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Abstract Background Signal transducer and activator of transcription 3 (STAT3), a multifaceted transcription factor, modulates host immune responses by activating cellular response to signaling ligands. STAT3 has a pivotal role in the pathophysiology of kidney injury by counterbalancing resident macrophage phenotypes under inflammation conditions. However, STAT3’s role in acute kidney injury (AKI), particularly in macrophage migration, and in chronic kidney disease (CKD) through fibrosis development, remains unclear. Methods Stattic (a JAK2/STAT3 inhibitor, 5 mg/kg or 10 mg/kg) was administered to evaluate the therapeutic effect on LPS-induced AKI (L-AKI) and LPS-induced CKD (L-CKD), with animals sacrificed 6–24 h and 14 days post-LPS induction, respectively. The immune mechanisms of STAT3 blockade were determined by comparing the macrophage phenotypes and correlated with renal function parameters. Also, the transcriptomic analysis was used to confirm the anti-inflammatory effect of L-AKI, and the anti-fibrotic role was further evaluated in the L-CKD model. Results In the L-AKI model, sequential increases in BUN and blood creatinine levels were time-dependent, with a marked elevation of 0–6 h after LPS injection. Notably, two newly identified macrophage subpopulations (CD11bhighF4/80low and CD11blowF4/80high), exhibited population changes, with an increase in the CD11bhighF4/80low population and a decrease in the CD11blowF4/80high macrophages. Corresponding to the FACS results, the tubular injury score, NGAL, F4/80, and p-STAT3 expression in the tubular regions were elevated. STAT3 inhibitor injection in L-AKI and L-CKD mice reduced renal injury and fibrosis. M2-type subpopulation with CD206 in CD11blowF4/80high population increased in the Stattic-treated group compared with that in the LPS-alone group in the L-AKI model. Additionally, STAT3 inhibitor reduced inflammation driven by LPS-stimulated macrophages and epithelial cells injury in the co-culture system. Transcriptomic profiling identified 3 common genes in the JAK-STAT, TLR, and TNF signaling pathways and 11 common genes in the LPS with macrophage response. The PI3K-AKT (IL-6, Akt3, and Pik3r1) and JAK-STAT pathways were determined as potential Stattic targets. Further confirmation through mRNA and protein expressions analyses showed that Stattic treatment reduced inflammation in the L-AKI and fibrosis in the L-CKD mice. Conclusions STAT3 blockade effectively mitigated inflammation by retrieving the CD11blowF4/80high population, further emphasizing the role of STAT3-associated macrophage-driven inflammation in kidney injury.

Abstract Variability in disease severity caused by a microbial pathogen is impacted by each infection representing a unique combination of host and pathogen genomes. Here, we show that the outcome of invasive Streptococcus pyogenes infection is regulated by an interplay between human STING genotype and bacterial NADase activity. S. pyogenes-derived c-di-AMP diffuses via streptolysin O pores into macrophages where it activates STING and the ensuing type I IFN response. However, the enzymatic activity of the NADase variants expressed by invasive strains suppresses STING-mediated type I IFN production. Analysis of patients with necrotizing S. pyogenes soft tissue infection indicates that a STING genotype associated with reduced c-di-AMP-binding capacity combined with high bacterial NADase activity promotes a ‘perfect storm’ manifested in poor outcome, whereas proficient and uninhibited STING-mediated type I IFN production correlates with protection against host-detrimental inflammation. These results reveal an immune-regulating function for bacterial NADase and provide insight regarding the host-pathogen genotype interplay underlying invasive infection and interindividual disease variability.

Prokaryotic chromosomes contain numerous small open reading frames (ORFs) of less than 200 bases. Since high-throughput proteomics methods often miss proteins containing fewer than 60 amino acids, it is difficult to decern if they encode proteins. Recent studies have revealed that many small proteins are membrane proteins with a single membrane-anchoring α-helix. As membrane anchoring or transmembrane motifs are accurately identifiable with high confidence using computational algorithms like Phobius and TMHMM, small membrane proteins (SMPS) can be predicted with high accuracy. This study employed a systematic approach, utilizing well-verified algorithms such as Orfipy, Phobius, and Blast to identify SMPs in prokaryotic organisms. Our main search parameters targeted candidate SMPs with an open reading frame between 60-180 nucleotides, a membrane-anchoring or transmembrane region 15 and 30 amino acids long, and sequence conservation among other microorganisms. Our findings indicate that each prokaryote possesses many SMPs, with some identified in the intergenic regions of currently annotated chromosomes. More extensively studied microorganisms, such as Escherichia coli and Bacillus subtilis, have more SMPs identified in their genomes compared to less studied microorganisms, suggesting the possibility of undiscovered SMPs in less studied microorganisms. In this study, we describe the common SMPs identified across various microorganisms and explore their biological roles. We have also developed a software pipeline and an accompanying online interface for discovering SMPs (http://cs.indstate.edu/pro-smp-finder). This resource aims to assist researchers in identifying new SMPs encoded in microbial genomes of interest.

