Simultaneous and Sensitive Detection of Multisite 5‑Methylcytosine Including Non-CpG Sites at Single-5mC-Resolution

The methylation status of multiplexed methylcytosine sites can be simultaneously monitored by ligation-depended PCR assay. The ability of quantitative detection of multiplexed sites in one PCR reaction makes it a good choice for detecting methylation at both CpG and non-CpG sites for research and diagnosis of disease compared with others. The assay can determine as low as 20 aM methylated DNA and has been successfully applied to the genomic DNA sample derived from cancer cell lines

Small Unnatural Amino Acid Carried Raman Tag for Molecular Imaging of Genetically Targeted Proteins

Raman has been implemented to image biological systems for decades. However, Raman microscopy along with Raman probes is restricted to image metabolites or a few intracellular organelles so far and lacks genetic specificity for imaging proteins of interest, which significantly hinders their application. Here, we report the Raman spectra-based protein imaging method, which incorporates a small phenyl ring enhanced Raman tag (total of ∼0.55 kDa) with a single unnatural amino acid (UAA) to genetically label specific proteins. We further demonstrate hyperspectral stimulated Raman scattering (SRS) imaging of the Histone3.3 protein in the nucleus, Sec61β protein in the endoplasmic reticulum of HeLa cells, and Huntingtin protein Htt74Q in mutant huntingtin-induced cells. Genetic encoding of a small, stable, sensitive, and narrow-band Raman tag took one key step forward to enable SRS or Raman imaging of specific proteins and could further facilitate quantitative Raman spectra-based supermultiplexing microscopy in the future

Small Unnatural Amino Acid Carried Raman Tag for Molecular Imaging of Genetically Targeted Proteins

Raman has been implemented to image biological systems for decades. However, Raman microscopy along with Raman probes is restricted to image metabolites or a few intracellular organelles so far and lacks genetic specificity for imaging proteins of interest, which significantly hinders their application. Here, we report the Raman spectra-based protein imaging method, which incorporates a small phenyl ring enhanced Raman tag (total of ∼0.55 kDa) with a single unnatural amino acid (UAA) to genetically label specific proteins. We further demonstrate hyperspectral stimulated Raman scattering (SRS) imaging of the Histone3.3 protein in the nucleus, Sec61β protein in the endoplasmic reticulum of HeLa cells, and Huntingtin protein Htt74Q in mutant huntingtin-induced cells. Genetic encoding of a small, stable, sensitive, and narrow-band Raman tag took one key step forward to enable SRS or Raman imaging of specific proteins and could further facilitate quantitative Raman spectra-based supermultiplexing microscopy in the future

Small Unnatural Amino Acid Carried Raman Tag for Molecular Imaging of Genetically Targeted Proteins

Raman has been implemented to image biological systems for decades. However, Raman microscopy along with Raman probes is restricted to image metabolites or a few intracellular organelles so far and lacks genetic specificity for imaging proteins of interest, which significantly hinders their application. Here, we report the Raman spectra-based protein imaging method, which incorporates a small phenyl ring enhanced Raman tag (total of ∼0.55 kDa) with a single unnatural amino acid (UAA) to genetically label specific proteins. We further demonstrate hyperspectral stimulated Raman scattering (SRS) imaging of the Histone3.3 protein in the nucleus, Sec61β protein in the endoplasmic reticulum of HeLa cells, and Huntingtin protein Htt74Q in mutant huntingtin-induced cells. Genetic encoding of a small, stable, sensitive, and narrow-band Raman tag took one key step forward to enable SRS or Raman imaging of specific proteins and could further facilitate quantitative Raman spectra-based supermultiplexing microscopy in the future

Image_1_Role of the ISKpn element in mediating mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae.pdf

