Progress in research on ablation of thyroid nodules.

The incidence of thyroid nodules is rising annually. Surgical treatment is effective, but often results in significant trauma, recurrent laryngeal nerve injury, hypoparathyroidism, and other complications. Recent years have seen significant breakthroughs in thyroid nodule ablation for treating thyroid diseases, although its application remains controversial. The objective was to review the development history and current research status of thyroid nodule ablation to provide a reference for future studies. The literature on thyroid nodule ablation was reviewed, analysing its advantages and disadvantages. The therapeutic effect of thyroid nodule ablation in treating benign thyroid lesions is noteworthy, but issues such as lax treatment indications and excessive medical treatment persist. Initial success has been achieved in treating thyroid malignant lesions, particularly papillary thyroid microcarcinoma (PTMC). However, the curative effect requires further follow-up verification.

A review on Gaucher disease: therapeutic potential of ß-glucocerebrosidase-targeted mRNA/saRNA approach.

Gaucher disease (GD), a rare hereditary lysosomal storage disorder, occurs due to a deficiency in the enzyme ß-glucocerebrosidase (GCase). This deficiency leads to the buildup of substrate glucosylceramide (GlcCer) in macrophages, eventually resulting in various complications. Among its three types, GD2 is particularly severe with neurological involvements. Current treatments, such as enzyme replacement therapy (ERT), are not effective for GD2 and GD3 due to their inability to cross the blood-brain barrier (BBB). Other treatment approaches, such as gene or chaperone therapies are still in experimental stages. Additionally, GD treatments are costly and can have certain side effects. The successful use of messenger RNA (mRNA)-based vaccines for COVID-19 in 2020 has sparked interest in nucleic acid-based therapies. Remarkably, mRNA technology also offers a novel approach for protein replacement purposes. Additionally, self-amplifying RNA (saRNA) technology shows promise, potentially producing more protein at lower doses. This review aims to explore the potential of a cost-effective mRNA/saRNA-based approach for GD therapy. The use of GCase-mRNA/saRNA as a protein replacement therapy could offer a new and promising direction for improving the quality of life and extending the lifespan of individuals with GD.

The potent BECN2-ATG14 coiled-coil interaction is selectively critical for endolysosomal degradation of GPRASP1/GASP1-associated GPCRs.

ABBREVIATIONS: AMBRA1 autophagy and beclin 1 regulator 1; ATG14 autophagy related 14; ATG5 autophagy related 5; ATG7 autophagy related 7; BECN1 beclin 1; BECN2 beclin 2; CC coiled-coil; CQ chloroquine CNR1/CB1R cannabinoid receptor 1 DAPI 4',6-diamidino-2-phenylindole; dCCD delete CCD; DRD2/D2R dopamine receptor D2 GPRASP1/GASP1 G protein-coupled receptor associated sorting protein 1 GPCR G-protein coupled receptor; ITC isothermal titration calorimetry; IP immunoprecipitation; KD knockdown; KO knockout; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NRBF2 nuclear receptor binding factor 2; OPRD1/DOR opioid receptor delta 1 PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15 phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K class III phosphatidylinositol 3-kinase; PtdIns3P phosphatidylinositol-3-phosphate; RUBCN rubicon autophagy regulator; SQSTM1/p62 sequestosome 1; UVRAG UV radiation resistance associated; VPS vacuolar protein sorting; WT wild type.

Strong immune responses and protection of PcrV and OprF-I mRNA vaccine candidates against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA) is a leading cause of hospital-acquired and ventilator-associated pneumonia. The multidrug-resistance (MDR) rate of PA is increasing making the management of PA a global challenge. Messenger RNA (mRNA) vaccines represent the most promising alternative to conventional vaccines and are widely studied for viral infection and cancer immunotherapy while rarely studied for bacterial infections. In this study, two mRNA vaccines encoding PcrV- the key component of the type III secretion system in Pseudomonas and the fusion protein OprF-I comprising outer membrane proteins OprF and OprI were constructed. The mice were immunized with either one of these mRNA vaccines or with the combination of both. Additionally, mice were vaccinated with PcrV, OprF, or the combination of these two proteins. Immunization with either mRNA-PcrV or mRNA-OprF-I elicited a Th1/Th2 mixed or slighted Th1-biased immune response, conferred broad protection, and reduced bacterial burden and inflammation in burn and systemic infection models. mRNA-PcrV induced significantly stronger antigen-specific humoral and cellular immune responses and higher survival rate compared with the OprF-I after challenging with all the PA strains tested. The combined mRNA vaccine demonstrated the best survival rate. Moreover, the mRNA vaccines showed the superiority over protein vaccines. These results suggest that mRNA-PcrV as well as the mixture of mRNA-PcrV and mRNA-OprF-I are promising vaccine candidates for the prevention of PA infection.