The second messenger molecule, c-di-AMP, plays a critical role in pathogenesis and virulence in S. pyogenes. We previously reported that deleting the c-di-AMP phosphodiesterase gene pde2 severely suppresses SpeB production at the transcriptional level. We performed transposon mutagenesis to gain insight into the mechanism of how Pde2 is involved in SpeB regulation. We identified one of the genes of the dlt operon, dltX, as a suppressor of the SpeB-null phenotype of the Δpde2 mutant. The dlt operon consists of five genes, dltX, dltA, dltB, dltC, and dltD in many Gram-positive bacteria, and its function is to incorporate D-alanine into lipoteichoic acids. DltX, a small membrane protein, is a newly identified member of the operon. The in-frame deletion of dltX or insertional inactivation of dltA in the Δpde2 mutant restored SpeB production, indicating that D-alanylation is crucial for the suppressor phenotype. These mutations did not affect the growth in lab media but showed increased negative cell surface charge and enhanced sensitivity to polymyxin B. Considering that dlt mutations change cell surface charge and sensitivity to cationic antimicrobial peptides, we examined the LiaFSR system that senses and responds to cell envelope stress. The ΔliaR mutation in the Δpde2 mutant also derepressed SpeB production, like the ΔdltX mutation. LiaFSR controls speB expression by regulating the expression of the transcriptional regulator SpxA2. However, the Dlt system did not regulate spxA2 expression. The SpeB phenotype of the Δpde2ΔdltX mutant in higher salt media differed from that of the Δpde2ΔliaR mutant, suggesting a unique pathway for the Dlt system in SpeB production, possibly related to ion transport or turgor pressure regulation.

Abstract We evaluate the diagnostic performance of deep learning artificial intelligence (AI) for bladder cancer, which used white-light images (WLIs) and narrow-band images, and tumor grade prediction of AI based on tumor color using the red/green/blue (RGB) method. This retrospective study analyzed 10,991 cystoscopic images of suspicious bladder tumors using a mask region-based convolutional neural network with a ResNeXt-101-32 × 8d-FPN backbone. The diagnostic performance of AI was evaluated by calculating sensitivity, specificity, and diagnostic accuracy, and its ability to detect cancers was investigated using the dice score coefficient (DSC). Using the support vector machine model, we analyzed differences in tumor colors according to tumor grade using the RGB method. The sensitivity, specificity, diagnostic accuracy and DSC of AI were 95.0%, 93.7%, 94.1% and 74.7%. In WLIs, there were differences in red and blue values according to tumor grade (p 90% for the diagnosis of chronic non-specific inflammation vs. carcinoma in situ using WLIs. The diagnostic performance of the AI-assisted diagnosis was of high quality, and the AI could distinguish the tumor grade based on tumor color.

This study evaluates the diagnostic accuracy and clinical utility of two artificial intelligence (AI) techniques: Kakao Brain Artificial Neural Network for Chest X-ray Reading (KARA-CXR), an assistive technology developed using large-scale AI and large language models (LLMs), and ChatGPT, a well-known LLM. The study was conducted to validate the performance of the two technologies in chest X-ray reading and explore their potential applications in the medical imaging diagnosis domain. The study methodology consisted of randomly selecting 2000 chest X-ray images from a single institution’s patient database, and two radiologists evaluated the readings provided by KARA-CXR and ChatGPT. The study used five qualitative factors to evaluate the readings generated by each model: accuracy, false findings, location inaccuracies, count inaccuracies, and hallucinations. Statistical analysis showed that KARA-CXR achieved significantly higher diagnostic accuracy compared to ChatGPT. In the ‘Acceptable’ accuracy category, KARA-CXR was rated at 70.50% and 68.00% by two observers, while ChatGPT achieved 40.50% and 47.00%. Interobserver agreement was moderate for both systems, with KARA at 0.74 and GPT4 at 0.73. For ‘False Findings’, KARA-CXR scored 68.00% and 68.50%, while ChatGPT scored 37.00% for both observers, with high interobserver agreements of 0.96 for KARA and 0.97 for GPT4. In ‘Location Inaccuracy’ and ‘Hallucinations’, KARA-CXR outperformed ChatGPT with significant margins. KARA-CXR demonstrated a non-hallucination rate of 75%, which is significantly higher than ChatGPT’s 38%. The interobserver agreement was high for KARA (0.91) and moderate to high for GPT4 (0.85) in the hallucination category. In conclusion, this study demonstrates the potential of AI and large-scale language models in medical imaging and diagnostics. It also shows that in the chest X-ray domain, KARA-CXR has relatively higher accuracy than ChatGPT.

