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Neurological Sciences : Official... Jun 2024Variations in the UBQLN2 gene are associated with a group of diseases with X-linked dominant inheritance and clinical phenotypes of amyotrophic lateral sclerosis (ALS)...
Variations in the UBQLN2 gene are associated with a group of diseases with X-linked dominant inheritance and clinical phenotypes of amyotrophic lateral sclerosis (ALS) and/or frontal temporal lobe dementia (FTD). Cases with UBQLN2 variations have been rarely reported worldwide. The reported cases exhibit strong clinical heterogeneity. Here, we report two adult-onset cases with UBQLN2 variations in Han Chinese. Whole exome sequencing revealed the hemizygous P506S (c.1516C > T) and the heterozygous P509S variation (c.1525C > T), both of which were located within the hotspot mutation region. The patient with the P506S variation was a 24-year-old male. The clinical feature was spastic paraplegia without lower motor neuron damage. The patient's mother was an asymptomatic heterozygote carrier with skewed X-chromosome inactivation. The patient with the P509S variation was a 63-year-old female. Clinical features included ALS and parkinsonism. F-fluorodopa PET-CT revealed presynaptic dopaminergic deficits in bilateral posterior putamen. These cases further highlight the clinical heterogeneity of UBQLN2 cases.
PubMed: 38943019
DOI: 10.1007/s10072-024-07674-7 -
Analytical and Bioanalytical Chemistry Jun 2024Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns...
Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns with antigens and antibodies used in various test kits render comparability assessments difficult. As the use of common, well-characterized reagents can help address this lack of standardization, the National Research Council Canada has produced two protein reference materials (RMs) for use in SARS-CoV-2 serology assays: biotinylated human angiotensin-converting enzyme 2 RM, ACE2-1, and SARS-CoV-2 Omicron BA.4/5 spike protein RM, OMIC-1. Reference values were assigned through a combination of amino acid analysis via isotope dilution liquid chromatography tandem mass spectrometry following acid hydrolysis, and ultraviolet-visible (UV-Vis) spectrophotometry at 280 nm. Vial-to-vial homogeneity was established using UV-Vis measurements, and protein oligomeric status, monitored by size exclusion liquid chromatography (LC-SEC), was used to evaluate transportation, storage, and freeze-thaw stabilities. The molar protein concentration in ACE2-1 was 25.3 ± 1.7 µmol L (k = 2, 95% CI) and consisted almost exclusively (98%) of monomeric ACE2, while OMIC-1 contained 5.4 ± 0.5 µmol L (k = 2) spike protein in a mostly (82%) trimeric form. Glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection facilitated calculation of corresponding mass concentrations. To confirm protein functionality, the binding of OMIC-1 to immobilized ACE2-1 was investigated with surface plasmon resonance and the resulting dissociation constant, K ~ 4.4 nM, was consistent with literature values.
PubMed: 38942955
DOI: 10.1007/s00216-024-05413-7 -
Scientific Reports Jun 2024Plekhm2 is a protein regulating endosomal trafficking and lysosomal distribution. We recently linked a recessive inherited mutation in PLEKHM2 to a familial form of...
Plekhm2 is a protein regulating endosomal trafficking and lysosomal distribution. We recently linked a recessive inherited mutation in PLEKHM2 to a familial form of dilated cardiomyopathy and left ventricular non-compaction. These patients' primary fibroblasts exhibited abnormal lysosomal distribution and autophagy impairment. We therefore hypothesized that loss of PLEKHM2 impairs cardiac function via autophagy derangement. Here, we characterized the roles of Plekhm2 in the heart using global Plekhm2 knockout (PLK2-KO) mice and cultured cardiac cells. Compared to littermate controls (WT), young PLK2-KO mice exhibited no difference in heart function or autophagy markers but demonstrated higher basal AKT phosphorylation. Older PLK2-KO mice had body and heart growth retardation and increased LC3II protein levels. PLK2-KO mice were more vulnerable to fasting and, interestingly, impaired autophagy was noted in vitro, in Plekhm2-deficient cardiofibroblasts but not in cardiomyocytes. PLK2-KO hearts appeared to be less sensitive to pathological hypertrophy induced by angiotensin-II compared to WT. Our findings suggest a role of Plekhm2 in murine cardiac autophagy. Plekhm2 deficiency impaired autophagy in cardiofibroblasts, but the autophagy in cardiomyocytes is not critically dependent on Plekhm2. The absence of Plekhm2 in mice appears to promote compensatory mechanism(s) enabling the heart to manage angiotensin-II-induced stress without detrimental consequences.
