibility of this strategy pave the way for the facile exploration of MOF materials across a wide range of applications, with the potential to significantly accelerate the design-to-application cycle of MOF-based devices.

Analysis of two consanguineous Chinese pedigrees affected with Hereditary prokallikrein deficiency and High molecular weight kininogen deficiency

To analyze the laboratory phenotype and genetic variants of two consanguineous Chinese pedigrees affected with Hereditary prokallikrein (PK) and High molecular weight kininogen (HMWK) deficiency and explore their molecular pathogenesis. A PK deficiency pedigree (10 individuals from 4 generations) and a HMWK deficiency pedigree (6 individuals from 3 generations) which were admitted to the First Affiliated Hospital of Wenzhou Medical University on December 3, 2021 and June 16, 2022, respectively were selected as the study subjects. Clinical data of the two pedigrees were collected, and the related coagulation indexes of the probands and their family members were determined. Genomic DNA of the two pedigrees was extracted from peripheral blood samples. All of the exons and flanking sequences of the KLKB1 and KNG1 genes of the probands were analyzed by direct sequencing. And the corresponding sites were sequenced among other family members. Bioinformatic software was used to analyze the conservation of variation sites and the effect of variant on the protein function. The plasma PK activity of proband 1, a 29-year-old female, and her brother were extremely low (< 1.0%). Proband 2 was a 66-year-old male with extremely low plasma HMWK activity (< 1.0%). Genetic sequencing revealed that the proband 1 and her brother had both harbored a homozygous c.417_418insCATTCTTA (p.Arg140Hisfs*3) insertional variant in exon 5 of the KLKB1 gene. Proband 2 had harbored a homozygous c.460C>A (p.Pro154Thr) missense variant in exon 4 of the KNG1 gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variants were respectively rated as pathogenic (PVS1+PM2_Supporting+PM4) and likely pathogenic (PS4+PM2_Supporting+PP3+PP4). The c.417_418insCATTCTTA (p.Arg140Hisfs*3) variant of the KLKB1 gene and the c.460C>A (p.Pro154Thr) variant of the KNG1 gene probably underlay the decreased PK and HMWK activities in the two pedigrees, respectively.

Recent Trends on Zinc Complexes Bearing Bi‐, Tri‐ and Tetra‐Dentate Schiff Base Ligands for Catalytic Ring‐Opening Polymerization of Lactides

AbstractNumerous zinc complexes based on N‐ and O‐donor Schiff‐base ligands have shown great promise in the ring‐opening polymerization (ROP) of lactides, which are cyclic dimers of lactic acid, to produce poly(lactic acid) (PLA). This is primarily because zinc is a borderline metal, making it non‐cytotoxic, inexpensive, abundant, biocompatible, and environmentally safe. Additionally, Schiff‐based ligands offer stability, stereoselectivity, desirable electronic and steric environments, and resistance to impurities. As a result, zinc complexes are easy to synthesize, colourless, have high reactivity and solubility in organic solvents, and are manageable under typical circumstances. Furthermore, these complexes are free to adopt a variety of coordination numbers since zinc does not have ligand field stabilization energy (LFSC). Zinc complexes possess high catalytic activity due to their distinctive properties, including high Lewis acidity, high electron transfer ability, stability concerning the reactive intermediate, and the ability to monitor polymerization by nuclear magnetic resonance (NMR) spectroscopy. Moreover, these complexes also have reasonable control over stereochemistry, number‐average molecular weight (Mn), and dispersity index (Ð). These advantages make it possible to produce colourless PLA with a high yield, high molecular weight, and narrow dispersity index in bulk and solvent‐based conditions.

Hemoglobin-PEG Interactions Probed by Small-Angle X-ray Scattering: Insights for Crystallization and Diagnostics Applications.

