In vitro activity of cefiderocol against European Pseudomonas aeruginosa and Acinetobacter spp., including isolates resistant to meropenem and recent ß-lactam/ß-lactamase inhibitor combinations.

Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter spp. represent major threats and have few approved therapeutic options. Non-|fermenting Gram-negative isolates were collected from hospitalized inpatients from 49 sites in 6 European countries between 01 January 2020 and 31 December 2020 and underwent susceptibility testing against cefiderocol and ß-lactam/ß-lactamase inhibitor combinations. Meropenem-resistant (MIC >8 mg/L), cefiderocol-susceptible isolates were analyzed by PCR, and cefiderocol-resistant isolates were analyzed by whole-genome sequencing to identify resistance mechanisms. Overall, 1,451 (950 P. aeruginosa; 501 Acinetobacter spp.) isolates were collected, commonly from the respiratory tract (42.0% and 39.3%, respectively). Cefiderocol susceptibility was higher than |ß|-|l|a|c|t|a|m|/|ß|-|l|a|c|t|a|mase| inhibitor combinations against P. aeruginosa (98.9% vs 83.3%-91.4%), and P. |aeruginosa resistant to meropenem (n = 139; 97.8% vs 12.2%-59.7%), ß-lactam/ß-lactamase inhibitor combinations (93.6%-98.1% vs 10.7%-71.8%), and both meropenem and ceftazidime-avibactam (96.7% vs 5.0%-||45.0%) or |ceftolozane-tazobactam (98.4% vs 8.1%-54.8%), respectively. Cefiderocol and sulbactam-durlobactam susceptibilities were high against Acinetobacter spp. (92.4% and 97.0%) and meropenem-resistant Acineto|bacter |spp. (n = 227; 85.0% and 93.8%) but lower against sulbactam-durlobactam- (n |= 15; 13.3%) and cefiderocol- (n = 38; 65.8%) resistant isolates, respectively. Among meropenem-resistant P. aeruginosa and Acinetobacter spp., the most common ß-||lactamase genes were metallo-ß-lactamases [30/139; blaVIM-2 (15/139)] and oxacillinases [215/227; blaOXA-23 (194/227)], respectively. Acquired ß-lactamase genes were identified in 1/10 and 32/38 of cefiderocol-resistant P. aeruginosa and Acinetobacter spp., and pirA-like or piuA mutations in 10/10 and 37/38, respectively. Conclusion: cefiderocol susceptibility was high against P. aeruginosa and Acinetobacter spp., including meropenem-resistant isolates and those resistant to recent ß-lactam/ß-lactamase inhibitor combinations common in first-line treatment of European non-fermenters. IMPORTANCE: This was the first study in which the in vitro activity of cefiderocol and non-licensed ß-lactam/ß-lactamase inhibitor combinations were directly compared against Pseudomonas aeruginosa and Acinetobacter spp., including meropenem- and ß-lactam/ß-lactamase inhibitor combination-resistant isolates. A notably large number of European isolates were collected. Meropenem resistance was defined according to the MIC breakpoint for high-dose meropenem, ensuring that data reflect antibiotic activity against isolates that would remain meropenem resistant in the clinic. Cefiderocol susceptibility was high against non-fermenters, and there was no apparent cross resistance between cefiderocol and ß-lactam/ß-lactamase inhibitor combinations, with the exception of sulbactam-durlobactam. These results provide insights into therapeutic options for infections due to resistant P. aeruginosa and Acinetobacter spp. and indicate how early susceptibility testing of cefiderocol in parallel with ß-lactam/ß-lactamase inhibitor combinations will allow clinicians to choose the effective treatment(s) from all available options. This is particularly important as current treatment options against non-fermenters are limited.

In Rheumatoid Arthritis, A Review of ncRNAs Related to NF-κB Signaling Pathways.