BackgroundColistin has emerged as a last-resort therapeutic against antibiotic-resistant bacterial infections, particularly those attributed to carbapenem-resistant Enterobacteriaceae (CRE) like CRKP. Yet, alarmingly, approximately 45% of multidrug-resistant Klebsiella pneumoniae strains now manifest resistance to colistin. Through our study, we discerned that the synergy between carbapenemase and IS elements amplifies resistance in Klebsiella pneumoniae, thereby narrowing the existing therapeutic avenues. This underscores the instrumental role of IS elements in enhancing colistin resistance through mgrB disruption.MethodsFrom 2021 to 2023, 127 colistin-resistant Klebsiella pneumoniae isolates underwent meticulous examination. We embarked on an exhaustive genetic probe, targeting genes associated with both plasmid-mediated mobile resistance-encompassing blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48-like, and mcr-1 to mcr-8-and chromosome-mediated resistance systems, including PhoP/Q, PmrA/B, and mgrB. PCR amplification revealed the presence of virulence-associated genes from the pLVPK plasmid, such as rmpA, rmpA2, iucA, iroB, and peg344. mgrB sequencing was delegated to Sangon Biotech, Shanghai, and the sequences procured were validated using BLAST. Our search for IS elements was navigated through the IS finder portal. Phenotypically, we harnessed broth microdilution (BMD) to ascertain the MICs of colistin. To sketch the clonal lineage of mgrB-mutated CoR-Kp isolates, sophisticated methodologies like MLST and PFGE were deployed. S1-PFGE unraveled the intrinsic plasmids in these isolates. Our battery of virulence assessment techniques ranged from the string test and capsular serotyping to the serum killing assay and the Galleria mellonella larval infection model.ResultsAmong the 127 analyzed isolates, 20 showed an enlarged mgrB PCR amplicon compared to wild-type strains. These emerged over a three-year period: three in 2021, thirteen in 2022, and four in 2023. Antimicrobial susceptibility tests revealed that these isolates consistently resisted several drugs, notably TCC, TZP, CAZ, and COL. Additionally, 85% resisted both DOX and TOB. The MICs for colistin across these strains ranged between 16 to 64 mg/L, with a median of 40 mg/L. From a genetic perspective, MLST unanimously categorized these mgrB-mutated CoR-hvKp isolates as ST11. PFGE further delineated them into six distinct clusters, with clusters A and D being predominant. This distribution suggests potential horizontal and clonal genetic transmission. Intriguingly, every mgrB-mutated CoR-hvKP isolate possessed at least two virulence genes akin to the pLVPK-like virulence plasmid, with iroB and rmpA2 standing out. Their virulence was empirically validated both in vitro and in vivo. A pivotal discovery was the identification of three distinct insertion sequence (IS) elements within or near the mgrB gene. These were:ISKpn26 in eleven isolates, mainly in cluster A, with various insertion sites including +74, +125, and an upstream −35.ISKpn14 in four isolates with insertions at +93, −35, and two upstream at −60.IS903B present in five isolates, marking positions like +74, +125, +116, and −35 in the promoter region. These diverse insertions, spanning six unique locations in or near the mgrB gene, underscore its remarkable adaptability.ConclusionOur exploration spotlights the ISKpn element’s paramount role in fostering mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae. Employing MLST and PFGE, we unearthed two primary genetic conduits: clonal and horizontal. A striking observation was the ubiquitous presence of the KPC carbapenemase gene in all the evaluated ST11 hypervirulent colistin-resistant Klebsiella pneumoniae strains, with a majority also harboring the NDM gene. The myriad mgrB gene insertion locales accentuate its flexibility and the overarching influence of IS elements, notably the pervasive IS5-like variants ISKpn26 and IS903B. Our revelations illuminate the escalating role of IS elements in antibiotic resistance within ST11 hypervirulent colistin-resistant Klebsiella pneumoniae, advocating for innovative interventions to counteract these burgeoning resistance paradigms given their profound ramifications for prevailing treatment modalities.</p

Precise Antibody-Independent m6A Identification via 4SedTTP-Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution

Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this “RT-silent” modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution

Table_2_Role of the ISKpn element in mediating mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae.XLSX