Protein-protein interactions, controlling protein aggregation in the solution phase, are crucial for the formulation of protein therapeutics and the use of proteins in diagnostic applications. Additives in the solution phase are factors that may enhance the protein's conformational stability or induce crystallization. Protein-PEG interactions do not always stabilize the native protein structure. Structural information is needed to validate excipients for protein stabilization in the development of protein therapeutics or use proteins in diagnostic assays. The present study investigates the impact of polyethylene glycol (PEG) molecular weight and concentration on the spatial structure of human hemoglobin (Hb) at neutral pH. Small-angle X-ray scattering (SAXS) in combination with size-exclusion chromatography is employed to characterize the Hb structure in solution without and with the addition of PEG. Our results evidence that human hemoglobin maintains a tetrameric conformation at neutral pH. The dummy atom model, reconstructed from the SAXS data, aligns closely with the known crystallographic structure of methemoglobin (metHb) from the Protein Data Bank. We established that the addition of short-chain PEG600, at concentrations of up to 10% (w/v), acts as a stabilizer for hemoglobin, preserving its spatial structure without significant alterations. By contrast, 5% (w/v) PEG with higher molecular weights of 2000 and 4000 leads to a slight reduction in the maximum particle dimension (Dmax), while the radius of gyration (Rg) remains essentially unchanged. This implies a reduced hydration shell around the protein due to the dehydrating effect of longer PEG chains. At a concentration of 10% (w/v), PEG2000 interacts with Hb to form a complex that does not distort the protein's spatial configuration. The obtained results provide a deeper understanding of PEG's influence on the Hb structure in solution and broader knowledge regarding protein-PEG interactions.

Molecular, structural, and functional characterization of delta subunit of T-complex protein-1 from Leishmania donovani.

Chaperonins/Heat shock protein 60 are ubiquitous multimeric protein complexes that assist in the folding of partially and/or misfolded proteins using metabolic energy into their native stage. The eukaryotic group II chaperonin, also referred as T-complex protein-1 ring complex (TRiC)/T-complex protein-1 (TCP1)/chaperonin containing T-complex protein (CCT), contains 8-9 paralogous subunits, arranged in each of the two rings of hetero-oligomeric complex. In Leishmania, till date, only one subunit, LdTCP1γ, has been well studied. Here, we report the molecular, structural, and functional characterization of TCP1δ subunit of Leishmania donovani (LdTCP1δ), the causative agent of Indian kala-azar. LdTCP1δ gene exhibited only 27.9% identity with LdTCP1γ and clustered in a separate branch in the phylogenic tree of LdTCP1 subunits. The purified recombinant protein formed a high molecular weight complex (0.75 MDa), arranged into 16-mer assembly, and performed in vitro chaperonin activity as assayed by ATP-dependent luciferase folding. LdTCP1δ exhibits 1.8-fold upregulated expression in metabolically active, rapidly dividing log phase promastigotes. Over-expression of LdTCP1δ in promastigotes results in increased infectivity and rate of multiplication of intracellular amastigotes. The study thus establishes the existence of an individual functionally active homo-oligomeric complex of LdTCP1δ chaperonin with its role in parasite infectivity and multiplication.

Aqueous extractable nonfibrillar and sarkosyl extractable fibrillar Alzheimer’s disease tau seeds have distinct properties

Pathological tau fibrils in progressive supranuclear palsy, frontotemporal dementia, chronic traumatic encephalopathy, and Alzheimer’s disease each have unique conformations, and post-translational modifications that correlate with unique disease characteristics. However, within Alzheimer’s disease (AD), both fibrillar (sarkosyl insoluble (AD SARK tau)), and nonfibrillar (aqueous extractable high molecular weight (AD HMW tau)) preparations have been suggested to be seed-competent. We now explore if these preparations are similar or distinct in their in vivo seeding characteristics. Using an in vivo amplification and time-course paradigm we demonstrate that, for AD HMW and AD SARK tau species, the amplified material is biochemically similar to the original sample. The HMW and SARK materials also show different clearance, propagation kinetics, and propagation patterns. These data indicate the surprising co-occurrence of multiple distinct tau species within the same AD brain, supporting the idea that multiple tau conformers – both fibrillar and nonfibrillar- can impact phenotype in AD.

