Structure and mechanism of a methyl transferase ribozyme

Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. We have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry. [Image: see text

Frequency tuning behaviour of terahertz quantum cascade lasers revealed by a laser beating scheme

In the terahertz frequency range, the commercialized spectrometers, such as the Fourier transform infrared and time domain spectroscopies, show spectral resolutions between a hundred megahertz and a few gigahertz. Therefore, the high precision frequency tuning ability of terahertz lasers cannot be revealed by these traditional spectroscopic techniques. In this work, we demonstrate a laser beating experiment to investigate the frequency tuning characteristics of terahertz quantum cascade lasers (QCLs) induced by temperature or drive current. Two terahertz QCLs emitting around 4.2 THz with identical active regions and laser dimensions (150 μm wide and 6 mm long) are employed in the beating experiment. One laser is operated as a frequency comb and the other one is driven at a lower current to emit a single frequency. To measure the beating signal, the single mode laser is used as a fast detector (laser self-detection). The laser beating scheme allows the high precision measurement of the frequency tuning of the single mode terahertz QCL. The experimental results show that in the investigated temperature and current ranges, the frequency tuning coefficients of the terahertz QCL are 6.1 MHz/0.1 K (temperature tuning) and 2.7 MHz/mA (current tuning) that cannot be revealed by a traditional terahertz spectrometer. The laser beating technique shows potential abilities in high precision linewidth measurements of narrow absorption lines and multi-channel terahertz communications

Prevalence of the NTEKPC-I on IncF Plasmids Among Hypervirulent Klebsiella pneumoniae Isolates in Jiangxi Province, South China

Infection caused by carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has become a tricky health care threat in China and KPC-2 enzyme is a main factor mediating resistance to carbapenems of K. pneumoniae. Here, we report the characterization of the genetic environment of the blaKPC-2 gene in CR-hvKP clinical isolates from South China. Forty-five non-duplicated CR-hvKP isolates collected in Jiangxi Province from 2018 to 2019 were analyzed. Each of them were multidrug-resistant due to the presence not only of blaKPC-2 gene but also of other resistance determinants, including Metallo-β-lactamases (NDM-1), extended-spectrum β-lactamases (TEM-1, CTX-M-14, SHV-1), and plasmid-mediated quinolone resistance determinants (qnrS, aac(6′)-Ib-cr). After plasmid analyses of PCR-based replicon typing (PBRT), mapping PCR, amplicon sequencing, and whole-genome sequencing (WGS) were used to analyze the genetic environment of the blaKPC-2 gene. PCR analysis of pLVPK-like plasmids, Southern Blot, and mouse lethality assay were used to characterize the virulence phenotype of K. pneumoniae. Multilocus sequence typing (MLST) analysis showed ST11 CR-hvKP was the predominant clone. In conclusion, this is the first analysis of diverse genetic structures blaKPC-2 gene in CR-hvKP isolates from south China. Both the NTEKPC-I on the IncF plasmids and pLVPK-like virulence plasmids make contributions to the formation of CR-hvKP especially ST11 which need more attention

Genetic characterization and passage instability of a novel hybrid virulence plasmid in a ST23 hypervirulent Klebsiella pneumoniae

Hypervirulent variants of Klebsiella pnuemoniae (hvKP), which causes life-threatening infections, is a global priority pathogen and frequently harbours virulence plasmids. The virulence plasmids have emerged as the predominant vehicles carrying the major pathogenic determinants of hypermucoviscosity and hypervirulence phenotypes. In the present study, we characterized a novel virulence plasmid in AP8555, an ST23 hvKP strain, which induced a metastatic infection and fatal septic shock in a critically ill patient. The serum killing assay, the quantitative biofilm formation assay, the G.mellonella infection model, and the mouse lethality assay demonstrated that AP8555 was almost as virulent as the hvKP strain NUTH-K2044. The plasmid pAP855 could be conjugated to Klebsiella quasipneumoniae ATCC700603 and E. coli J53 at a frequency of 7.2× 10−5 and 8.7× 10−7, respectively. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHI1B/IncFIB/IncFII and presented high similarity to the pK2044 plasmid. In contrast, a 130-kb large-fragment insertion was observed on the plasmid, which introduced a genetic hybrid zone with multiple conjugation-related genes of type IV secretion systems (T4SS) and CcdAB toxin-antitoxin systems (TAS) to the plasmid. In the transconjugants, the presence of pAP855 had a negative impact on bacterial fitness, but enhancing the virulence-associated phenotypes. In vitro evolution experiments showed that pAP855 in the transconjugants could not be stably inherited after 10 days of passage. Our study not only reports a novel hybrid plasmid but also highlights the putative pathway of conjugative virulence plasmid formation and evolution by means of genetic rearrangement through sequence insertion. These findings indicate that structural versatility could contribute to the dissemination of cointegrate virulence plasmid, although the plasmid incurred a fitness cost. Therefore, continuous monitoring the acquisition of conjugative virulence plasmids may have critical value for plasmid research and increase awareness of hvKP

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

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

Research progress on treatment of renal anemia in patients with chronic kidney disease

Renal anemia is a prevalent complication of chronic kidney disease （CKD）， which severely affects the prognosis of patients. The pathogenesis of renal anemia is complicated， and the core of which is erythropoietin deficiency and iron metabolism disorder. For years， renal anemia has been mainly treated based on erythropoiesis-stimulating and iron agents. Blood transfusion is used as a remedial treatment. Other therapeutic interventions include alleviating malnutrition， supplementing folic acid and vitamin B12， adequate dialysis and controlling hyperparathyroidism， etc.， which yield low efficacy. In recent years， clinical application of hypoxia-inducible factor prolyl hydroxylase inhibitors has brought another innovation in the treatment of renal anemia， and some potential approaches for the treatment of renal anemia have been reported. In this article， recent research progresses upon the treatment of renal anemia in CKD patients were reviewed， aiming provide new ideas for clinical treatment of this disease

Interaction analysis and loop pairing for MIMO processes described by T–S fuzzy models

This paper presents a loop pairing method for determining the control configuration for multi-input–multi-output (MIMO) processes represented by Takagi–Sugeno (T–S) fuzzy models. The method is simple with straightforward calculation and it provides more accurate results compared with existing fuzzy pairing approaches, since both steady-state and dynamic information for the system is utilized. Each individual loop in the MIMO process is represented by a T–S fuzzy model based on the data and the models are then assembled to form the MIMO model. Simple formulae are derived to calculate the steady-state and dynamic information for the loops. In this way, interactions among the loops can be assessed and loop pairing can be determined according to the relative normalized gain array (RNGA) criterion. Two examples are provided to show that loop pairing decisions obtained from T–S fuzzy models are the same as those obtained from precise mathematical models. This demonstrates the effectiveness of the proposed interaction measure and the loop pairing method

An online data-driven fault diagnosis method for air handling units by rule and convolutional neural networks

The stable operation of air handling units (AHU) is critical to ensure high efficiency and to extend the lifetime of the heating, ventilation, and air conditioning (HVAC) systems of buildings. In this paper, an online data-driven diagnosis method for AHU in an HVAC system is proposed and elaborated. The rule-based method can roughly detect the sensor condition by setting threshold values according to prior experience. Then, an efficient feature selection method using 1D convolutional neural networks (CNNs) is proposed for fault diagnosis of AHU in HVAC systems according to the system's historical data obtained from the building management system. The new framework combines the rule-based method and CNNs-based method (RACNN) for sensor fault and complicated fault. The fault type of AHU can be accurately identified via the offline test results with an accuracy of 99.15% and fast online detection within 2 min. In the lab, the proposed RACNN method was validated on a real AHU system. The experimental results show that the proposed RACNN improves the performance of fault diagnosis.Published versionThis research is supported under the RIE2020 Industry Alignment Fund — Industry Collaboration Projects (IAF-ICP) Funding Initiative, as well as cash and in-kind contribution from Surbana Jurong Pte Ltd