BackgroundColistin has emerged as a last-resort therapeutic against antibiotic-resistant bacterial infections, particularly those attributed to carbapenem-resistant Enterobacteriaceae (CRE) like CRKP. Yet, alarmingly, approximately 45% of multidrug-resistant Klebsiella pneumoniae strains now manifest resistance to colistin. Through our study, we discerned that the synergy between carbapenemase and IS elements amplifies resistance in Klebsiella pneumoniae, thereby narrowing the existing therapeutic avenues. This underscores the instrumental role of IS elements in enhancing colistin resistance through mgrB disruption.MethodsFrom 2021 to 2023, 127 colistin-resistant Klebsiella pneumoniae isolates underwent meticulous examination. We embarked on an exhaustive genetic probe, targeting genes associated with both plasmid-mediated mobile resistance-encompassing blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48-like, and mcr-1 to mcr-8-and chromosome-mediated resistance systems, including PhoP/Q, PmrA/B, and mgrB. PCR amplification revealed the presence of virulence-associated genes from the pLVPK plasmid, such as rmpA, rmpA2, iucA, iroB, and peg344. mgrB sequencing was delegated to Sangon Biotech, Shanghai, and the sequences procured were validated using BLAST. Our search for IS elements was navigated through the IS finder portal. Phenotypically, we harnessed broth microdilution (BMD) to ascertain the MICs of colistin. To sketch the clonal lineage of mgrB-mutated CoR-Kp isolates, sophisticated methodologies like MLST and PFGE were deployed. S1-PFGE unraveled the intrinsic plasmids in these isolates. Our battery of virulence assessment techniques ranged from the string test and capsular serotyping to the serum killing assay and the Galleria mellonella larval infection model.ResultsAmong the 127 analyzed isolates, 20 showed an enlarged mgrB PCR amplicon compared to wild-type strains. These emerged over a three-year period: three in 2021, thirteen in 2022, and four in 2023. Antimicrobial susceptibility tests revealed that these isolates consistently resisted several drugs, notably TCC, TZP, CAZ, and COL. Additionally, 85% resisted both DOX and TOB. The MICs for colistin across these strains ranged between 16 to 64 mg/L, with a median of 40 mg/L. From a genetic perspective, MLST unanimously categorized these mgrB-mutated CoR-hvKp isolates as ST11. PFGE further delineated them into six distinct clusters, with clusters A and D being predominant. This distribution suggests potential horizontal and clonal genetic transmission. Intriguingly, every mgrB-mutated CoR-hvKP isolate possessed at least two virulence genes akin to the pLVPK-like virulence plasmid, with iroB and rmpA2 standing out. Their virulence was empirically validated both in vitro and in vivo. A pivotal discovery was the identification of three distinct insertion sequence (IS) elements within or near the mgrB gene. These were:ISKpn26 in eleven isolates, mainly in cluster A, with various insertion sites including +74, +125, and an upstream −35.ISKpn14 in four isolates with insertions at +93, −35, and two upstream at −60.IS903B present in five isolates, marking positions like +74, +125, +116, and −35 in the promoter region. These diverse insertions, spanning six unique locations in or near the mgrB gene, underscore its remarkable adaptability.ConclusionOur exploration spotlights the ISKpn element’s paramount role in fostering mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae. Employing MLST and PFGE, we unearthed two primary genetic conduits: clonal and horizontal. A striking observation was the ubiquitous presence of the KPC carbapenemase gene in all the evaluated ST11 hypervirulent colistin-resistant Klebsiella pneumoniae strains, with a majority also harboring the NDM gene. The myriad mgrB gene insertion locales accentuate its flexibility and the overarching influence of IS elements, notably the pervasive IS5-like variants ISKpn26 and IS903B. Our revelations illuminate the escalating role of IS elements in antibiotic resistance within ST11 hypervirulent colistin-resistant Klebsiella pneumoniae, advocating for innovative interventions to counteract these burgeoning resistance paradigms given their profound ramifications for prevailing treatment modalities.</p

Table_1_Role of the ISKpn element in mediating mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae.XLSX