Rheumatoid arthritis (RA) is an autoimmune disease with no known cure that results in joint deformities and dysfunction, significantly impacting the quality of life of patients. The abnormal NF-ＫB signaling pathway in RA has emerged as a crucial research area for the development of RA therapies, with non-coding RNAs (ncRNAs) serving as a potentially meaningful avenue to regulate it. Thus, understanding the role of ncRNAs in RA and the identification of new therapeutic targets have become pressing issues in the field. In this review, we aim to summarize recent studies on ncRNAs that regulate the NF-ＫB signaling pathway in RA, including miRNAs, lncRNAs, and circRNAs, as well as the mechanisms by which drugs modulate NF-Ｋ B activity. By highlighting these recent advances, we hope to promote further research into targeted RA therapy and provide novel directions and ideas for researchers in the field.

Machine-Learning-Based Classification Model to Address Diagnostic Challenges in Transbronchial Lung Biopsy.

BACKGROUND: When obtaining specimens from pulmonary nodules in TBLB, distinguishing between benign samples and mis-sampling from a tumor presents a challenge. Our objective is to develop a machine-learning-based classifier for TBLB specimens. METHODS: Three pathologists assessed six pathological findings, including interface bronchitis/bronchiolitis (IB/B), plasma cell infiltration (PLC), eosinophil infiltration (Eo), lymphoid aggregation (Ly), fibroelastosis (FE), and organizing pneumonia (OP), as potential histologic markers to distinguish between benign and malignant conditions. A total of 251 TBLB cases with defined benign and malignant outcomes based on clinical follow-up were collected and a gradient-boosted decision-tree-based machine learning model (XGBoost) was trained and tested on randomly split training and test sets. RESULTS: Five pathological changes showed independent, mild-to-moderate associations (AUC ranging from 0.58 to 0.75) with benign conditions, with IB/B being the strongest predictor. On the other hand, FE emerged to be the sole indicator of malignant conditions with a mild association (AUC = 0.66). Our model was trained on 200 cases and tested on 51 cases, achieving an AUC of 0.78 for the binary classification of benign vs. malignant on the test set. CONCLUSION: The machine-learning model developed has the potential to distinguish between benign and malignant conditions in TBLB samples excluding the presence or absence of tumor cells, thereby improving diagnostic accuracy and reducing the burden of repeated sampling procedures for patients.

Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile.

IMPORTANCE: Clostridioides difficile is the widespread anaerobic spore-forming bacterium that is a major cause of potentially lethal nosocomial infections associated with antibiotic therapy worldwide. Due to the increase in severe forms associated with a strong inflammatory response and higher recurrence rates, a current imperative is to develop synergistic and alternative treatments for C. difficile infections. In particular, phage therapy is regarded as a potential substitute for existing antimicrobial treatments. However, it faces challenges because C. difficile has highly active CRISPR-Cas immunity, which may be a specific adaptation to phage-rich and highly crowded gut environment. To overcome this defense, C. difficile phages must employ anti-CRISPR mechanisms. Here, we present the first anti-CRISPR protein that inhibits the CRISPR-Cas defense system in this pathogen. Our work offers insights into the interactions between C. difficile and its phages, paving the way for future CRISPR-based applications and development of effective phage therapy strategies combined with the engineering of virulent C. difficile infecting phages.

Epigenetic Mechanisms in Vascular Inflammation: Modulation of Endothelial Adhesion Molecules and Endothelium-Leukocyte Adhesion.

The endothelium, an essential component of the vascular system, plays a critical role in the inflammatory response. Under pro-inflammatory stimuli, endothelial cells undergo activation and dysfunction, leading to the release of inflammatory mediators and upregulation of cell adhesion molecules. These changes facilitate the adhesion, rolling, and transmigration of leukocytes into the subendothelial space. Emerging evidence suggests that epigenetic mechanisms, including nucleic acid methylation, post-translational histone modifications, and non-coding RNA, contribute significantly to the regulation of vascular inflammation and expression of cell adhesion molecules. Understanding the epigenetic molecular signatures that govern these processes may provide new insights into the development of therapeutic strategies to combat vascular inflammation and associated diseases. This review aims to summarize the current knowledge on the epigenetic mechanisms involved in modulating the intricate processes underlying vascular inflammation, with a specific focus on the expression of endothelial adhesion molecules and endothelium-leukocyte adhesion.