Aromatic (co)polycarbonates bearing pendant 2,3-dimethylmaleimido group based upon a new phthalimidine-containing “cardo” bisphenol

A new “cardo” bisphenol viz., 1-(2-(1,1-bis(4-hydroxyphenyl)-3-oxoisoindolin-2-yl)ethyl)-3,4-dimethyl-1H-pyrrole-2,5-dione (PPH-MA) was synthesized in a two-step reaction sequence starting from phenolphthalein. PPH-MA was utilized as a step-growth monomer for the synthesis of a homo- and fourco-polycarbonates bearing pendant 2,3-dimethylmaleimido groups (PC-MAs) via solution polycondensation of PPH-MA or various mol % compositions of PPH-MA and bisphenol-A, respectively, with triphosgene.1H NMR spectroscopy confirmed the chemical structure and composition of PC-MAs. Inherent viscosity and number average molecular weight values of PC-MAs were in the range 0.45–0.64 dL g−1 and 18,300 − 36,200 g mol−1, respectively, indicating the formation of polymers of medium to reasonably high molecular weights. Tough, transparent and flexible films of PC-MAs could be cast from chloroform solution. X-ray diffraction studies indicated the amorphous nature of PC-MAs. The 10% weight loss temperature (T10) values of PC-MAs were in the range 373–443 °C indicating their good thermal stability.

Chitosan-graft-poly(vinyl acetate) for wood-adhesive applications

Poly(vinyl acetate), PVAc, adhesives are commonly used for wood bonding; however, they are fossil-based and the final products usually do not have a sufficient water resistance for more durable applications. In this study we prepared an adhesive formulation by grafting VAc from chitosan using emulsion polymerization, chitosan-graft-PVAc. Thereby, we could decrease the fossil-based content of the adhesive and at the same time significantly improve the water resistance. Chitosan by itself has very good bonding properties as a wood adhesive, especially regarding water resistance; however, very low solid contents of the adhesive formulation can be achieved due to a very high viscosity of chitosan adhesives. In our chitosan-graft-PVAc adhesives, we explored two chitosan samples with different molecular weights, by using as-received chitosan and hydrolyzing it to a lower molecular weight. The chitosan fractions in the adhesives prepared with a higher molecular weight chitosan were 15, 20 and 25 wt%. However, due to the high viscosity, a solid content higher than 17 wt% could not be achieved for these adhesives. Sufficient bond strengths were achieved when the adhesive was applied in 122 g/m2 solid spread rate. In order to decrease the viscosity, we used hydrolyzed chitosan, with a lower molecular weight, to allow for a higher adhesive solid content, 34 wt%, and for a higher chitosan fraction, 40 wt%. In the adhesive with 40 wt% chitosan and 17 wt% solid content, all VAc was grafted from chitosan. This decreased the molecular mobility of the chains, leading to a lower susceptibility to plastic creep in the adhesive which contributes to the final bond strength. The dry and wet strengths of the specimens bonded with adhesives containing chitosan were higher than the strength of the specimens bonded with the reference PVAc adhesive.

Controlled two-step synthesis of n-type conjugated ladder polymers using a flow reactor