BackgroundColistin has emerged as a last-resort therapeutic against antibiotic-resistant bacterial infections, particularly those attributed to carbapenem-resistant Enterobacteriaceae (CRE) like CRKP. Yet, alarmingly, approximately 45% of multidrug-resistant Klebsiella pneumoniae strains now manifest resistance to colistin. Through our study, we discerned that the synergy between carbapenemase and IS elements amplifies resistance in Klebsiella pneumoniae, thereby narrowing the existing therapeutic avenues. This underscores the instrumental role of IS elements in enhancing colistin resistance through mgrB disruption.MethodsFrom 2021 to 2023, 127 colistin-resistant Klebsiella pneumoniae isolates underwent meticulous examination. We embarked on an exhaustive genetic probe, targeting genes associated with both plasmid-mediated mobile resistance-encompassing blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48-like, and mcr-1 to mcr-8-and chromosome-mediated resistance systems, including PhoP/Q, PmrA/B, and mgrB. PCR amplification revealed the presence of virulence-associated genes from the pLVPK plasmid, such as rmpA, rmpA2, iucA, iroB, and peg344. mgrB sequencing was delegated to Sangon Biotech, Shanghai, and the sequences procured were validated using BLAST. Our search for IS elements was navigated through the IS finder portal. Phenotypically, we harnessed broth microdilution (BMD) to ascertain the MICs of colistin. To sketch the clonal lineage of mgrB-mutated CoR-Kp isolates, sophisticated methodologies like MLST and PFGE were deployed. S1-PFGE unraveled the intrinsic plasmids in these isolates. Our battery of virulence assessment techniques ranged from the string test and capsular serotyping to the serum killing assay and the Galleria mellonella larval infection model.ResultsAmong the 127 analyzed isolates, 20 showed an enlarged mgrB PCR amplicon compared to wild-type strains. These emerged over a three-year period: three in 2021, thirteen in 2022, and four in 2023. Antimicrobial susceptibility tests revealed that these isolates consistently resisted several drugs, notably TCC, TZP, CAZ, and COL. Additionally, 85% resisted both DOX and TOB. The MICs for colistin across these strains ranged between 16 to 64 mg/L, with a median of 40 mg/L. From a genetic perspective, MLST unanimously categorized these mgrB-mutated CoR-hvKp isolates as ST11. PFGE further delineated them into six distinct clusters, with clusters A and D being predominant. This distribution suggests potential horizontal and clonal genetic transmission. Intriguingly, every mgrB-mutated CoR-hvKP isolate possessed at least two virulence genes akin to the pLVPK-like virulence plasmid, with iroB and rmpA2 standing out. Their virulence was empirically validated both in vitro and in vivo. A pivotal discovery was the identification of three distinct insertion sequence (IS) elements within or near the mgrB gene. These were:ISKpn26 in eleven isolates, mainly in cluster A, with various insertion sites including +74, +125, and an upstream −35.ISKpn14 in four isolates with insertions at +93, −35, and two upstream at −60.IS903B present in five isolates, marking positions like +74, +125, +116, and −35 in the promoter region. These diverse insertions, spanning six unique locations in or near the mgrB gene, underscore its remarkable adaptability.ConclusionOur exploration spotlights the ISKpn element’s paramount role in fostering mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae. Employing MLST and PFGE, we unearthed two primary genetic conduits: clonal and horizontal. A striking observation was the ubiquitous presence of the KPC carbapenemase gene in all the evaluated ST11 hypervirulent colistin-resistant Klebsiella pneumoniae strains, with a majority also harboring the NDM gene. The myriad mgrB gene insertion locales accentuate its flexibility and the overarching influence of IS elements, notably the pervasive IS5-like variants ISKpn26 and IS903B. Our revelations illuminate the escalating role of IS elements in antibiotic resistance within ST11 hypervirulent colistin-resistant Klebsiella pneumoniae, advocating for innovative interventions to counteract these burgeoning resistance paradigms given their profound ramifications for prevailing treatment modalities.</p

Additional file 1: Figure S1. of Curcumin slows osteoarthritis progression and relieves osteoarthritis-associated pain symptoms in a post-traumatic osteoarthritis mouse model

Non-encapsulated curcumin reduces MMP-13 mRNA expression in a dose-and time-dependent manner. Human primary chondrocytes were treated with curcumin at indicated concentrations (0–200 μM) for 6 hours (A) or treated with curcumin at 100 μM at different durations (0–48 hours) (B) in the presence of IL-1β. *P <0.05, t test or one-way ANOVA with Tukey post-hoc test, n = 3/group. (DOCX 116 kb