Functional PAM sequence for DNA interference by CRISPR-Cas I-B system of Leptospira interrogans and the role of LinCas11b encoded within lincas8b.

Pathogenic species of Leptospira are recalcitrant for genetic manipulation using conventional tools, and therefore there is a need to explore techniques of higher efficiency. Application of endogenous CRISPR-Cas tool is emerging and efficient; nevertheless, it is limited by a poor understanding of interference machinery in the bacterial genome and its associated protospacer adjacent motif (PAM). In this study, interference machinery of CRISPR-Cas subtype I-B (Lin_I-B) from L. interrogans was experimentally validated in E. coli using the various identified PAM (TGA, ATG, ATA). The overexpression of the Lin_I-B interference machinery in E. coli demonstrated that LinCas5, LinCas6, LinCas7, and LinCas8b can self-assemble on cognate CRISPR RNA to form an interference complex (LinCascade). Moreover, a robust interference of target plasmids containing a protospacer with a PAM suggested a functional LinCascade. We also recognized a small open reading frame within lincas8b that independently co-translates into LinCas11b. A mutant variant LinCascade-Cas11b that lacks LinCas11b co-expression erred to mount target plasmid interference. At the same time, LinCas11b complementation in LinCascade-Cas11b rescued target plasmid interference. Thus, the present study establishes Leptospira subtype I-B interference machinery to be functional and, soon, may pave the way for scientists to harness it as a programmable endogenous genetic manipulation tool.

PEGylation of the antimicrobial peptide LyeTx I-b maintains structure-related biological properties and improves selectivity.

The biological activity of antimicrobial peptides and proteins is closely related to their structural aspects and is sensitive to certain post-translational modifications such as glycosylation, lipidation and PEGylation. However, PEGylation of protein and peptide drugs has expanded in recent years due to the reduction of their toxicity. Due to their size, the PEGylation process can either preserve or compromise the overall structure of these biopolymers and their biological properties. The antimicrobial peptide LyeTx I-bcys was synthesized by Fmoc strategy and coupled to polyethylene glycol 2.0 kDa. The conjugates were purified by HPLC and characterized by MALDI-ToF-MS analysis. Microbiological assays with LyeTx I-bcys and LyeTx I-bPEG were performed against Staphylococcus aureus (ATCC 33591) and Escherichia coli (ATCC 25922) in liquid medium. MIC values of 2.0 and 1.0 µM for LyeTx I-bcys and 8.0 and 4.0 µM for LyeTx I-bPEG were observed against S. aureus and E. coli, respectively. PEGylation of LyeTx I-bcys (LyeTx I-bPEG) decreased the cytotoxicity determined by MTT method for VERO cells compared to the non-PEGylated peptide. In addition, structural and biophysical studies were performed to evaluate the effects of PEGylation on the nature of peptide-membrane interactions. Surface Plasmon Resonance experiments showed that LyeTx I-b binds to anionic membranes with an association constant twice higher than the PEGylated form. The three-dimensional NMR structures of LyeTx I-bcys and LyeTx I-bPEG were determined and compared with the LyeTx I-b structure, and the hydrodynamic diameter and zeta potential of POPC:POPG vesicles were similar upon the addition of both peptides. The mPEG-MAL conjugation of LyeTx I-bcys gave epimers, and it, together with LyeTx I-bPEG, showed clear α-helical profiles. While LyeTx I-bcys showed no significant change in amphipathicity compared to LyeTx I-b, LyeTx I-bPEG was found to have a slightly less clear separation between hydrophilic and hydrophobic faces. However, the similar conformational freedom of LyeTx I-b and LyeTx I-bPEG suggests that PEGylation does not cause significant structural changes. Overall, our structural and biophysical studies indicate that the PEGylation does not alter the mode of peptide interaction and maintains antimicrobial activity while minimizing tissue toxicity, which confirmed previous results obtained in vivo. Interestingly, significantly improved proteolytic resistance to trypsin and proteinase K was observed after PEGylation.