The majority of bacteria in the natural environment organize themselves into communal biofilms. Biofilm formation benefits bacteria conferring resistance to harmful molecules (e.g., antibiotics, disinfectants, and host immune factors) and coordinating their gene expression through quorum sensing (QS). A primary signaling molecule promoting bacterial biofilm formation is the universal second messenger cyclic di-GMP. This dinucleotide predominantly controls the gene expression of motility, adhesins, and capsule production to coordinate biofilm formation. Cyclic di-GMP is synthesized by diguanylate cyclases (DGCs) that have a GGDEF domain and is degraded by phosphodiesterases (PDEs) containing either an EAL or an HD-GYP domain. Since high cellular c-di-GMP concentrations are correlated with promoting the ability of bacteria to form biofilms, numerous research endeavors to identify chemicals capable of inhibiting the c-di-GMP synthesis activity of DGCs have been performed in order to inhibit bacterial biofilm formation. This review describes currently identified chemical inhibitors that disturb the activity of DGCs and the methods of screening and assay for their discovery.

Thrust and total impulse performance of a SparkJet actuator are affected by pressure waves inside the cavity, so it is important to understand these effects. The two objectives of the present study are to characterize the jet performance of a SparkJet actuator and to investigate pressure waves inside the cavity and correlate them with actuator performance. To this end, a SparkJet actuator is solved numerically based on equilibrium gas assumption. Loss of thrust occurs because of the pressure drop caused by the pressure wave, flow separation at the orifice throat, and vortex above the orifice exit. A fast Fourier transform of thrust reveals pressure wave components at two natural frequencies that originate from the pressure waves inside the cavity. The reflected pressure waves inside the cavity affect natural frequencies. The pressure wave reflected in the r-axis direction is affected by the energy deposition aspect ratio which influenced the total impulse by as much as ∼3.21%. The pressure wave reflected in the z-axis direction is affected by the effective height distance. However, this rarely affects the total impulse directly. The use of an appropriate taper angle affects the total impulse by as much as ∼8.49%. Approximately, 11% of the total impulse is originated from oscillations by the reflected wave in the r-axis direction, whereas 4% of it is from the reflected wave in the z-axis direction. This work contributes to improving the design of electrode and cavity configurations for SparkJet actuators and showing the importance of oscillations made by reflected pressure waves.

Abstract Objectives To identify risk stratification biomarkers to enrich for the subset of Staphylococcus aureus bacteraemia patients who develop deep‐seated tissue infections with high morbidity and mortality to guide clinical trial enrolment and clinical management. Methods We evaluated the prognostic value of eight biomarkers for persistent bacteraemia, mortality and endovascular infection foci in a validation cohort of 160 patients with S. aureus bacteraemia enrolled consecutively over 3 years. Results High levels of IL‐17A, IL‐10 or soluble E‐selectin at bacteraemia diagnosis correlated with the duration of positive blood cultures. When thresholds defined in an independent cohort were applied, these biomarkers were robust predictors of persistent bacteraemia or endovascular infection. High serum levels of IL‐17A and IL‐10 often preceded the radiographic diagnosis of infective endocarditis, suggesting potential utility for prioritising diagnostic radiographic imaging. High IL‐8 was prognostic for all‐cause mortality, while IL‐17A and IL‐10 were superior to clinical metrics in discriminating between attributable mortality and non‐attributable mortality. High IL‐17A and IL‐10 identified more patients who developed microbiological failure or mortality than were identified by infective endocarditis diagnosis. Conclusion These biomarkers offer potential utility to identify patients at risk of persistent bacteraemia to guide diagnostic imaging and clinical management. Low biomarker levels could be used to rule out the need for more invasive TEE imaging in patients at lower risk of infective endocarditis. These biomarkers could enable clinical trials by enriching for patients with the greatest need for novel therapies.