Table_3_Role of the ISKpn element in mediating mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae.XLSX

BackgroundColistin has emerged as a last-resort therapeutic against antibiotic-resistant bacterial infections, particularly those attributed to carbapenem-resistant Enterobacteriaceae (CRE) like CRKP. Yet, alarmingly, approximately 45% of multidrug-resistant Klebsiella pneumoniae strains now manifest resistance to colistin. Through our study, we discerned that the synergy between carbapenemase and IS elements amplifies resistance in Klebsiella pneumoniae, thereby narrowing the existing therapeutic avenues. This underscores the instrumental role of IS elements in enhancing colistin resistance through mgrB disruption.MethodsFrom 2021 to 2023, 127 colistin-resistant Klebsiella pneumoniae isolates underwent meticulous examination. We embarked on an exhaustive genetic probe, targeting genes associated with both plasmid-mediated mobile resistance-encompassing blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48-like, and mcr-1 to mcr-8-and chromosome-mediated resistance systems, including PhoP/Q, PmrA/B, and mgrB. PCR amplification revealed the presence of virulence-associated genes from the pLVPK plasmid, such as rmpA, rmpA2, iucA, iroB, and peg344. mgrB sequencing was delegated to Sangon Biotech, Shanghai, and the sequences procured were validated using BLAST. Our search for IS elements was navigated through the IS finder portal. Phenotypically, we harnessed broth microdilution (BMD) to ascertain the MICs of colistin. To sketch the clonal lineage of mgrB-mutated CoR-Kp isolates, sophisticated methodologies like MLST and PFGE were deployed. S1-PFGE unraveled the intrinsic plasmids in these isolates. Our battery of virulence assessment techniques ranged from the string test and capsular serotyping to the serum killing assay and the Galleria mellonella larval infection model.ResultsAmong the 127 analyzed isolates, 20 showed an enlarged mgrB PCR amplicon compared to wild-type strains. These emerged over a three-year period: three in 2021, thirteen in 2022, and four in 2023. Antimicrobial susceptibility tests revealed that these isolates consistently resisted several drugs, notably TCC, TZP, CAZ, and COL. Additionally, 85% resisted both DOX and TOB. The MICs for colistin across these strains ranged between 16 to 64 mg/L, with a median of 40 mg/L. From a genetic perspective, MLST unanimously categorized these mgrB-mutated CoR-hvKp isolates as ST11. PFGE further delineated them into six distinct clusters, with clusters A and D being predominant. This distribution suggests potential horizontal and clonal genetic transmission. Intriguingly, every mgrB-mutated CoR-hvKP isolate possessed at least two virulence genes akin to the pLVPK-like virulence plasmid, with iroB and rmpA2 standing out. Their virulence was empirically validated both in vitro and in vivo. A pivotal discovery was the identification of three distinct insertion sequence (IS) elements within or near the mgrB gene. These were:ISKpn26 in eleven isolates, mainly in cluster A, with various insertion sites including +74, +125, and an upstream −35.ISKpn14 in four isolates with insertions at +93, −35, and two upstream at −60.IS903B present in five isolates, marking positions like +74, +125, +116, and −35 in the promoter region. These diverse insertions, spanning six unique locations in or near the mgrB gene, underscore its remarkable adaptability.ConclusionOur exploration spotlights the ISKpn element’s paramount role in fostering mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae. Employing MLST and PFGE, we unearthed two primary genetic conduits: clonal and horizontal. A striking observation was the ubiquitous presence of the KPC carbapenemase gene in all the evaluated ST11 hypervirulent colistin-resistant Klebsiella pneumoniae strains, with a majority also harboring the NDM gene. The myriad mgrB gene insertion locales accentuate its flexibility and the overarching influence of IS elements, notably the pervasive IS5-like variants ISKpn26 and IS903B. Our revelations illuminate the escalating role of IS elements in antibiotic resistance within ST11 hypervirulent colistin-resistant Klebsiella pneumoniae, advocating for innovative interventions to counteract these burgeoning resistance paradigms given their profound ramifications for prevailing treatment modalities.</p