Transcriptional analysis of CRISPR I-B arrays of Leptospira interrogans serovar Lai and its processing by Cas6.

In the genome of various Leptospira interrogans serovars, the subtype I-B locus of CRISPR-Cas possesses either one or multiple CRISPR arrays. In silico database (CRISPRCasdb) for predicting CRISPR-Cas reveals seven CRISPR arrays in L. interrogans serovar Lai positioned between the two independent cas-operons. Here, we present the redefined repeat-spacer boundaries of the CRISPR subtype I-B locus of serovar Lai. Such refinement of boundaries of arrays in serovar Lai was done after comparison with the characterized array of another serovar Copenhageni and the manual analysis of CRISPR flanking sequences. Using the reverse transcription-PCR (RT-PCR), we account that the seven CRISPR are transcriptionally active in serovar Lai. Our RT-PCR and quantitative real-time PCR analysis of transcripts in serovar Lai indicated that seven CRISPR of subtype I-B transcribe together as a single precursor unit. Moreover, the cleavage of the two miniature pre-crRNA of the subtype I-B by Cas6 demonstrates the biogenesis of the expected size of mature crRNA essential for the guided interference of foreign DNA. This study features insight into transcription direction and the crRNA biogenesis in serovar Lai essential for RNA-mediated interference of invading nucleic acids.

Exploiting the Type I-B CRISPR Genome Editing System in Thermoanaerobacterium aotearoense SCUT27 and Engineering the Strain for Enhanced Ethanol Production.

Thermoanaerobacterium aotearoense strain SCUT27 is a potential industrial biofuel-producing strain because of its broad substrate spectrum, especially the ability to co-use glucose and xylose. The bottleneck hindering the development of strain SCUT27 is the lack of selective markers for polygene manipulation in this thermophilic bacterium. In this study, the endogenous type I-B clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system was developed for multiplex genome editing of strain SCUT27. The protospacer-adjacent motif was identified by in silico analysis and verified with orotidine-5'-phosphate decarboxylase (pyrF) or lactate dehydrogenase (ldh) as the editing target. The type I-B CRISPR/Cas system was functional in strain SCUT27 with 58.3% to 100% editing efficiency. A multiplex genome editing method based on thymidine kinase (tdk) as a negative selection marker was developed, and strain SCUT27/Δtdk/Δldh/ΔargR, in which ldh and the arginine repressor (argR) were knocked out successively, was successfully obtained. Strain SCUT27/Δtdk/Δldh/ΔargR exhibited prominent advantages over wild-type SCUT27 in ethanol production, with significantly improved ability to metabolize xylose. IMPORTANCE Thermophilic microbes have attracted great attention as potential candidates for production of biofuels and chemicals from lignocellulose because of their thermal tolerance and wide substrate spectra. The ability to edit multiple genes using the native type I-B CRISPR/Cas system would speed up engineering of Thermoanaerobacterium aotearoense strain SCUT27 for higher ethanol production from lignocellulosic hydrolysates. Here, we produced a mutant strain, T. aotearoense SCUT27/Δtdk/Δldh/ΔargR, using the native CRISPR/Cas system. The engineered strain showed satisfactory performance with improved ethanol productivity from various lignocellulosic hydrolysates. Our data lay the foundations for development of this thermophilic microbe into an excellent ethanol producer using lignocellulosic hydrolysates. The methods described here may also provide a reference to develop multigene editing methods for other microorganisms.

Hepatocyte-Specific Knock-Out of Nfib Aggravates Hepatocellular Tumorigenesis via Enhancing Urea Cycle.