The RRM-mediated RNA binding activity in T. brucei RAP1 is essential for VSG monoallelic expression.

Trypanosoma brucei is a protozoan parasite that causes human African trypanosomiasis. Its major surface antigen VSG is expressed from subtelomeric loci in a strictly monoallelic manner. We previously showed that the telomere protein TbRAP1 binds dsDNA through its 737RKRRR741 patch to silence VSGs globally. How TbRAP1 permits expression of the single active VSG is unknown. Through NMR structural analysis, we unexpectedly identify an RNA Recognition Motif (RRM) in TbRAP1, which is unprecedented for RAP1 homologs. Assisted by the 737RKRRR741 patch, TbRAP1 RRM recognizes consensus sequences of VSG 3'UTRs in vitro and binds the active VSG RNA in vivo. Mutating conserved RRM residues abolishes the RNA binding activity, significantly decreases the active VSG RNA level, and derepresses silent VSGs. The competition between TbRAP1's RNA and dsDNA binding activities suggests a VSG monoallelic expression mechanism in which the active VSG's abundant RNA antagonizes TbRAP1's silencing effect, thereby sustaining its full-level expression.

Development of a Library of Disulfide Bond-Containing Cationic Lipids for mRNA Delivery.

Lipid nanoparticles (LNPs) are the commonly used delivery tools for messenger RNA (mRNA) therapy and play an indispensable role in the success of COVID-19 mRNA vaccines. Ionizable cationic lipids are the most important component in LNPs. Herein, we developed a series of new ionizable lipids featuring bioreducible disulfide bonds, and constructed a library of lipids derived from dimercaprol. LNPs prepared from these ionizable lipids could be stored at 4 °C for a long term and are non-toxic toward HepG2 and 293T cells. In vivo experiments demonstrated that the best C4S18A formulations, which embody linoleoyl tails, show strong firefly luciferase (Fluc) mRNA expression in the liver and spleen via intravenous (IV) injection, or at the local injection site via intramuscular injection (IM). The newly designed ionizable lipids can be potentially safe and high-efficiency nanomaterials for mRNA therapy.

Designing multi-epitope mRNA construct as a universal influenza vaccine candidate for future epidemic/pandemic preparedness.

Despite the availability of prevention and treatment strategies and advancing immunization approaches, the influenza virus remains a global threat that continues to plague humanity with unpredictable pandemics. Due to the unusual genetic variability and segmented genome, the reassortment between different strains of influenza is facilitated and the viruses continuously evolve and adapt to the host cell's immunity. This underlies the seasonal vaccine mismatches that decrease the vaccine efficacy and increase the risk of outbreaks. Thus, the development of a universal vaccine covering all the influenza A and B strains would reduce the pervasiveness of the influenza virus. In the current study, a potentially universal influenza multi-epitope vaccine was designed based on the experimentally tested conserved T cell and B cell epitopes of hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and matrix-2 proton channel (M2) of the virus. The immune simulation and molecular docking of the vaccine construct with TLR2, TLR3, and TLR4 elicited the favorable immunogenicity of the vaccine and the formation of stable complexes, respectively. Ultimately, based on the immunoinformatics analysis, the universal mRNA multi-epitope vaccine designed in this study might have a protection potential against the various subtypes of influenza A and B.

TbRAP1 has an unusual duplex DNA binding activity required for its telomere localization and VSG silencing.

Localization of Repressor Activator Protein 1 (RAP1) to the telomere is essential for its telomeric functions. RAP1 homologs either directly bind the duplex telomere DNA or interact with telomere-binding proteins. We find that Trypanosoma brucei RAP1 relies on a unique double-stranded DNA (dsDNA) binding activity to achieve this goal. T. brucei causes human sleeping sickness and regularly switches its major surface antigen, variant surface glycoprotein (VSG), to evade the host immune response. VSGs are monoallelically expressed from subtelomeres, and TbRAP1 is essential for VSG regulation. We identify dsDNA and single-stranded DNA binding activities in TbRAP1, which require positively charged 737RKRRR741 residues that overlap with TbRAP1's nuclear localization signal in the MybLike domain. Both DNA binding activities are electrostatics-based and sequence nonspecific. The dsDNA binding activity can be substantially diminished by phosphorylation of two 737RKRRR741-adjacent S residues and is essential for TbRAP1's telomere localization, VSG silencing, telomere integrity, and cell proliferation.

The hydrolytic water molecule of Class A ß-lactamase relies on the acyl-enzyme intermediate ES* for proper coordination and catalysis.

Serine-based ß-lactamases of Class A, C and D all rely on a key water molecule to hydrolyze and inactivate ß-lactam antibiotics. This process involves two conserved catalytic steps. In the first acylation step, the ß-lactam antibiotic forms an acyl-enzyme intermediate (ES*) with the catalytic serine residue. In the second deacylation step, an activated water molecule serves as nucleophile (WAT_Nu) to attack ES* and release the inactivated ß-lactam. The coordination and activation of WAT_Nu is not fully understood. Using time-resolved x-ray crystallography and QM/MM simulations, we analyzed three intermediate structures of Class A ß-lactamase PenP as it slowly hydrolyzed cephaloridine. WAT_Nu is centrally located in the apo structure but becomes slightly displaced away by ES* in the post-acylation structure. In the deacylation structure, WAT_Nu moves back and is positioned along the Bürgi-Dunitz trajectory with favorable energetic profile to attack ES*. Unexpectedly, WAT_Nu is also found to adopt a catalytically incompetent conformation in the deacylation structure forming a hydrogen bond with ES*. Our results reveal that ES* plays a significant role in coordinating and activating WAT_Nu through subtle yet distinct interactions at different stages of the catalytic process. These interactions may serve as potential targets to circumvent ß-lactamase-mediated antibiotic resistance.

Modified Penicillin Molecule with Carbapenem-Like Stereochemistry Specifically Inhibits Class C ß-Lactamases.

Bacterial ß-lactamases readily inactivate most penicillins and cephalosporins by hydrolyzing and "opening" their signature ß-lactam ring. In contrast, carbapenems resist hydrolysis by many serine-based class A, C, and D ß-lactamases due to their unique stereochemical features. To improve the resistance profile of penicillins, we synthesized a modified penicillin molecule, MPC-1, by "grafting" carbapenem-like stereochemistry onto the penicillin core. Chemical modifications include the trans conformation of hydrogen atoms at C-5 and C-6 instead of cis, and a 6-α hydroxyethyl moiety to replace the original 6-ß aminoacyl group. MPC-1 selectively inhibits class C ß-lactamases, such as P99, by forming a nonhydrolyzable acyl adduct, and its inhibitory potency is ∼2 to 5 times higher than that for clinically used ß-lactamase inhibitors clavulanate and sulbactam. The crystal structure of MPC-1 forming the acyl adduct with P99 reveals a novel binding mode for MPC-1 that resembles carbapenem bound in the active site of class A ß-lactamases. Furthermore, in this novel binding mode, the carboxyl group of MPC-1 blocks the deacylation reaction by occluding the critical catalytic water molecule and renders the acyl adduct nonhydrolyzable. Our results suggest that by incorporating carbapenem-like stereochemistry, the current collection of over 100 penicillins and cephalosporins can be modified into candidate compounds for development of novel ß-lactamase inhibitors.

Crystallographic Snapshots of Class A ß-Lactamase Catalysis Reveal Structural Changes That Facilitate ß-Lactam Hydrolysis.

ß-Lactamases confer resistance to ß-lactam-based antibiotics. There is great interest in understanding their mechanisms to enable the development of ß-lactamase-specific inhibitors. The mechanism of class A ß-lactamases has been studied extensively, revealing Lys-73 and Glu-166 as general bases that assist the catalytic residue Ser-70. However, the specific roles of these two residues within the catalytic cycle remain not fully understood. To help resolve this, we first identified an E166H mutant that is functional but is kinetically slow. We then carried out time-resolved crystallographic study of a full cycle of the catalytic reaction. We obtained structures that represent apo, ES*-acylation, and ES*-deacylation states and analyzed the conformational changes of His-166. The "in" conformation in the apo structure allows His-166 to form a hydrogen bond with Lys-73. The unexpected "flipped-out" conformation of His-166 in the ES*-acylation structure was further examined by molecular dynamics simulations, which suggested deprotonated Lys-73 serving as the general base for acylation. The "revert-in" conformation in the ES*-deacylation structure aligns His-166 toward the water molecule that hydrolyzes the acyl adduct. Finally, when the acyl adduct is fully hydrolyzed, His-166 rotates back to the "in" conformation of the apo-state, restoring the Lys-73/His-166 interaction. Using His-166 as surrogate, our study identifies distinct conformational changes within the active site during catalysis. We suggest that the native Glu-166 executes similar changes in a less constricted way. Taken together, this structural series improves our understanding of ß-lactam hydrolysis in this important class of enzymes.

Perturbing the general base residue Glu166 in the active site of class A ß-lactamase leads to enhanced carbapenem binding and acylation.

Most class A ß-lactamases cannot hydrolyze carbapenem antibiotics effectively. The molecular mechanism of this catalytic inefficiency has been attributed to the unique stereochemistry of carbapenems, including their 6-α-hydroxyethyl side chain and the transition between two tautomeric states when bound at the active site. Previous studies have shown that the 6-α-hydroxyethyl side chain of carbapenems can interfere with catalysis by forming hydrogen bonds with the deacylation water molecule to reduce its nucleophilicity. Here our studies of a class A noncarbapenemase PenP demonstrate that substituting the general base residue Glu166 with Ser or other residues leads to a significant enhancement of the acylation kinetics by ∼100-500 times toward carbapenems like meropenem. The structures of PenP and Glu166Ser both in apo form and in complex with meropenem reveal that Glu166 is critical for the formation of a hydrogen bonding network within the active site that locks Asn170 in an orientation to impose steric clash with the 6-α-hydroxyethyl side chain of meropenem. The Glu166Ser substitution weakens this network and enables Asn170 to adopt an alternative conformation to avoid steric clash and accommodate faster acylation kinetics. Furthermore, the weakened hydrogen bonding network caused by the Glu166Ser substitution allows the 6-α-hydroxyethyl moiety to adopt a catalytically favorable orientation as seen in class A carbapenemases. In summary, our data identify a previously unreported role of the universally conserved general base residue Glu166 in impeding the proper binding of carbapenems by restricting their 6-α-hydroxyethyl group.

The content of amino acids in artificial diet influences the development and reproduction of brown planthopper, Nilaparvata lugens (STÅL).

Nitrogen availability from dietary protein has profound effects on the physiology and ecology of insect herbivores. The amount of amino acids consumed by Nilaparvata lugens impacts its phenotypic characteristics and reproduction. In this work, we hypothesized that amino acids deficiency leads to physiological trade-offs between survival and reproduction. We investigated the effect of larval nutrition on larval period, wing dimorphism, egg production, ovarian development, lifespan, and stored nutrients. Larvae were reared on the standard medium and an amino acid deficient medium (AADM), adults were reared on the standard medium. Nymphs reared on AADM had shorter larval period (20.78 d/23.09 d), higher brachypterous forms (34.06%/16.52%), the adults females were fed back on standard medium after emergency, they featured extended preoviposition period (11.41/13.45 d), declining number of laid eggs (2.27/37.44), ovarian dysplasia, and shorter lifespan compared with control group. Adults from both dietary treatment groups had approximately the same proportion of total lipids and protein nutrients carried over from larvae feeding into adulthood. We infer that N. lugens makes a physiological trade-off between survival and reproduction by suppressing ovarian development. This is probably a common strategy during times of nutritional deficiency in nature.

Engineering Clostridia Neurotoxins with elevated catalytic activity.

BoNT/B and TeNT cleave substrate VAMP2 at the same scissile bond, yet these two toxins showed different efficiency on substrate hydrolysis and had different requirements for the recognition of P2' site of VAMP2, E(78). These differences may be due to their different composition of their substrate recognition pockets in the active site. Swapping of LC/T S1' pocket residue, L(230), with the corresponding isoleucine in LC/B increased LC/T activity by ∼25 fold, while swapping of LC/B S1' pocket residue, S(201), with the corresponding proline in LC/T increased LC/B activity by ∼10 fold. Optimization of both S1 and S1' pocket residues of LC/T, LC/T (K(168)E, L(230)I) elevated LC/T activity by more than 100-fold. The highly active LC/T derivative engineered in this study has the potential to be used as a more effective tool to study mechanisms of exocytosis in central neuron. The LC/B derivative with elevated activity has the potential to be developed into novel therapy to minimize the impact of immunoresistance during BoNT/B therapy.