Nicotinamide adenine dinucleotide (NADH) is an important cofactor involved in metabolic redox reactions in living cells. The detection of NADH in living animal cells is a challenge. We developed a one-step monitoring method for NADH via an electrocatalytic reaction that uses a surface-modified, screen-printed electrode (SPE) having a redox active monolayer 4′-mercapto-N-phenlyquinone diamine (NPQD) formed by a self-assembled monolayer (SAM) of an aromatic thiol, 4-aminothiophenol (4-ATP). This electrode has a limit of detection (LOD) of 0.49 μM and a sensitivity of 0.0076 ± 0.0006 μM/μA in cell culture media, which indicates that it retains its selectivity. The applicability of this NADH sensor was demonstrated for the first time by cell viability monitoring via NADH-sensing in cell culture supernatants.

Abstract Background Small noncoding regulatory RNAs (sRNAs) are post-transcriptional regulators, regulating mRNAs, proteins, and DNA in bacteria. One class of sRNAs, trans-acting sRNAs, are the most abundant sRNAs transcribed from the intergenic regions (IGRs) of the bacterial genome. In Streptococcus pyogenes, a common and potentially deadly pathogen, many sRNAs have been identified, but only a few have been studied. The goal of this study is to identify trans-acting sRNAs that can be substrates of RNase III. The endoribonuclease RNase III cleaves double stranded RNAs, which can be formed during the interaction between an sRNA and target mRNAs. Results For this study, we created an RNase III null mutant of Streptococcus pyogenes and its RNA sequencing (RNA-Seq) data were analyzed and compared to that of the wild-type. First, we developed a custom script that can detect intergenic regions of the S. pyogenes genome. A differential expression analysis with Cufflinks and Stringtie was then performed to identify the intergenic regions whose expression was influenced by the RNase III gene deletion. Conclusion This analysis yielded 12 differentially expressed regions with >|2| fold change and p ≤ 0.05. Using Artemis and Bamview genome viewers, these regions were visually verified leaving 6 putative sRNAs. This study not only expanded our knowledge on novel sRNAs but would also give us new insight into sRNA degradation.

Background and Objective: Procedural thromboembolism after a mechanical thrombectomy (MT) for an acute ischemic stroke (AIS) has rarely been studied. It may occur from the artery-to-artery embolization of atherosclerotic plaque in the aortic arch. We investigated the relationship between aortic arch calcification (AoAC) on a chest X-ray and procedural thromboembolism on diffusion-weighted imaging (DWI) after an MT. Materials and Methods: From January 2017 to December 2020, 131 patients underwent DWI within two days following an MT for an AIS. Procedural thromboembolism was defined as new DWI-positive lesions in other territories from the occluded artery on DWI within two days after MT. Results: Procedural thromboembolism was observed in 30 (22.9%) patients. Procedural thromboembolism was associated with old age (72.3 ± 9.44 vs. 65.7 ± 12.8 years, p = 0.003), a longer procedural time (77.6 ± 37.6 vs. 60.1 ± 29.7 min, p = 0.024), and AoAC (calcification (73.3%) vs. no calcification (29.7%), p < 0.001). Multivariable logistic regression analysis showed that procedural thromboembolism was independently associated with AoAC (adjusted odds ratio (OR): 6.107, adjusted 95% confidence interval (CI): 2.374–15.705, p < 0.001) and a longer procedural time (adjusted OR: 1.015, adjusted 95% CI: 1.001–1.030, p = 0.031). Conclusions: Procedural thromboembolism after an MT for an AIS was related to AoAC on a chest X-ray and a longer procedural time. Our results suggest that although rapid recanalization is the most crucial goal of an MT for an AIS, the importance of the careful advance of the guiding catheter through the aortic arch should not be underestimated to reduce the risk of procedural thromboembolism, especially in patients with AoAC on a chest X-ray.

The construction project of the Kijang research reactor (KJRR), which is the second research reactor in Korea, has been launched. The KJRR was designed to use, for the first time, U–Mo fuel. Plate-type U–7 wt.% Mo/Al–5 wt.% Si, referred to as U–7Mo/Al–5Si, dispersion fuel with a uranium loading of 8.0 gU/cm3, was selected to achieve higher fuel efficiency and performance than are possible when using U3Si2/Al dispersion fuel. To qualify the U–Mo fuel in terms of plate geometry, the first miniplates [HANARO Miniplate (HAMP-1)], containing U–7Mo/Al–5Si dispersion fuel (8 gU/cm3), were fabricated at the Korea Atomic Energy Research Institute and recently irradiated at HANARO. The PIE (Post-irradiation Examination) results of the HAMP-1 irradiation test were analyzed in depth in order to verify the safe in-pile performance of the U–7Mo/Al–5Si dispersion fuel under the KJRR irradiation conditions. Nondestructive analyses included visual inspection, gamma spectrometric mapping, and two-dimensional measurements of the plate thickness and oxide thickness. Destructive PIE work was also carried out, focusing on characterization of the microstructural behavior using optical microscopy and scanning electron microscopy. Electron probe microanalysis was also used to measure the elemental concentrations in the interaction layer formed between the U–Mo kernels and the matrix. A blistering threshold test and a bending test were performed on the irradiated HAMP-1 miniplates that were saved from the destructive tests. Swelling evaluation of the U–Mo fuel was also conducted using two methods: plate thickness measurement and meat thickness measurement.

Rejection is an important issue in kidney transplant. Although with adequate trough level of tacrolimus, acute rejection occurs, and we are focused on these cases. We hypothesized that the lower concentration of tacrolimus in the peripheral blood mononuclear cell would be a cause of rejection; in this regard, we describe ABCB1, which regulates intracellular concentration of tacrolimus. The effect of inflammation on the intracellular concentration of tacrolimus was evaluated, as was the association between that concentration and ABCB1 and CD44 activities. Seven kidney recipients experiencing acute rejection were prospectively enrolled. Both the whole blood concentration of tacrolimus and intracellular concentration of tacrolimus were measured at the time of enrollment and after stabilization. A human T lymphoblastoid cell line (Jurkat T cell) was treated with various concentrations of tacrolimus for 21 h and then stimulated for 3 h. The levels of mRNA interleukin-2, interleukin-8, and interferon-γ decreased dose dependently by tacrolimus. Furthermore, a fluorescence-activated cell sorter was used to count cells expressing CD44 and ABCB1; changes in intracellular concentration of tacrolimus were explored after tacrolimus treatment and stimulation. Also, B6 splenocytes were tested in the same manner as previous Jurkat T cell experiments. The tacrolimus ratio of three patients was lower at the time of acute rejection than when patients were stabilized. In vitro, the intracellular concentration of tacrolimus decreased after stimulation. Under the same conditions, CD44 + ABCB1 + cells increased in proportion on tacrolimus treatment and stimulation. This work supports the hypothesis that inflammation reduces the intracellular tacrolimus level, possibly via drug efflux mediated by CD44 + ABCB1 + and inflammation could lead to acute rejection.

The discovery of small noncoding regulatory RNAs (sRNAs) in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen’s survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described.

Background and objective: Procedural thromboembolisms after mechanical thrombectomy (MT) for acute ischemic stroke has rarely been studied. We retrospectively evaluated factors associated with procedural thromboembolisms after MT using diffusion-weight imaging (DWI) within 2 days of MT. Materials and Methods: From January 2018 to March 2020, 78 patients with acute ischemic stroke who underwent MT were evaluated using DWI. Procedural thromboembolisms were defined as new cerebral infarctions in other territories from the occluded artery on DWI after MT. Results: Procedural thromboembolisms were observed on DWI in 16 patients (20.5%). Procedural thromboembolisms were associated with old age (73.8 ± 8.18 vs. 66.8 ± 11.2 years, p = 0.021), intravenous (IV) thrombolysis (12 out of 16 (75.0%) vs. 25 out of 62 (40.3%), p = 0.023), heparinization (4 out of 16 (25.0%) vs. 37 out of 62 (59.7%), p = 0.023), and longer procedural time (90.9 ± 35.6 vs. 64.4 ± 33.0 min, p = 0.006). Multivariable logistic regression analysis revealed that procedural thromboembolisms were independently associated with procedural time (adjusted odds ratio (OR); 1.020, 95% confidence interval (CI); 1.002–1.039, p = 0.030) and IV thrombolysis (adjusted OR; 4.697, 95% CI; 1.223–18.042, p = 0.024). The cutoff value of procedural time for predicting procedural thromboembolisms was ≥71 min (area under the curve; 0.711, 95% CI; 0.570–0.851, p = 0.010). Conclusions: Procedural thromboembolisms after MT for acute ischemic stroke are significantly associated with longer procedural time and IV thrombolysis. This study suggests that patients with IV thrombolysis and longer procedural time (≥71 min) are at a higher risk of procedural thromboembolisms after MT for acute ischemic stroke.

ABSTRACT: A city experiencing a cycle from growth to decline cannot maintain sustainable development without the type of urban identity that could be consolidated by culture-led urban regeneration. A plan for urban regeneration in a declining urban area should be practiced partially or on the whole according to the characteristics of the community. By transforming a low-value and deteriorated area into a highly valued district, the local community can simultaneously restore its social pride, revive the local economy, and realize an urban identity.Firstly, this paper examines urban decline in order to better understand urban regeneration and the need for multidisciplinary management, and also, by considering the necessity for and universal types of urban regeneration, investigates the characteristics of culture-led urban regeneration as a tool for realizing socio-economic revival and urban identity. In particular, this study suggests the action techniques and benchmarking points for urban regeneration by analyzing cases of culture-led urban regeneration in Korea. Three subjects were considered as case studies in this paper: 1) Hanok village in Jeonju city, which changed from a twilight zone to a tourist attraction; 2) Changdong district in Changwon city, which recovered from an area of declining and dark alleyways that had been the hub for arts and culture in the 1970s to become a new artist village; and 3) Cheongju city, which is being transformed from an idle industrial facility into a cultural space. This thesis suggests the implementation process of culture-led urban regeneration to find an urban identity through analysis of the causes of urban decline, the methods of regeneration, and the results of urban regeneration in the three aforementioned cases. In the conclusion section of this paper, the implementation process for culture-led urban regeneration is summarized as consisting of 5 phases: Phase 1, the diagnosis of decline; Phase 2, understanding the reasons for decline and local characteristics; Phase 3, making a database and establishing a direction; Phase 4, applying various techniques suitable for Phase 3; and Phase 5, monitoring and feedback. KEYWORDS: Urban Regeneration, culture-led, historic and cultural resources, urban identity

The yeast Rgt1 repressor inhibits transcription of the glucose transporter (HXT) genes in the absence of glucose. It does so by recruiting the general corepressor complex Ssn6-Tup1 and the HXT corepressor Mth1. In the presence of glucose, Rgt1 is phosphorylated by the cAMP-activated protein kinase A (PKA) and dissociates from the HXT promoters, resulting in expression of HXT genes. In this study, using Rgt1 chimeras that bind DNA constitutively, we investigate how glucose regulates Rgt1 function. Our results show that the DNA-bound Rgt1 constructs repress expression of the HXT1 gene in conjunction with Ssn6-Tup1 and Mth1, and that this repression is lifted when they dissociate from Ssn6-Tup1 in high glucose conditions. Mth1 mediates the interaction between the Rgt1 constructs and Ssn6-Tup1, and glucose-induced downregulation of Mth1 enables PKA to phosphorylate the Rgt1 constructs. This phosphorylation induces dissociation of Ssn6-Tup1 from the DNA-bound Rgt1 constructs, resulting in derepression of HXT gene expression. Therefore, Rgt1 removal from DNA occurs in response to glucose but is not necessary for glucose induction of HXT gene expression, suggesting that glucose regulates Rgt1 function by primarily modulating the Rgt1 interaction with Ssn6-Tup1.

Due to recent advances of bioinformatics and high throughput sequencing technology, discovery of regulatory noncoding RNAs in bacteria has been increased to a great extent. Based on this bandwagon, many studies searching for trans-acting small noncoding RNAs in streptococci have been performed intensively, especially in the important human pathogen, group A and B streptococci. However, studies for cis-encoded noncoding antisense RNAs in streptococci have been scarce. A recent study shows antisense RNAs are involved in virulence gene regulation in group B streptococcus, S. agalactiae. This suggests antisense RNAs could have important roles in the pathogenesis of streptococcal pathogens. In this review, we describe recent discoveries of chromosomal cis-encoded antisense RNAs in streptococcal pathogens and other low GC Gram (+) bacteria to provide a guide for future studies.

The transport of glucose across the plasma membrane is mediated by members of the glucose transporter family. In this study, we investigated glucose uptake through the yeast hexose transporter 1 (Hxt1) by measuring incorporation of 2-NBDG, a non-metabolizable, fluorescent glucose analog, into the yeast Saccharomyces cerevisiae. We find that 2-NBDG is not incorporated into the hxt null strain lacking all glucose transporter genes and that this defect is rescued by expression of wild type Hxt1, but not of Hxt1 with mutations at the putative glucose-binding residues, inferred from the alignment of yeast and human glucose transporter sequences. Similarly, the growth defect of the hxt null strain on glucose is fully complemented by expression of wild type Hxt1, but not of the mutant Hxt1 proteins. Thus, 2-NBDG, like glucose, is likely to be transported into the yeast cells through the glucose transport system. Hxt1 is internalized and targeted to the vacuole for degradation in response to glucose starvation. Among the mutant Hxt1 proteins, Hxt1N370A and HXT1W473A are resistant to such degradation. Hxt1N370A, in particular, is able to neither uptake 2-NBDG nor restore the growth defect of the hxt null strain on glucose. These results demonstrate 2-NBDG as a fluorescent probe for glucose uptake in the yeast cells and identify N370 as a critical residue for the stability and function of Hxt1.

Small cyclic nucleotide derivatives are employed as second messengers by both prokaryotes and eukaryotes to regulate diverse cellular processes responding to various signals. In bacteria, c-di-AMP has been discovered most recently, and some Gram-positive pathogens including S. pyogenes use this cyclic nucleotide derivative as a second messenger instead of c-di-GMP, a well-studied important bacterial second messenger. GdpP, c-di-AMP phosphodiesterase, is responsible for degrading c-di-AMP inside cells, and the cellular role of GdpP in S. pyogenes has not been examined yet. To test the cellular role of GdpP, we created a strain with a nonpolar inframe deletion of the gdpP gene, and examined the properties of the strain including virulence. From this study, we demonstrated that GdpP influences the biogenesis of SpeB, the major secreted cysteine protease, at a post-translational level, susceptibility to the beta lactam antibiotic ampicillin, and is necessary for full virulence in a murine subcutaneous infection model.

Streptococcus pyogenes (Group A Streptococcus or GAS) is a Gram-positive bacterial pathogen that has shown complex modes of regulation of its virulence factors to cause diverse diseases. Bacterial small RNAs are regarded as novel widespread regulators of gene expression in response to environmental signals. Recent studies have revealed that several small RNAs (sRNAs) have an important role in S. pyogenes physiology and pathogenesis by regulating gene expression at the translational level. To search for new sRNAs in S. pyogenes, we performed a genomewide analysis through computational prediction followed by experimental verification. To overcome the limitation of low accuracy in computational prediction, we employed a combination of three different computational algorithms (sRNAPredict, eQRNA and RNAz). A total of 45 candidates were chosen based on the computational analysis, and their transcription was analyzed by reverse-transcriptase PCR and Northern blot. Through this process, we discovered 7 putative novel trans-acting sRNAs. Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals. Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis. This study provides a significant resource for future study of small RNAs and their roles in physiology and pathogenesis of S. pyogenes.

The capsule of Streptococcus pyogenes serves as an adhesin as well as an anti-phagocytic factor by binding to CD44 on keratinocytes of the pharyngeal mucosa and the skin, the main entry sites of the pathogen. We discovered that S. pyogenes HSC5 and MGAS315 strains are further thermoregulated for capsule production at a post-transcriptional level in addition to the transcriptional regulation by the CovRS two-component regulatory system. When the transcription of the hasABC capsular biosynthetic locus was de-repressed through mutation of the covRS system, the two strains, which have been used for pathogenesis studies in the laboratory, exhibited markedly increased capsule production at sub-body temperature. Employing transposon mutagenesis, we found that CvfA, a previously identified membrane-associated endoribonuclease, is required for the thermoregulation of capsule synthesis. The mutation of the cvfA gene conferred increased capsule production regardless of temperature. However, the amount of the capsule transcript was not changed by the mutation, indicating that a post-transcriptional regulator mediates between CvfA and thermoregulated capsule production. When we tested naturally occurring invasive mucoid strains, a high percentage (11/53, 21%) of the strains exhibited thermoregulated capsule production. As expected, the mucoid phenotype of these strains at sub-body temperature was due to mutations within the chromosomal covRS genes. Capsule thermoregulation that exhibits high capsule production at lower temperatures that occur on the skin or mucosal surface potentially confers better capability of adhesion and invasion when S. pyogenes penetrates the epithelial surface.