Nuclear Factor I B (NFIB) has been reported to promote tumor growth, metastasis, and liver regeneration, but its mechanism in liver cancer is not fully elucidated. The present study aims to reveal the role of NFIB in hepatocellular carcinogenesis. In our study, we constructed hepatocyte-specific NFIB gene knockout mice with CRISPR/Cas9 technology (Nfib-/-; Alb-cre), and induced liver cancer mouse model by intraperitoneal injection of DEN/CCl4. First, we found that Nfib-/- mice developed more tumor nodules and had heavier livers than wild-type mice. H&E staining indicated that the liver histological severity of Nfib-/- group was more serious than that of WT group. Then we found that the differentially expressed genes in the tumor tissue between Nfib-/- mice and wild type mice were enriched in urea cycle. Furthermore, ASS1 and CPS1, the core enzymes of the urea cycle, were significantly upregulated in Nfib-/- tumors. Subsequently, we validated that the expression of ASS1 and CPS1 increased after knockdown of NFIB by lentivirus in normal hepatocytes and also promoted cell proliferation in vitro. In addition, ChIP assay confirmed that NFIB can bind with promoter region of both ASS1 and CPS1 gene. Our study reveals for the first time that hepatocyte-specific knock-out of Nfib aggravates hepatocellular tumor development by enhancing the urea cycle.

NBCe1 Regulates Odontogenic Differentiation of Human Dental Pulp Stem Cells via NF-κB.

Background and Objectives: Dental pulp stem cells (DPSCs) play an important role in the repair of tooth injuries. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a Na＋-coupled HCO3- transporter encoded by the solute carrier 4A4 (SLC4A4) gene and plays a crucial role in maintaining the pH of DPSCs. Our previous research confirmed that NBCe1 is highly expressed in odontoblasts during the development of the tooth germ. Therefore, in this study, we aimed to investigate the effect of NBCe1 on odontogenic differentiation of DPSCs and further clarify the underlying mechanisms. Methods and Results: DPSCs were isolated and identified, and the selective NBCe1 inhibitor S0859 was used to treat DPSCs. We used a cell counting Kit-8 assay to detect cell proliferative ability, and intracellular pH was assessed using confocal microscopy. Odontogenic differentiation of DPSCs was analyzed using real-time PCR and Alizarin Red S staining, and the NF-κB pathway was assessed using western blotting. Our results indicated that 10 µM S0859 was the optimal concentration for DPSC induction. Intracellular pH was decreased upon treatment with S0859. The mRNA expressions of DSPP, DMP1, RUNX2, OCN, and OPN were upregulated in the NBCe1 inhibited group compared to the controls. Moreover, NBCe1 inhibition significantly activated the NF-κB pathway, and a NF-κB inhibitor reduced the effect of NBCe1 on DPSC differentiation. Conclusions: NBCe1 inhibition significantly promotes odontogenic differentiation of DPSCs, and this process may be regulated by activating the NF-κB signaling pathway.

Warren Miller y el Modelo TDI-B de toma de decisiones reproductivas: Una revisión teórica / Warren Miller and the T-D-I-B reproductive decision-making model: A theoretical review / Warren Miller e o Modelo T-D-I-B de tomada de decisões reprodutivas: Uma revisão teórica

El presente estudio teórico objetiva sistematizar los aspectos más importantes del modelo Trait-Desires-Intentions-Behaviors (T-D-I-B) de Warren Miller que explica las decisiones reproductivas. Organizamos la información en base a cuatro ejes temáticos: 1) Elementos biográficos de Warren Miller y sus primeras producciones científicas sobre el comportamiento reproductivo; 2) Descripción del modelo T-D-I-B; 3) Definición del modelo diádico donde las estructuras motivacionales de dos socios interactúan a medida que se desarrolla la secuencia de cuatro pasos de cada individuo y; 4) Exposición de la evidencia empírica del modelo T-D-I-B y estudios actuales realizados en Irán, Polonia, Brasil y Perú.

The present theoretical study aims to systematize the most important aspects of Warren Miller's Trait-Desires-Intentions-Behaviors (T-D-I-B) model that explains reproductive decisions. We organize the information based on four thematic axes: 1) Biographical elements of Warren Miller and his first scientific productions on reproductive behavior; 2) Description of the T-D-I-B model; 3) Definition of the dyadic model where the motivational structures of two partners interact as each individual's four-step sequence develops and 4) Exposure of the empirical evidence of the T-D-I-B model and current studies carried out in Iran, Poland, Brazil and Peru.

O presente estudo teórico visa sistematizar os aspectos mais importantes do modelo Traço-Desejo-Intenções-Comportamentos (T-D-I-B) de Warren Miller, que explica as decisões reprodutivas. Organizamos as informações com base em quatro eixos temáticos: 1) Elementos biográficos de Warren Miller e suas primeiras produções científicas sobre comportamento reprodutivo; 2) Descrição do modelo T-D-I-B; 3) Definição do modelo diádico em que as estruturas motivacionais de dois parceiros interagem conforme a sequência de quatro etapas de cada indivíduo se desenvolve; 4) Exposição da evidência empírica do modelo T-D-I-B e estudos atuais realizados no Irã, Polônia, Brasil e Peru.

EDA-NOCV analysis of carbene-borylene bonded dinitrogen complexes for deeper bonding insight: A fair comparison with a metal-dinitrogen system.

Binding of dinitrogen (N2 ) to a transition metal center (M) and followed by its activation under milder conditions is no longer impossible; rather, it is routinely studied in laboratories by transition metal complexes. In contrast, binding of N2 by main group elements has been a challenge for decades, until very recently, an exotic cAAC-borylene (cAAC = cyclic alkyl(amino) carbene) species showed similar binding affinity to kinetically inert and non-polar dinitrogen (N2 ) gas under ambient conditions. Since then, N2 binding by short lived borylene species has made a captivating news in different journals for its unusual features and future prospects. Herein, we carried out different types of DFT calculations, including EDA-NOCV analysis of the relevant cAAC-boron-dinitrogen complexes and their precursors, to shed light on the deeper insight of the bonding secret (EDA-NOCV = energy decomposition analysis coupled with natural orbital for chemical valence). The hidden bonding aspects have been uncovered and are presented in details. Additionally, similar calculations have been carried out in comparison with a selected stable dinitrogen bridged-diiron(I) complex. Singlet cAAC ligand is known to be an exotic stable species which, combined with the BAr group, produces an intermediate singlet electron-deficient (cAAC)(BAr) species possessing a high lying HOMO suitable for overlapping with the high lying π*-orbital of N2 via effective π-backdonation. The BN2 interaction energy has been compared with that of the FeN2 bond. Our thorough bonding analysis might answer the unasked questions of experimental chemists about how boron compounds could mimic the transition metal of dinitrogen binding and activation, uncovering hidden bonding aspects. Importantly, Pauling repulsion energy also plays a crucial role and decides the binding efficiency in terms of intrinsic interaction energy between the boron-center and the N2 ligand.

Assembly of Cas7 subunits of Leptospira on the mature crRNA of CRISPR-Cas I-B is modulated by divalent ions.

The spirochete Leptospira interrogans serovar Copenhageni harbors the genetic elements of the CRISPR-Cas type I-B system in its genome. CRISPR-Cas is a CRISPR RNA (crRNA) mediated adaptive immune system in most prokaryotes against mobile genetic elements (MGEs). To eliminate the intruding MGEs, CRISPR-Cas type I systems utilize a Cascade (CRISPR-associated complex for antiviral defense) complex composed of Cas5, Cas6, Cas7, and Cas8 bound with a crRNA. The Cas7 is essentially known to constitute the major component of the Cascade complex. The present study reports the biochemical characterization of the Cas7 (LinCas7) from the CRISPR-Cas type I-B system of L. interrogans serovar Copenhageni. The pure recombinant LinCas7 (rLinCas7) exists as a monomer in the solution by size exclusion chromatography. The rLinCas7 demonstrates an endoDNase activity dependent upon divalent Mg2+ ions, monovalent ions, pH, temperature, and substrate size. Analysis of ribonucleoprotein composite (rLinCas7-crRNA) by electron microscopy and native-PAGE demonstrated that rLinCas7 could oligomerize on the mature CRISPR RNA (crRNA) framework in the presence of Mg2+ ions. The ribonucleoprotein composite attains a helical shape similar to the backbone of the Cascade complex. However, in the absence of Mg2+ ions, rLinCas7 acts as an RNase. The fluorescence spectroscopy disclosed a weak interaction (Kd = 26.81 mM) between rLinCas7 and Mg2+ ions, leading to an overall conformational change in rLinCas7 that modulates the rLinCas7's activity on DNA and RNA substrates. The nuclease activity of LinCas7 characterized in this study aids to the functional divergences among proteins of the Cas7 family from different CRISPR-Cas systems in various organisms.

A Facile "Double-Catalysts" Approach to Directionally Fabricate Pyridinic NB-Pair-Doped Crystal Graphene Nanoribbons/Amorphous Carbon Hybrid Electrocatalysts for Efficient Oxygen Reduction Reaction.

Carbon material is a promising electrocatalyst for the oxygen reduction reaction (ORR). Doping of heteroatoms, the most widely used modulating strategy, has attracted many efforts in the past decade. Despite all this, the catalytic activity of heteroatoms-modulated carbon is hard to compare to that of metal-based electrocatalysts. Here, a "double-catalysts" (Fe salt, H3 BO3 ) strategy is presented to directionally fabricate porous structure of crystal graphene nanoribbons (GNs)/amorphous carbon doped by pyridinic NB pairs. The porous structure and GNs accelerate ion/mass and electron transport, respectively. The N percentage in pyridinic NB pairs accounts for ≈80% of all N species. The pyridinic NB pair drives the ORR via an almost 4e- transfer pathway with a half-wave potential (0.812 V vs reversible hydrogen electrode (RHE)) and onset potential (0.876 V vs RHE) in the alkaline solution. The ORR catalytic performance of the as-prepared carbon catalysts approximates commercial Pt/C and outperforms most prior carbon-based catalysts. The assembled Zn-air battery exhibits a high peak power density of 94 mW cm-2 . Density functional theory simulation reveals that the pyridinic NB pair possesses the highest catalytic activity among all the potential configurations, due to the highest charge density at C active sites neighboring B, which enhances the interaction strength with the intermediates in the p-band center.

Effects of architecture structure on volatile organic compound and polycyclic aromatic hydrocarbon diffusion in Singapore's Integrated Transport Hubs.

Millions of passengers wait for buses at Integrated Transport Hubs (ITH) daily in metropolitan cities. Environmental exposure and associated risk for passengers is of great public concern. In this study, eight volatile organic compounds (VOCs) and the 16 EPA priority polycyclic aromatic hydrocarbons (PAHs) were analyzed in airborne samples collected from indoor waiting areas (Indoor) and bus parks of nine Singapore ITH, which comprises of two types of architectural structure (i.e., fully sheltered and open/partially enclosed). The median concentrations of total VOCs (TVOCs), total gaseous PAHs (TgPAHs) and total airborne particles-adsorbed PAH (TpPAHs) concentrations in Indoor were 30.42 µg/m3, 18.99 ng/m3 and 1.38 ng/m3; respectively. A strong correlation (r ≥ 0.75, p < 0.001) was observed between Indoor and bus parks air compounds. The "Indoor" to bus park pollutant concentration ratio (I/B ratio) showed lower values in the bus interchanges with fully sheltered bus parks (TVOCs: 0.98; TgPAHs: 0.76; TpPAHs: 0.71) than those with open/partially enclosed ones (TVOCs: 1.28; TgPAHs: 1.31; TpPAHs: 0.90). This result suggests that fully sheltered structure may cause the accumulation of air pollutants. The daily VOC and PAH exposure for commuters were further estimated by considering inhalation and dermal doses using Monte Carlo simulation (n = 100,000). Overall, the result showed that the risk is still within international guideline values. In sum, the effect of architecture structure on the migration of air pollutants should be taken into consideration in future transport hub design to reduce pollutant exposure to commuters.

MiR-212-3p functions as a tumor suppressor gene in group 3 medulloblastoma via targeting nuclear factor I/B (NFIB).

Haploinsufficiency of chromosome 17p and c-Myc amplification distinguish group 3 medulloblastomas which are associated with early metastasis, rapid recurrence, and swift mortality. Tumor suppressor genes on this locus have not been adequately characterized. We elucidated the role of miR-212-3p in the pathophysiology of group 3 tumors. First, we learned that miR-212-3p undergoes epigenetic silencing by histone modifications in group 3 tumors. Restoring its expression reduced cancer cell proliferation, migration, colony formation, and wound healing in vitro and attenuated tumor burden and improved survival in vivo. MiR-212-3p also triggered c-Myc destabilization and degradation, leading to elevated apoptosis. We then isolated an oncogenic target of miR-212-3p, i.e. NFIB, a nuclear transcription factor implicated in metastasis and recurrence in various cancers. Increased expression of NFIB was confirmed in group 3 tumors and associated with poor survival. NFIB silencing reduced cancer cell proliferation, migration, and invasion. Concurrently, reduced medullosphere formation and stem cell markers (Nanog, Oct4, Sox2, CD133) were noted. These results substantiate the tumor-suppressive role of miR-212-3p in group 3 MB and identify a novel oncogenic target implicated in metastasis and tumor recurrence.

Pegylated LyeTx I-b peptide is effective against carbapenem-resistant Acinetobacter baumannii in an in vivo model of pneumonia and shows reduced toxicity.

The World Health Organization (WHO) has been warning about the importance of developing new drugs against superbugs. Antimicrobial peptides are an alternative in this context, most of them being involved in innate immunity, acting in various ways, and some even showing synergism with commercial antimicrobial agents. LyeTx I-b is a synthetic peptide derived from native LyeTx I, originally isolated from Lycosa erythrognatha spider venom. Although LyeTx I-b is active against several multidrug-resistant bacteria, it shows some hemolytic and cytotoxic effects. To overcome this hindrance, in the present study we PEGylated LyeTx I-b and evaluated its toxicity and in vitro and in vivo activities on pneumonia caused by multi-resistant Acinetobacter baumannii. PEGylated LyeTx I-b (LyeTx I-bPEG) maintained the same MIC value as the non- PEGylated peptide, showed anti-biofilm activity, synergistic effect with commercial antimicrobial agents, and did not induce resistance. Moreover, in vivo experiments showed its activity against pneumonia. Additionally, LyeTx I-bPEG reduced hemolysis up to 10 times, was approximately 2 times less cytotoxic to HEK-293 cells and 4 times less toxic to mice in acute toxicity models, compared to LyeTx I-b. Our results show LyeTx I-bPEG as a promising antimicrobial candidate, significantly active against pneumonia caused by multidrug-resistant A. baumannii.

LyeTx I-b Peptide Attenuates Tumor Burden and Metastasis in a Mouse 4T1 Breast Cancer Model.

Cationic anticancer peptides have exhibited potent anti-proliferative and anti-inflammatory effects in neoplastic illness conditions. LyeTx I-b is a synthetic peptide derived from Lycosa erythrognatha spider venom that previously showed antibiotic activity in vitro and in vivo. This study focused on the effects of LyeTxI-b on a 4T1 mouse mammary carcinoma model. Mice with a palpable tumor in the left flank were subcutaneously or intratumorally injected with LyeTx I-b (5 mg/kg), which significantly decreased the tumor volume and metastatic nodules. Histological analyses showed a large necrotic area in treated primary tumors compared to the control. LyeTxI-b reduced tumor growth and lung metastasis in the 4T1 mouse mammary carcinoma model with no signs of toxicity in healthy or cancerous mice. The mechanism of action of LyeTx I-b on the 4T1 mouse mammary carcinoma model was evaluated in vitro and is associated with induction of apoptosis and cell proliferation inhibition. Furthermore, LyeTx I-b seems to be an efficient regulator of the 4T1 tumor microenvironment by modulating several cytokines, such as TGF-ß, TNF-α, IL-1ß, IL-6, and IL-10, in primary tumor and lung, spleen, and brain. LyeTx I-b also plays a role in leukocytes rolling and adhesion into spinal cord microcirculation and in the number of circulating leukocytes. These data suggest a potent antineoplastic efficacy ofLyeTx I-b.

Protospacer-Adjacent Motif Specificity during Clostridioides difficile Type I-B CRISPR-Cas Interference and Adaptation.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.