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Parkinsonism & Related Disorders May 2024Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease characterized by increasingly worsening ataxia and non-ataxia features, negatively impacting...
INTRODUCTION
Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease characterized by increasingly worsening ataxia and non-ataxia features, negatively impacting patients' quality of life. This study was designed to test formally evaluate whether oral trehalose was effective in SCA3 patients.
METHODS
In this double-blind, randomized controlled trial, SCA3 patients received either 100 g oral trehalose or 30 g maltose to improve ataxia severity over six months. We also measured other clinical (non-ataxia), patient-reported (quality of life, motivations), and safety endpoints. An unscheduled interim analysis was conducted using two-way ANOVAs to analyze the interaction between time (baseline, 3-months, 6-months) and intervention (Trehalose vs. Placebo).
RESULTS
Fifteen participants (Trehalose = 7 vs. Placebo = 8) completed the study at the time of interim analysis. There was no interaction effect on the ataxia severity, and available data suggested an estimated sample size of 132 (66 per arm) SCA3 patients required to demonstrate changes in a 6-month trial. There were significant interaction effects for executive function (ƞ = 0.28-0.43). Safety data indicated that 100 g oral trehalose was well-tolerated.
CONCLUSION
We performed an unplanned interim analysis due to a slow recruitment rate. The new estimated sample size was deemed unfeasible, leading to premature termination of the clinical trial. In this small, current sample of SCA3 patients, 100 g oral trehalose did not differentially impact on ataxia severity compared to placebo. Interestingly, our findings may suggest an improvement in executive function. Future efforts will require a large multi-country, multi-center study to investigate the potential effect of trehalose.
PubMed: 38843619
DOI: 10.1016/j.parkreldis.2024.107013 -
Virus Research Aug 2024African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with...
African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with unknown functions. E184L has been reported as one of the immunogenic ASFV proteins that may contribute to ASFV pathogenesis and immune evasion. However, the antigenic epitopes of E184L are not yet characterized. In this study, recombinant E184L protein was expressed in prokaryotic expression system and four monoclonal antibodies (mAbs), designated as 1A10, 2D2, 3H6, and 4C10 were generated. All four mAbs reacted specifically with ASFV infected cells. To identify the epitopes of the mAbs, a series of overlapped peptides of E184L were designed and expressed as maltose binding fusion proteins. Accordingly, the expressed fusion proteins were probed with each E184L mAb separately by using Western blot. Following a fine mapping, the minimal linear epitope recognized by mAb 1A10 was identified as IQRQGFL, and mAbs 2D2, 3H6, and 4C10 recognized a region located between DPTEFF. Alignment of amino acids of E184L revealed that the two linear epitopes are highly conserved among different ASFV isolates. Furthermore, the potential application of the two epitopes in ASFV diagnosis was assessed through epitope-based ELISA using 24 ASFV positive and 18 negative pig serum and the method were able to distinguish positive and negative samples, indicating the two epitopes are dominant antigenic sites. To our knowledge, this is the first study to characterize the B cell epitopes of the antigenic E184L protein of ASFV, offering valuable tools for future research, as well as laying a foundation for serological diagnosis and epitope-based marker vaccine development.
Topics: African Swine Fever Virus; Antibodies, Monoclonal; Epitopes, B-Lymphocyte; Animals; Epitope Mapping; Antibodies, Viral; Swine; African Swine Fever; Mice; Viral Proteins; Antigens, Viral; Mice, Inbred BALB C
PubMed: 38838820
DOI: 10.1016/j.virusres.2024.199412 -
Molecular Biology Reports Jun 2024Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has...
BACKGROUND
Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research, partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene.
METHODS AND RESULTS
Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16 bp.
CONCLUSIONS
This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.
Topics: Triticum; CRISPR-Cas Systems; Gene Editing; Microinjections; Mutation; Pollen
PubMed: 38824203
DOI: 10.1007/s11033-024-09644-w -
Molecular Imaging and Biology May 2024A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains...
PURPOSE
A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains selectively colonize solid tumors and can deliver genetically encoded cargo molecules to the tumor cells. Here, we engineered bacteria to express monomeric streptavidin (mSA) in tumors, and developed a novel tumor pre-targeting system by visualizing the presence of tumor-associated mSA using a biotinylated imaging probe.
PROCEDURES
We constructed a plasmid expressing mSA fused to maltose-binding protein and optimized the ribosome binding site sequence to increase solubility and expression levels. E. coli MG1655 was transformed with the recombinant plasmid, expression of which is driven by the pBAD promotor. Expression of mSA was induced by L-arabinose 4 days after injection of bacteria into mice bearing CT26 mouse colon carcinoma cells. Selective accumulation of mSA in tumor tissues was visualized by optical imaging after administration of a biotinylated fluorescent dye. Counting of viable bacterial cells was also performed.
RESULTS
Compared with a conventional system, the novel expression system resulted in significantly higher expression of mSA and sustained binding to biotin. Imaging signals in tumor tissues were significantly stronger in the mSA-expressing group than in non-expressing group (P = 0.0005). Furthermore, the fluorescent signal in tumor tissues became detectable again after multiple inductions with L-arabinose. The bacterial counts in tumor tissues showed no significant differences between conditions with and without L-arabinose (P = 0.45). Western blot analysis of tumor tissues confirmed expression and binding of mSA to biotin.
CONCLUSIONS
We successfully engineered tumor-targeting bacteria carrying a recombinant plasmid expressing mSA, which was targeted to, and expressed in, tumor tissues. These data demonstrate the potential of this novel tumor pre-targeting system when combined with biotinylated imaging probes or therapeutic agents.
PubMed: 38814379
DOI: 10.1007/s11307-024-01915-z -
The Journal of International Medical... May 2024To investigate the hepatic effects of high-dose intravenous (IV) iron, including those on liver function and the degree of fibrosis, in a rat model of cirrhosis.
OBJECTIVE
To investigate the hepatic effects of high-dose intravenous (IV) iron, including those on liver function and the degree of fibrosis, in a rat model of cirrhosis.
METHODS
We evenly allocated 25 Sprague-Dawley rats into five groups: normal rats (control group), cirrhotic rats receiving IV normal saline (liver cirrhosis [LC] group), and cirrhotic rats receiving 20, 40, or 80 mg/kg IV ferric carboxymaltose (LC-iron20, LC-iron40, and LC-iron80 group, respectively). Biochemical parameters were compared at 0, 7, 14, 21, and 28 days. The degrees of hepatic fibrosis and iron deposition were evaluated. Inflammatory and oxidative stress markers were also compared.
RESULTS
There were no significant differences in the 28-day serum alanine aminotransferase levels among the LC-iron20, LC-iron40, and LC-iron80 groups (69 ± 7, 1003 ± 127, 1064 ± 309, 919 ± 346, and 820 ± 195 IU/L in the control, LC, LC-iron20, LC-iron40, and LC-iron80 groups, respectively). Hepatic iron accumulation increased in a dose-dependent manner, but the degree of hepatic fibrosis was comparable among the groups. The inflammatory and oxidative stress marker levels did not differ significantly according to the IV iron dose.
CONCLUSIONS
Administration of IV iron at various high doses appears safe in our rat model of cirrhosis.
Topics: Animals; Rats, Sprague-Dawley; Liver; Oxidative Stress; Male; Liver Cirrhosis; Disease Models, Animal; Rats; Ferric Compounds; Iron; Injections, Intravenous; Alanine Transaminase; Maltose; Biomarkers; Liver Function Tests; Dose-Response Relationship, Drug
PubMed: 38811356
DOI: 10.1177/03000605241253733 -
Journal of Bacteriology Jun 2024The EnvZ-OmpR two-component system of regulates the expression of the and porin genes in response to medium osmolarity. However, certain mutations in confer...
The EnvZ-OmpR two-component system of regulates the expression of the and porin genes in response to medium osmolarity. However, certain mutations in confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel and pleiotropic alleles, and , among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike , pleiotropic mutations in have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (Δ) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential β-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a Δ mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic / alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity.
Topics: Escherichia coli Proteins; Escherichia coli; Gene Expression Regulation, Bacterial; Bacterial Outer Membrane Proteins; Anti-Bacterial Agents; Alleles; Bacterial Proteins; Porins; Mutation; Stress, Physiological; Phosphorylation; Multienzyme Complexes; Trans-Activators
PubMed: 38809006
DOI: 10.1128/jb.00172-24 -
Food Chemistry Oct 2024Different quantification methods for in vitro amylolysis were compared for individual chickpea and lentil cotyledon cells (ICC) as a relevant case study. For the first... (Comparative Study)
Comparative Study
Different quantification methods for in vitro amylolysis were compared for individual chickpea and lentil cotyledon cells (ICC) as a relevant case study. For the first time, much-applied spectrophotometric methods relying on the quantification of certain functional groups (i.e., DNS, GOPOD) were compared to chromatographic quantification of starch metabolites (HPLC-ELSD). The estimated rate constant and linked initial rates of amylolysis were highly correlated for DNS, GOPOD, and HPLC-ELSD. However, absolute amylolysis levels depended on the applied method and sample-specific metabolite formation patterns. Multiresponse modelling was employed to further investigate HPLC-ELSD metabolite formation patterns. This delivered insight into the relative importance of different amylolysis reactions during in vitro digestion of pulse ICC, proving that maltotriose and maltose formation determined the overall amylolysis rate in this case. Multiresponse reaction rate constants of maltotriose and maltose formation were highly correlated to single response amylolysis rate constants (and initial rates) obtained for all three quantification methods.
Topics: Starch; Cotyledon; Digestion; Lens Plant; Cicer; Chromatography, High Pressure Liquid; Kinetics; Models, Biological; Trisaccharides
PubMed: 38805919
DOI: 10.1016/j.foodchem.2024.139762 -
Molecular Pharmaceutics Jul 2024Drying protein-based drugs, usually via lyophilization, can facilitate storage at ambient temperature and improve accessibility but many proteins cannot withstand drying...
Drying protein-based drugs, usually via lyophilization, can facilitate storage at ambient temperature and improve accessibility but many proteins cannot withstand drying and must be formulated with protective additives called excipients. However, mechanisms of protection are poorly understood, precluding rational formulation design. To better understand dry proteins and their protection, we examine adenylate kinase (AdK) lyophilized alone and with the additives trehalose, maltose, bovine serum albumin, cytosolic abundant heat soluble protein D, histidine, and arginine. We apply liquid-observed vapor exchange NMR to interrogate the residue-level structure in the presence and absence of additives. We pair these observations with differential scanning calorimetry data of lyophilized samples and AdK activity assays with and without heating. We show that the amino acids do not preserve the native structure as well as sugars or proteins and that after heating the most stable additives protect activity best.
Topics: Freeze Drying; Escherichia coli; Adenylate Kinase; Trehalose; Serum Albumin, Bovine; Excipients; Calorimetry, Differential Scanning; Maltose; Histidine; Arginine; Magnetic Resonance Spectroscopy
PubMed: 38805365
DOI: 10.1021/acs.molpharmaceut.4c00356 -
Protein Science : a Publication of the... Jun 2024The conjugation of proteins with polymers offers immense biotechnological potential by creating novel macromolecules. This article presents experimental findings on the...
The conjugation of proteins with polymers offers immense biotechnological potential by creating novel macromolecules. This article presents experimental findings on the structural properties of maltose-binding protein (MBP) conjugated with linear biodegradable polyphosphoester polymers with different molecular weights. We studied isotopic effects on both proteins and polymers. Circular dichroism and fluorescence spectroscopy and small-angle neutron scattering reveal that the conjugation process destabilizes the protein, affecting the secondary more than the tertiary structure, even at room temperature, and that the presence of two domains in the MBP may contribute to its observed instability. Notably, unfolding temperatures differ between native MBP and the conjugates. In particular, this study sheds light on the complex interplay of factors such as the deuteration influencing protein stability and conformational changes in the conjugation processes. The perdeuteration influences the hydrogen bond network and hydrophobic interactions in the case of the MBP protein. The perdeuteration of the protein influences the hydrogen bond network and hydrophobic interactions. This is evident in the decreased thermal stability of deuterated MBP protein, in the conjugate, especially with high-molecular-mass polymers.
Topics: Maltose-Binding Proteins; Protein Stability; Deuterium; Polymers; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions
PubMed: 38801224
DOI: 10.1002/pro.5032 -
Food Chemistry Oct 2024Maltose, cellobiose, and lactose dissolved in 0.01 mol/L arginine solution or 0.01 mol/L phosphate buffer (pH 7.0) at 0.2 mol/L were reacted batchwise at 110 °C....
Maltose, cellobiose, and lactose dissolved in 0.01 mol/L arginine solution or 0.01 mol/L phosphate buffer (pH 7.0) at 0.2 mol/L were reacted batchwise at 110 °C. The yields of the corresponding keto-disaccharides were 23.4% and 17.6% for maltose, 19.3% and 13.0% for cellobiose, and 20.0% and 13.7% for lactose in arginine solution at 30 min and phosphate buffer at 150 min, respectively. All the disaccharides were hydrolyzed in the range of 0.5 to 3.5%, the degree of hydrolysis being greater in arginine solution than in phosphate buffer. The monosaccharides produced by hydrolysis were slightly isomerized to other monosaccharides. A decrease in pH and an increase in absorbance at 280 and 420 nm continued even at the almost constant yields of the main products. The browning was particularly pronounced in the latter half of the reaction, suggesting a change of the by-products to substances with high absorption coefficient.
Topics: Arginine; Disaccharides; Hydrolysis; Isomerism; Phosphates; Hydrogen-Ion Concentration
PubMed: 38797100
DOI: 10.1016/j.foodchem.2024.139707