Short-term culture for rapid identification by mass spectrometry and automated antimicrobial susceptibility testing from positive bottles.

BACKGROUND: Early and appropriate antibiotic treatment improves the clinical outcome of patients with sepsis. There is an urgent need for rapid identification (ID) and antimicrobial susceptibility testing (AST) of bacteria that cause bloodstream infection (BSI). Rapid ID and AST can be achieved by short-term incubation on solid medium of positive blood cultures using MALDI-TOF mass spectrometry (MS) and the BD M50 system. The purpose of this study is to evaluate the performance of rapid method compared to traditional method. METHODS: A total of 124 mono-microbial samples were collected. Positive blood culture samples were short-term incubated on blood agar plates and chocolate agar plates for 5 ∼ 7 h, and the rapid ID and AST were achieved through Zybio EXS2000 MS and BD M50 System, respectively. RESULTS: Compared with the traditional 24 h culture for ID, this rapid method can shorten the cultivation time to 5 ∼ 7 h. Accurate organism ID was achieved in 90.6% of Gram-positive bacteria (GP), 98.5% of Gram-negative bacteria (GN), and 100% of fungi. The AST resulted in the 98.5% essential agreement (EA) and 97.1% category agreements (CA) in NMIC-413, 99.4% EA and 98.9% CA in PMIC-92, 100% both EA and CA in SMIC-2. Besides, this method can be used for 67.2% (264/393) of culture bottles during routine work. The mean turn-around time (TAT) for obtaining final results by conventional method is approximately 72.6 ± 10.5 h, which is nearly 24 h longer than the rapid method. CONCLUSIONS: The newly described method is expected to provide faster and reliable ID and AST results, making it an important tool for rapid management of blood cultures (BCs). In addition, this rapid method can be used to process most positive blood cultures, enabling patients to receive rapid and effective treatment.

Gardnerella Vaginalis Bacteremia in a Pregnant Woman with Vaginal Myomectomy.

BACKGROUND: This case involves a 28-year-old pregnant woman (39w+2) who was admitted to obstetrics due to abdominal tightness and bacteremia with Gardnerella vaginalis which developed after caesarean section and vaginal myomectomy. METHODS: A blood culture was performed, and the bacteria were identified through mass spectrometry. RESULTS: Mass spectrometry data indicated that the infection bacteria were Gardnerella vaginalis. The patient's temperature returned to normal after oral ampicillin in combination with clindamycin. CONCLUSIONS: Gardnerella vaginalis bacteremia is very rare in clinical practice, and the combination of ampicillin and clindamycin has a good therapeutic effect. This study may provide a reference for the diagnosis and treatment of Gardnerella vaginalis bacteremia.

Use of non-canonical amino acids in genetic code expansion-based therapeutics: Effects on mouse gut microbiota.

Vaccines and cell therapeutics based on genetic code expansion are emerging. A crucial step in these therapeutic technologies is the oral administration of non-canonical amino acids (ncAAs) to control pathogen growth and therapeutic protein levels in vivo. Investigating the toxicity effects of ncAAs can help identify more suitable candidates for developing genetic code expansion-based vaccines and cell therapeutics. In this study, we determined the effects of three ncAAs, namely, 4-acetyl-phenylalanine (pAcF), 4-iodo-phenylalanine (pIoF), and 4-methoxy-phenylalanine (pMeoF), commonly used in genetic code expansion-based vaccines and cell therapeutics, on the main organs, serum biochemical parameters, and gut microbiota in mice. We observed that pIoF and pMeoF significantly altered serum biochemical parameters to some extent. Moreover, the alterations in the mouse gut microbial composition were considerably greater after the oral administration of pIoF and pMeoF than after that of pAcF, compared with that in the control mice. These findings suggest that pAcF is more suitable than pIoF and pMeoF for application in genetic code expansion-based vaccines and cell therapeutics as it disturbs the physiological and gut microecological balance in mice to a lesser extent.

A novel "off-on" fluorescent probe for the detection of nickel ions and its clinical application.

Nickel serves as an essential micronutrient for the human body, playing a vital role in various enzymatic processes. However, excessive nickel entering the environment can cause pollution and pose serious risks to animals, plants, and human health. High concentrations of nickel ions in the human body increase the risk of various diseases, highlighting the need for accurate measurement of nickel ions levels. In this study, we designed a sequence-specific cleavage probe for nickel (II) ion called SSC-Ni. Similar to the TaqMan probe, SSC-Ni is an off-on fluorescent probe with an exceptionally low background fluorescence signal. It exhibits high detection specificity, making it highly selective for nickel ions, and the detection limit of the probe towards Ni2+ is as low as 82 nM. The SSC-Ni probe can be utilized for convenient and cost-effective high-throughput quantitative detection of nickel ions in serum. Its user-friendly operation and affordability make it a practical solution. By addressing the lack of simple and effective nickel ion detection methods, this probe has the potential to contribute significantly to environmental monitoring and the protection of human health.