The coplanar backbone of conjugated ladder polymers (cLPs) enhances electron transfer and π-π interactions, making cLPs promising materials for various applications in organic electronics and optoelectronics. Nevertheless, the limited solubility and intricate synthetic processes for cLPs make it challenging to control their molecular weight and corresponding properties, and pose as significant obstacles to their widespread application. Here, an n-type cLP, PNDI-2BocL, was synthesized using a flow reactor for the first time, with controlled molecular weights. The polymerization reaction conditions to synthesize a precursor polymer, PNDI-2Boc, were first optimized in flow, followed by sequential ladderization in batch through the injection of an acid solution. The reaction time of polymerization was varied from 7.5 to 15 min, yielding soluble PNDI-2BocL with a number-averaged molecular weight (Mn) ranging from 9.8 to 54.0 kg/mol with a deviation of 11 %. Additionally, 15 min of polymerization and ladderization were successfully conducted in a single flow system sequentially for the first time. In the molecular weight-dependent studies, higher optical absorption at longer wavelengths, increased degrees of crystallinity, and higher electron mobilities in organic field-effect transistors (OFETs) were observed for higher molecular weight PNDI-2BocL. Our results highlight the importance of controlling the molecular weights of cLPs and demonstrate that the flow synthesis technique provides a viable solution for synthesizing soluble cLPs in a controlled, reproducible, and rapid manner.

Influence of doping soda-lime-silica glasses with Gd2O3, In2O3, and La2O3 on the enhancing of their physical, opto-mechanical and radiation shielding properties

A series of soda-lime-silicate glasses doped Gd2O3, In2O3, and La2O3 rare earth ions was prepared using the melt-quenching technique. The prepared glass samples are designed depending on a chemical composition of (15-X)Na2O – 30CaO – 55SiO2 –XM2O3, where X = 1.0 mol% and M = Ga (NCSG), In (NCSI), and La (NCSL). The amorphous state of the synthesized samples confirmed by XRD analysis, and structural changes were studied via density (ρg), molar volume (Vm), Oxygen molar volume (Vo), and Oxygen packing density (OPD). The mechanical and shielding attenuation parameters of the produced glasses were also projected. The ρg of prepared glasses increased from 2.742 to 2.829 g/cm3 as a result of M2O3-dopant with high molecular weight rare earth oxides. Whereas, the physical parameters Vm, Vo, and OPD were behave in the same trend. Elastic moduli were improved when Na2O replaced with M2O3. The M2O3/Na2O replacement led to increase stiffer and rigidity of the prepared glasses. The NCSL sample was possessed the lowest optical energy band (Eopt.) values (3.45 eV and 2.63 eV for direct and indirect transitions). The NCSL glass sample possessed the highest mass (MAC) and linear (LAC) absorption capacity. In addition, the same glass sample verified the lowest values of half-value layer (HVL) and mean free path (MFP). Thus, the sample coded as NCSL sample including La2O3 is a superior for optical and radiation shielding capacities among all studied glasses. Therefore, the studied glasses can be applied for several optical and radiation shielding applications.

Benchtop-Stable Carbyl Iminopyridyl NiII Complexes for Olefin Polymerization.

Design of catalysts for Ni-catalyzed olefin polymerization predominantly focuses on ligand design rather than the activation process when attempting to achieve a broader scope of polyolefin micro- and macrostructures. Air-stable alkyl-or aryl-functionalized NiII precatalysts were designed which eliminate the need of in situ alkylating processes and are activated solely by halide abstraction to generate the cationic complex for olefin polymerization. These complexes represent an emerging class of olefin polymerization catalysts, enabling the study of various cocatalysts forming either inner- or outer-sphere ion pairs. It is demonstrated that an organoboron cocatalyst activation produces a well-defined ion pair, which in contrast to ill-defined organoaluminum cocatalysts, can directly activate the complex by halide abstraction to yield comparatively higher molecular weight homo/copolymers. Under high ethylene pressure, broader branching densities and the gradual incorporation of short-chain branches were achieved, circumventing the need for elaborate ligand design and copolymerization with α-olefins. The underlying chain-walking mechanism and ion pair interactions were further elucidated by DFT calculations. A phenyl group on the bridging carbon functioned as a rotational barrier, producing higher molecular weight polymers compared to methyl-substituted analogs. Here, we provide a perspective to manipulate the iminopyridyl NiII system, leveraging ion pair interactions and ligand design to govern polyolefin molecular weights and microstructures.

Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs.

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes, but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR, which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends, capturing the target region for sequencing on PacBio's Sequel, Sequel II, or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized, even for repeats in the thousands. Key features • This protocol is adapted from PacBio's previous "no-amp targeted sequencing utilizing the CRISPR-Cas9 system." • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type, blood, or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio.

Plant Defense Mechanisms against Polycyclic Aromatic Hydrocarbon Contamination: Insights into the Role of Extracellular Vesicles.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants that pose significant environmental and health risks. These compounds originate from both natural phenomena, such as volcanic activity and wildfires, and anthropogenic sources, including vehicular emissions, industrial processes, and fossil fuel combustion. Their classification as carcinogenic, mutagenic, and teratogenic substances link them to various cancers and health disorders. PAHs are categorized into low-molecular-weight (LMW) and high-molecular-weight (HMW) groups, with HMW PAHs exhibiting greater resistance to degradation and a tendency to accumulate in sediments and biological tissues. Soil serves as a primary reservoir for PAHs, particularly in areas of high emissions, creating substantial risks through ingestion, dermal contact, and inhalation. Coastal and aquatic ecosystems are especially vulnerable due to concentrated human activities, with PAH persistence disrupting microbial communities, inhibiting plant growth, and altering ecosystem functions, potentially leading to biodiversity loss. In plants, PAH contamination manifests as a form of abiotic stress, inducing oxidative stress, cellular damage, and growth inhibition. Plants respond by activating antioxidant defenses and stress-related pathways. A notable aspect of plant defense mechanisms involves plant-derived extracellular vesicles (PDEVs), which are membrane-bound nanoparticles released by plant cells. These PDEVs play a crucial role in enhancing plant resistance to PAHs by facilitating intercellular communication and coordinating defense responses. The interaction between PAHs and PDEVs, while not fully elucidated, suggests a complex interplay of cellular defense mechanisms. PDEVs may contribute to PAH detoxification through pollutant sequestration or by delivering enzymes capable of PAH degradation. Studying PDEVs provides valuable insights into plant stress resilience mechanisms and offers potential new strategies for mitigating PAH-induced stress in plants and ecosystems.

Efficient Production of an Alginate Lyase in Bacillus subtilis with Combined Strategy: Vector and Host Selection, Promoter and Signal Peptide Screening, and Modification of a Translation Initiation Region.

Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis (B. subtilis), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, PnprE exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SPydhT demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis. These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis.

Dihydroxyphenylalanine-conjugated high molecular weight polyethylenimine for targeted delivery of Plasmid

High molecular weight polyethylenimine (HMW PEI; branched 25 kDa PEI) has been widely investigated for gene delivery due to its high transfection efficiency. However, the toxicity and lack of targeting to specific cells have limited its clinical application. In the present investigation, L-3, 4‐dihydroxyphenylalanine (L-DOPA) was conjugated on HMW PEI in order to target L-type amino acid transporter 1 (LAT-1) and modulate positive charge density on the surface of polymer/plasmid complexes (polyplexes). The results of biophysical characterization revealed that the PEI conjugates are able to form nanoparticles ≤ 180 nm with the zeta potential ranging from + 9.5–12.4 mV. These polyplexes could condense plasmid DNA and protect it against nuclease digestion at the carrier to plasmid ratios higher than 4. L-DOPA conjugated PEI derivatives were complexed with a plasmid encoding human interleukin-12 (hIL-12). Targeted polyplexes showed up to 2.5 fold higher transfection efficiency in 4T1 murine mammary cancer cell line, which expresses LAT-1, than 25 kDa PEI polyplexes prepared in the same manner. The cytotoxicity of these polyplexes was also substantially lower than the unmodified parent HMW PEI. These results support the use of L-3, 4‐dihydroxyphenylalanine derivatives of PEI in any attempt to develop a LAT-1 targeted gene carrier.