Topics: Animals; Autophagy; Mice, Knockout; Fibroblasts; Mice; Myocytes, Cardiac; Protein Serine-Threonine Kinases; Myocardium; Cells, Cultured; Phosphorylation
PubMed: 38942823
DOI: 10.1038/s41598-024-65670-5 -
Communications Biology Jun 2024Antimicrobial resistance (AMR) poses a serious threat to the clinical management of typhoid fever. AMR in Salmonella Typhi (S. Typhi) is commonly associated with the H58...
Antimicrobial resistance (AMR) poses a serious threat to the clinical management of typhoid fever. AMR in Salmonella Typhi (S. Typhi) is commonly associated with the H58 lineage, a lineage that arose comparatively recently before becoming globally disseminated. To better understand when and how H58 emerged and became dominant, we performed detailed phylogenetic analyses on contemporary genome sequences from S. Typhi isolated in the period spanning the emergence. Our dataset, which contains the earliest described H58 S. Typhi organism, indicates that ancestral H58 organisms were already multi-drug resistant (MDR). These organisms emerged spontaneously in India in 1987 and became radially distributed throughout South Asia and then globally in the ensuing years. These early organisms were associated with a single long branch, possessing mutations associated with increased bile tolerance, suggesting that the first H58 organism was generated during chronic carriage. The subsequent use of fluoroquinolones led to several independent mutations in gyrA. The ability of H58 to acquire and maintain AMR genes continues to pose a threat, as extensively drug-resistant (XDR; MDR plus resistance to ciprofloxacin and third generation cephalosporins) variants, have emerged recently in this lineage. Understanding where and how H58 S. Typhi originated and became successful is key to understand how AMR drives successful lineages of bacterial pathogens. Additionally, these data can inform optimal targeting of typhoid conjugate vaccines (TCVs) for reducing the potential for emergence and the impact of new drug-resistant variants. Emphasis should also be placed upon the prospective identification and treatment of chronic carriers to prevent the emergence of new drug resistant variants with the ability to spread efficiently.
Topics: Salmonella typhi; Typhoid Fever; Phylogeny; Humans; Anti-Bacterial Agents; Drug Resistance, Multiple, Bacterial; Haplotypes; Mutation; Genome, Bacterial
PubMed: 38942806
DOI: 10.1038/s42003-024-06451-8 -
PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.Nature Communications Jun 2024The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a...
The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.
Topics: Humans; Gene Editing; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Francisella; Bacterial Proteins; Leber Congenital Amaurosis; Streptococcus pyogenes; HEK293 Cells; Mutation; RNA, Guide, CRISPR-Cas Systems; Protein Engineering; Genome, Human
PubMed: 38942756
DOI: 10.1038/s41467-024-49233-w -
Science Bulletin Jun 2024Currently approved vaccines have been successful in preventing the severity of COVID-19 and hospitalization. These vaccines primarily induce humoral immune responses;... (Review)
Review
Currently approved vaccines have been successful in preventing the severity of COVID-19 and hospitalization. These vaccines primarily induce humoral immune responses; however, highly transmissible and mutated variants, such as the Omicron variant, weaken the neutralization potential of the vaccines, thus, raising serious concerns about their efficacy. Additionally, while neutralizing antibodies (nAbs) tend to wane more rapidly than cell-mediated immunity, long-lasting T cells typically prevent severe viral illness by directly killing infected cells or aiding other immune cells. Importantly, T cells are more cross-reactive than antibodies, thus, highly mutated variants are less likely to escape lasting broadly cross-reactive T cell immunity. Therefore, T cell antigen-based human coronavirus (HCoV) vaccines with the potential to serve as a supplementary weapon to combat emerging SARS-CoV-2 variants with resistance to nAbs are urgently needed. Alternatively, T cell antigens could also be included in B cell antigen-based vaccines to strengthen vaccine efficacy. This review summarizes recent advancements in research and development of vaccines containing T cell antigens or both T and B cell antigens derived from proteins of SARS-CoV-2 variants and/or other HCoVs based on different vaccine platforms.