Background: Knee osteoarthritis (OA) is a prevalent degenerative joint disease significantly impacting global health. Early and accurate diagnosis is crucial for effective management, but traditional methods often rely on subjective assessments. This study evaluates the efficacy of a deep learning model implemented through a no-code AI platform for diagnosing and grading knee OA from plain radiographs. Methods: We utilized the Osteoarthritis Initiative (OAI) dataset, comprising knee X-ray data from 1526 patients. The data were split into training (47.0%), validation (26.5%), and test (26.5%) sets. We employed a ResNet101 model on the DEEP:PHI no-code AI platform for image analysis. The model was trained to classify knee OA into five grades (0–4) based on the Kellgren–Lawrence scale. Results: Our AI model demonstrated high accuracy in distinguishing between different OA grades, with particular strength in early-stage detection. The model achieved optimal performance at 20 epochs, suggesting efficient learning dynamics. Grad-CAM visualizations were used to enhance the interpretability of the model's decision-making process. Conclusions: This study demonstrates the potential of AI, implemented through a no-code platform, to accurately diagnose and grade knee OA from radiographs. The use of a no-code AI platform such as DEEP:PHI represents a step towards democratizing AI in healthcare, enabling the rapid development and deployment of sophisticated medical AI applications without extensive coding expertise. This approach could significantly enhance the early detection and management of knee OA, potentially improving patient outcomes and streamlining clinical workflows. [ABSTRACT FROM AUTHOR]

In hypersonic computational fluid dynamics, the two-temperature (2-T) model is widely used to simulate thermochemical nonequilibrium. The 2-T model incorporates translational-rotational and electron-electronic-vibrational energies, assuming that the integrated energies have the equivalent temperature. In this study, multi-T models are constructed to accurately predict the effects on heat flux and free electrons due to the separation of energy modes under hypersonic environments. The three-temperature (3-T) model separates the electron-electronic energy from the electron-electronic-vibrational energy of the 2-T model. The 3-T model can accurately predict the distribution and temperature of free electrons by separating the energy of free electrons, which has different characteristics from heavy particles. The four-temperature model treats rotational energy as a nonequilibrium energy mode, distinct from translational-rotational energy. While the rotational temperature reaches equilibrium rapidly at low temperatures, at high-temperature regime rotational temperature shows a relaxation time similar to that of vibrational temperature, which cannot be ignored. To develop multi-T models, electron-vibrational relaxation and translational-rotational relaxation, which are omitted in the 2-T model, are considered. Various flight test and ground facility conditions are analyzed to verify the effects of electron and heat flux under circumstances that include shock, expansion, and shock wave boundary layer interaction. The results of the multi-T models show significant differences in electron temperature and distribution caused by electron-electronic nonequilibrium. Additionally, rotational nonequilibrium increases the shock standoff distance and alters the electron distribution at high altitudes. The heat flux difference across multi-T models is found to be negligible, except in the high degree of ionization condition. [ABSTRACT FROM AUTHOR]

Streptococcus pyogenes, commonly known as Group A Streptococcus (GAS), is a significant human pathogen responsible for a wide range of diseases, from mild pharyngitis to severe conditions such as necrotizing fasciitis and toxic shock syndrome. The increasing antibiotic resistance, especially against macrolide antibiotics, poses a challenge to the effective treatment of these infections. This paper reviews the current state and mechanisms of antibiotic resistance in S. pyogenes. Furthermore, molecular targets for developing anti-virulence agents, which aim to attenuate virulence rather than killing it outright, are explored. This review specifically focuses on virulence regulators, proteins that coordinate the expression of multiple virulence factors in response to environmental signals, playing a crucial role in the pathogen's ability to cause disease. Key regulatory systems, such as RopB, Mga, CovRS, and the c-di-AMP signaling system, are discussed for their roles in modulating virulence gene expression. Additionally, potential molecular target sites for the development of anti-virulence agents are suggested. By concentrating on these regulatory pathways, it is proposed that anti-virulence strategies could enhance the effectiveness of existing antibiotics and reduce the selective pressure that drives the development of resistance. [ABSTRACT FROM AUTHOR]