DVDR-SRGAN: Differential Value Dense Residual Super-Resolution Generative Adversarial Network.

In the field of single-image super-resolution reconstruction, GAN can obtain the image texture more in line with the human eye. However, during the reconstruction process, it is easy to generate artifacts, false textures, and large deviations in details between the reconstructed image and the Ground Truth. In order to further improve the visual quality, we study the feature correlation between adjacent layers and propose a differential value dense residual network to solve this problem. We first use the deconvolution layer to enlarge the features, then extract the features through the convolution layer, and finally make a difference between the features before being magnified and the features after being extracted so that the difference can better reflect the areas that need attention. In the process of extracting the differential value, using the dense residual connection method for each layer can make the magnified features more complete, so the differential value obtained is more accurate. Next, the joint loss function is introduced to fuse high-frequency information and low-frequency information, which improves the visual effect of the reconstructed image to a certain extent. The experimental results on Set5, Set14, BSD100, and Urban datasets show that our proposed DVDR-SRGAN model is improved in terms of PSNR, SSIM, and LPIPS compared with the Bicubic, SRGAN, ESRGAN, Beby-GAN, and SPSR models.

ESRGAN-DP: Enhanced super-resolution generative adversarial network with adaptive dual perceptual loss.

The proposal of perceptual loss solves the over-smoothing problem of images caused by pixel-wise loss and improves the visual quality of the images, but it also inevitably produces a large number of artifacts and distortions in images. The reason for this phenomenon is that the perceptual features only rely on a single pre-trained visual geometry group (VGG) network, which results in the features of the image being unable to be fully extracted, thus limiting the reasoning ability of the model. To fundamentally reduce the generation of artifacts and distortions, this paper proposes the Dual Perceptual Loss (DP Loss). First, we improve the perceptual feature extraction method so that it no longer only extracts single-type VGG features. In addition, a residual network (ResNet) feature that has a complementary relationship with the VGG feature can also be extracted. Then, we propose a dynamic weighting method to eliminate the magnitude difference between perceptual losses. Finally, to obtain the excellent effect of image reconstruction, enhanced super-resolution generative adversarial network (ESRGAN) with strong learning capability is used in this paper to adapt the complexity of DP Loss. The abundant experimental studies and evaluations are conducted on benchmark datasets. Results are encouraging and better than those previously reported on these datasets. The code is available at https://github.com/Sunny6-6-6/ESRGAN-DP.

Gram-GAN: Image Super-Resolution Based on Gram Matrix and Discriminator Perceptual Loss.

The solution of a high-resolution (HR) image corresponding to a low-resolution (LR) image is not unique in most cases. However, single-LR-single-HR supervision is widely adopted in single-image super-resolution (SISR) tasks, which leads to inflexible inference logic of the model and poor generalization ability. To improve the flexibility of model inference, we constructed a novel form of supervision, except for the ground truth (GT). Specifically, considering the structural properties of natural images, we propose using extra supervision to focus on the textural similarity of the images. As textural similarity does not account for the position information of images, a Gram matrix was constructed to break the limitations of spatial position and focus on the textural information. Besides the use of traditional perceptual loss, we propose a discriminator perceptual loss based on the two-network architecture of generative adversarial networks (GAN). The difference between the discriminator features used in this loss and the traditional visual geometry group (VGG) features is that the discriminator features can describe the relevant information from the perspective of super-resolution. Quantitative and qualitative experiments were performed to demonstrate the effectiveness of the proposed method.

Simultaneous detection of human norovirus GI, GII and SARS-CoV-2 by a quantitative one-step triplex RT-qPCR.

Background: There are many similarities in the clinical manifestations of human norovirus and SARS-CoV-2 infections, and nucleic acid detection is the gold standard for diagnosing both diseases. In order to expedite the identification of norovirus and SARS-CoV-2, a quantitative one-step triplex reverse transcription PCR (RT-qPCR) method was designed in this paper. Methods: A one-step triplex RT-qPCR assay was developed for simultaneous detection and differentiation of human norovirus GI (NoV-GI), GII (NoV-GII) and SARS-CoV-2 from fecal specimens. Results: The triplex RT-qPCR assay had high detection reproducibility (CV < 1%) and sensitivity. The lower limits of detection (LLOD95) of the triplex RT-qPCR assay for each target site were 128.5-172.8 copies/mL, and LLOD95 of the singleplex RT-qPCR assay were 110.3-142.0 copies/mL. Meanwhile, among the detection of clinical oropharyngeal swabs and fecal specimens, the results of the singleplex and triplex RT-qPCR assay showed high agreement. Conclusion: The triplex RT-qPCR assay for simultaneous detection of NoV-GI, NoV-GII and SARS-CoV-2 from fecal specimens has high clinical application value.