PubMed: 38942698
DOI: 10.1016/j.scib.2024.02.041 -
Progress in Molecular Biology and... 2024Repurposing pharmaceuticals is a technique used to find new, alternate clinical applications for approved drug molecules. It may include altering the drug formulation,... (Review)
Review
Repurposing pharmaceuticals is a technique used to find new, alternate clinical applications for approved drug molecules. It may include altering the drug formulation, route of administration, dose or the dosage regimen. The process of repurposing medicines starts with screening libraries of previously approved drugs for the targeted disease condition. If after an the initial in silico, in vitro or in vivo experimentation, the molecule has been found to be active against a particular target, the molecule is considered as a good candidate for clinical trials. As the safety profile of such molecules is available from the previous data, significant time and resources are saved. These advantages of drug repurposing approach make it especially helpful for finding treatments for rapidly evolving conditions including bacterial infections. An ever-increasing incidence of antimicrobial resistance, owing to the mutations in bacterial genome, leads to therapeutic failure of many approved antibiotics. Repurposing the approved drug molecules for use as antibiotics can provide an effective means for the combating life-threatening bacterial diseases. A number of drugs have been considered for drug repurposing against bacterial infections. These include, but are not limited to, Auranofin, Closantel, and Toremifene that have been repurposed for various infections. In addition, the reallocation of route of administration, redefining dosage regimen and reformulation of dosage forms have also been carried out for repurposing purpose. The current chapter addresses the drug discovery and development process with relevance to repurposing against bacterial infections.
Topics: Drug Repositioning; Humans; Bacterial Infections; Animals; Anti-Bacterial Agents
PubMed: 38942533
DOI: 10.1016/bs.pmbts.2024.03.031 -
RNA (New York, N.Y.) Jun 2024Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for...
Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences Single Molecule Real-Time sequencing. By employing reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second strand synthesis. Deletion and insertion rates increase for all RTs during second strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.
PubMed: 38942481
DOI: 10.1261/rna.080002.124 -
The Science of the Total Environment Jun 2024Microplastics (MPs) have found extensive application globally due to their low cost, flexibility and light weight. Microplastic pollution is a growing environmental... (Review)
Review
Microplastics (MPs) have found extensive application globally due to their low cost, flexibility and light weight. Microplastic pollution is a growing environmental concern that poses significant threats to aquatic ecosystems worldwide, including African freshwater systems. Nevertheless, although Africa houses some of the deepest and largest freshwater rivers and lakes in the world such as Lake Tanganyika and Victoria, River Congo and the Nile, there is limited information available regarding the presence of MPs in these inland waters. Selected published data on MPs in African freshwater systems, including sediments, biota, rivers, and lakes, were incorporated in this review. The study discovered that the sampling technique employed has a major impact on the morphological characteristics and abundance of MPs in African freshwater systems. Fibers and fragments were the most common shapes; black, white, and transparent were the most prevalent colors; and polyethene terephthalate, polystyrene, and polypropylene were the frequently dominant polymers. As the distance between the sampling sites increased geographically, the polymer similarities declined. MPs have been found to translocate into body cells and tissues where they are capable of causing genetic mutations, cytotoxicity, oxidative stress and neurotoxicity. In Africa, MPs are poorly managed and monitored, and there has been insufficient research done on the possibility that they could be present in drinking water. Considering the fact that humans in the continent are exposed to freshwater and aquatic organisms, the risk assessment routes are currently unvalidated, therefore it was recommended that African nations should strengthen their capacity for plastic management and environmental monitoring. This review provides up to date information on the occurrence, prevalence, ecotoxicity and management of MPs across African freshwater systems.
PubMed: 38942312
DOI: 10.1016/j.scitotenv.2024.174092 -
Journal of Virological Methods Jun 2024The most widely used invitro diagnostic qualitative screening method for dengue virus infection is the lateral flow immunoassay technique. Testing of dengue...
The most widely used invitro diagnostic qualitative screening method for dengue virus infection is the lateral flow immunoassay technique. Testing of dengue non-structural antigen NS1 offers specificity in determining the active infection while testing of IgM and IgG helps in differentiating the primary and secondary dengue infections. The ELISA functions as the golden standard for dengue testing and PCR credits for the most accurate determination tool at the genetic level. The RT-PCR endorsed NS1 gene and in ELISA or LFIA NS1 antigen is used as the marker owing to the specificity and lesser chances of mutation effects. This study evaluated the performance of AG-Q Dengue NS1 LFIA kit in comparison with RT-PCR quantification cycle (Cq) Values and ELISA NS1 quantitation. The study also focused on differentiating the samples among dengue serotypes using the RealStar Dengue Type RT-PCR Kit 1.0. Dengue serotype 2 is the prominent viral strain in Kerala region succeeded by serotype 3 and 1 with a prevalence rate of 64%, 20% and 6% respectively. Dengue serotype 4 was not reported during this study period. 10% co-infection with DENV 1 & DENV 2 was also reported. The AG-Q Dengue NS1 kit stood as efficient in screening by providing positive results with samples having RT-PCR Cq values up to 43 and ELISA NS1 quantification minimum of 14 Panbio units.
PubMed: 38942174
DOI: 10.1016/j.jviromet.2024.114991