Comprehensive Analysis Identified ASF1B as an Independent Prognostic Factor for HBV-Infected Hepatocellular Carcinoma.

Purpose: Hepatitis B (HBV)-infected hepatocellular carcinoma is one of the most common cancers, and it has high incidence and mortality rates worldwide. The incidence of hepatocellular carcinoma has been increasing in recent years, and existing treatment modalities do not significantly improve prognosis. Therefore, it is important to find a biomarker that can accurately predict prognosis. Methods: This study was analyzed using the The Cancer Genome Atlas (TCGA) database and validated by the International Cancer Genome Consortium (ICGC) database. The STRING database was used to construct a gene co-expression network and visualize its functional clustering using Cytoscape. A prognostic signature model was constructed to observe high and low risk with prognosis, and independent prognostic factors for HBV-infected hepatocellular carcinoma were identified by Cox regression analysis. The independent prognostic factors were then analyzed for expression and survival, and their pathway enrichment was analyzed using gene set enrichment analysis (GSEA). Results: 805 differentially expressed genes (DEGs) were obtained by differential analysis. Protein-protein interaction (PPI) showed that DEGs were mostly clustered in functional modules, such as cellular matrix response, cell differentiation, and tissue development. Prognostic characterization models showed that the high-risk group was associated with poor prognosis, while Cox regression analysis identified ASF1B as the only independent prognostic factor. As verified by expression and prognosis, ASF1B was highly expressed in HBV-infected hepatocellular carcinoma and led to a poor prognosis. GSEA showed that high ASF1B expression was involved in cell cycle-related signaling pathways. Conclusion: Bioinformatic analysis identified ASF1B as an independent prognostic factor in HBV-infected hepatocellular carcinoma, and its high expression led to a poor prognosis. Furthermore, it may promote hepatocellular carcinoma progression by affecting cell cycle-related signaling pathways.

Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.△A and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase.

Incorporation of noncanonical amino acids (ncAAs) into proteins has been proven to be a powerful tool to manipulate protein structure and function, and to investigate many biological processes. Improving the yields of ncAA-containing proteins is of great significance in industrial-scale applications. Escherichia coli C321.ΔA was generated by the replacement of all known amber codons and the deletion of RF1 in the genome and has been proven to be an ideal host for ncAA-containing protein expression using genetic code expansion. In this study, we investigated the transcriptome and proteome profiles of this first codon reassignment strain and found that some functions and metabolic pathways were differentially expressed when compared with those of its parent strain. Genes involved in carbohydrate and energy metabolism were remarkably downregulated. Our results may provide important clues about the growth defects in E. coli C321.ΔA. Furthermore, we improved the yields of ncAA-containing proteins in E. coli C321.ΔA by integrating the T7 RNA polymerase system.

Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13.

The interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13NTD) and Ub monomer has been characterized, while it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13NTD. The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have determined the complex structure between Rpn13NTD and K48-diUb. In this structure, Rpn13NTD simultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13NTD similarly as the Ub monomer in the known Rpn13NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13NTD. Thus, a charge-reversal mutation in Rpn13NTD weakens the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, physical blockage of the access of the distal Ub to Rpn13NTD with a proximity-attached Ub monomer can disrupt the interaction between Rpn13 and K48-diUb. Taken together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.

Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus.

Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS147, IS29, IS27, and IS8) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS147 and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29 is dispensable. The large insert IS147 contains Ca3 and Ca4, two calcium-binding Dx[DN]xDG motifs that are conserved in many pyrolysin-like proteases. Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation. Mature insert-deletion variants were characterized and showed that IS29 and IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS29 and the other in IS27 Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Interestingly, autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation. Additionally, a single mutation in IS29 increases resistance to salt-induced autocleavage and further increases enzyme thermostability. Our results suggest that these extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments.IMPORTANCE Pyrolysin-like proteases belong to the subtilase superfamily and are characterized by large inserts and long C-terminal extensions; however, the role of the inserts in enzyme function is unclear. Our results demonstrate that four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease.