Search

ABSTRACT The gut microbiota is essential for providing colonization resistance against pathogens. Dietary sugars markedly shift the composition of the intestinal microbiota and alter host susceptibility to enteric infections. Here, we demonstrate the effect of L-arabinose on bacterial infection by using a mouse infection model with Salmonella enterica serovar Typhimurium (S. Tm). In the presence of microbiota, L-arabinose induces a dramatic expansion of Enterobacteriaceae, thereby decreasing the microbiota diversity and causing more severe systemic infection. However, L-arabinose supplementation does not alter the disease progression of Salmonella infection in a microbiota-depleted mouse model. More importantly, short-term supplementation of L-arabinose fails to exert anti-diabetic effects in Salmonella-infected hyperglycemia mice and still promotes infection. Overall, our work reveals that a high intake of dietary L-arabinose supports a bloom of Enterobacteriaceae in Salmonella-infected gut, further accelerating the process of systemic infection.IMPORTANCEL-arabinose is a promising natural sweetener and food additive for the regulation of hyperglycemia. Since diabetic subjects are more susceptible to infections, the safety of dietary L-arabinose in diabetic patients experiencing infection remains a concern. Our findings reveal that L-arabinose exacerbates Salmonella infection outcome by inducing gut microbiota dysbiosis in mice. High dietary intake of L-arabinose may be deleterious for diabetic individuals undergoing infection.

Abstract Background Most studies had shown a linear relationship between serum albumin (sALB) and the prevalence of diabetic retinopathy (DR). Thus, the purpose of this study is to investigate whether their relationship is non-linear. Methods We included 426 patients with type 2 diabetes who were hospitalized in Guangdong Provincial People’s Hospital from December 2017 to November 2018. The outcome was the prevalence of DR. A two-piecewise logistics regression model was performed to identify the non-linear relationship between sALB and the prevalence of DR. The inflection point was calculated to determine the saturation effect through the maximum likelihood ratio and a recursive algorithm. Results DR was diagnosed in 167 of 426 type 2 diabetic patients. The relationship between sALB and DR was nonlinear. When sALB was less than 38.10 g/L, a significant negative association was observed (OR = 0.82; 95% CI, 0.72–0.94; P = 0.0037), while no significant association was observed when sALB was greater than 38.10 g/L (OR = 1.12; 95% CI, 0.92–1.35; P = 0.2637). Conclusions The relationship between sALB and the prevalence of DR is non-linear. sALB is negatively associated with the prevalence of DR when sALB is less than 38.10 g/L. Our findings need to be confirmed by further prospective research.

Abstract Blood urea nitrogen (BUN) is an indicator of renal function and catabolic status in human body. Diabetic retinopathy (DR) is a major microvascular complication of diabetes mellitus (DM) and a serious threat to the vision of diabetic patients. We included 426 type 2 diabetic patients who visited the endocrinology department of Guangdong Provincial People’s Hospital and received an ophthalmology consultation from December 2017 to November 2018. The outcome was the probability of DR in participants. Multivariable logistics analysis was used to confirm the relationship between BUN and the probability of DR. And interaction tests were conducted to find the effects of DM duration on their association. A total of 167 of 426 patients with type 2 diabetes had DR, with a probability of 39.20%. After adjusting for potential confounders, a positive association between BUN and the probability of DR (OR = 1.12; 95% CI 1.03–1.21; P = 0.0107). And a test for interaction between DM duration and BUN on the probability of DR was significant (P = 0.0295). We suggested that in patients with type 2 diabetes, BUN was positively associated with the probability of DR and the association was influenced by DM duration.

Abstract Microbial keratitis, a nonviral corneal infection caused by bacteria, fungi, and protozoa, is an urgent condition in ophthalmology requiring prompt treatment in order to prevent severe complications of corneal perforation and vision loss. It is difficult to distinguish between bacterial and fungal keratitis from image unimodal alone, as the characteristics of the sample images themselves are very close. Therefore, this study aims to develop a new deep learning model called knowledge-enhanced transform-based multimodal classifier that exploited the potential of slit-lamp images along with treatment texts to identify bacterial keratitis (BK) and fungal keratitis (FK). The model performance was evaluated in terms of the accuracy, specificity, sensitivity and the area under the curve (AUC). 704 images from 352 patients were divided into training, validation and testing set. In the testing set, our model reached the best accuracy was 93%, sensitivity was 0.97(95% CI [0.84,1]), specificity was 0.92(95% CI [0.76,0.98]) and AUC was 0.94(95% CI [0.92,0.96]), exceeding the benchmark accuracy of 0.86. The diagnostic average accuracies of BK ranged from 81 to 92%, respectively and those for FK were 89–97%. It is the first study to focus on the influence of disease changes and medication interventions on infectious keratitis and our model outperformed the benchmark models and reaching the state-of-the-art performance.

ABSTRACT Salmonella could survive and replicate in macrophages, where it encounters multiple stresses. Deficiency of a DNA adenine methyltransferase impairs the survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in hydrogen peroxide. To investigate whether DNA methylation is involved in the expression of oxidative stress-responsive genes, we combined RNA-seq and single-molecule real-time sequencing to integrate transcriptome and methylome analysis. Here, we show that (i) the entire amount of m6A GATC remains stable during oxidative stress; (ii) no significant association is observed between DNA methylation and transcription level in most genes; (iii) coincidence of level change between transcription and m6A GATC in the regulatory regions is identified in 49 genes under oxidative stress. Some of them are known to contribute to bacterial defenses against oxidative stress through reducing H2O2 levels, directing aberrant protein product degradation and inducing outer membrane protein expression. Specifically, the transcription level of ahpCF is negatively correlated to the m6A GATC level in its regulatory region. The transcription of smpB is elevated along with the decrease in m6A level at position 2,879,954 on the minus strand. In contrast, mRNA levels of STM14_2773 and ycfR are positively correlated with m6A GATC content in their regulatory regions. Highly stable DNA methylome and coupled change of m6A GATC with gene expression in specific positions suggest that DNA methylation homeostasis at genome-wide and plasticity in specific regions are crucial for bacterial response to oxidative stress. These findings provide new insights into the epigenetic regulatory mechanisms of gene expression in S. Typhimurium in the host microenvironment. IMPORTANCE The intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) comes across a wide variety of stresses from entry to dissemination, such as reactive oxygen species. To adapt itself to oxidative stress, Salmonella must adopt various and complex strategies. In this study, we revealed that DNA adenine methyltransferase was essential for S. Typhimurium to survive in hydrogen peroxide. We then screened out oxidative stress-responsive genes that were potentially regulated by DNA methylation in S. Typhimurium. Our results show that the DNA methylome is highly stable throughout the genome, and the coupled change of m6A GATC with gene expression is identified in only a few positions, which suggests the complexity of the DNA methylation and gene expression regulation networks. The results may shed light on our understanding of m6A-mediated gene expression regulation in bacteria.

Herpes simplex virus type 1 (HSV-1) Keratitis (HSK) is a highly prevalent eye disease worldwide, characterized by lifelong recurrent episodes and a major risk of leading to blindness. Detecting HSV-1 promptly and accurately can initiate a timely and appropriate therapeutic regimen, minimizing tissue damage and preventing vision impairment. Currently, PCR is the most reliable method for identifying HSV-1, but its utilization for point-of-care (POC) HSV-1 detection is limited due to the need for sophisticated equipment, particularly in areas with limited resources. Here, we propose a new method for on-site HSV detection by using LAMP-Cas12 diagnostic technology and gold nanoparticles. This technique possesses comparable sensitivity to qPCR, and its detection results could be easily read and interpreted without the need for complex equipment. In detecting HSV in clinical tear specimens, this strategy achieved a 93.9 % consistency in positive detection and a 100 % consistency in negative detection compared to qPCR. Our strategy innovates the technique of current HSV-1 detections and is expected to play a crucial role in POC diagnosis of HSK in the future.

Abstract Background Severe ocular surface disorders are one of the major blinding diseases, and a paucity of original tissue obscures successful reconstruction. We developed a new surgical technique of direct oral mucosal epithelial transplantation (OMET) to reconstruct severely damaged ocular surfaces in 2011. This study elaborates on the clinical efficacy of OMET. Methods A retrospective review of patients with severe ocular surface disorders who underwent OMET from 2011 to 2021 at the Department of Ophthalmology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine was conducted. Patients who were followed up for at least 3 months postoperatively and had sufficient pre or postoperative records were included. Surgical efficacy was evaluated by comparing the best-corrected visual acuity (BCVA), corneal transparency, neovascularization grade, and symblepharon grade. Additionally, postoperative ocular surface impression cytology was used to study the morphology of the newborn epithelial cells. Results Forty-eight patients (49 eyes; mean age: 42.55 ± 12.40 years, range:12–66 years) were enrolled in the study. The etiology included chemical burns (30 eyes), thermal burns (16 eyes), explosive injuries (1 eye), Stevens-Johnson syndrome (1 eye), and multiple pterygiums (1 eye). The mean follow-up period was 25.97 ± 22.99 months. Postoperatively, 29 eyes (59.18%) showed improved corneal transparency, 26 eyes (53.06%) had improved BCVA, 47 eyes (95.92%) had a stable epithelium until the final follow-up, 44 eyes (89.80%) had a reduced neovascularization grade. Of the 20 eyes with preoperative symblepharon, 15 (75%) were completely resolved, and five (25%) were partially resolved. Impression cytological studies showed no postoperative conjunctival invasion onto the corneal surface. Conclusions OMET is a safe and effective surgical technique for reconstruction in severe ocular surface disorder by maintaining a stable epithelium and reducing the neovascularization and symblepharon grade.

Lactic acid bacteria (LAB) are ubiquitous microorganisms that can colonize the intestine and participate in the physiological metabolism of the host. LAB can produce a variety of metabolites, including organic acids, bacteriocin, amino acids, exopolysaccharides and vitamins. These metabolites are the basis of LAB function and have a profound impact on host health. The intestine is colonized by a large number of gut microorganisms with high species diversity. Metabolites of LAB can keep the balance and stability of gut microbiota through aiding in the maintenance of the intestinal epithelial barrier, resisting to pathogens and regulating immune responses, which further influence the nutrition, metabolism and behavior of the host. In this review, we summarize the metabolites of LAB and their influence on the intestine. We also discuss the underlying regulatory mechanisms and emphasize the link between LAB and the human gut from the perspective of health promotion.

Post-transcriptional global carbon storage regulator A (CsrA) is a sequence-specific RNA-binding protein involved in the regulation of multiple bacterial processes. Hemolysin is an important virulence factor in the enterohemorrhagic Escherichia coli O157:H7 (EHEC). Here, we show that CsrA plays a dual role in the regulation of hemolysis in EHEC. CsrA significantly represses plasmid-borne enterohemolysin (EhxA)-mediated hemolysis and activates chromosome-borne hemolysin E (HlyE)-mediated hemolysis through different mechanisms. RNA structure prediction revealed a well-matched stem-loop structure with two potential CsrA binding sites located on the 5' untranslated region (UTR) of ehxB, which encodes a translocator required for EhxA secretion. CsrA inhibits EhxA secretion by directly binding to the RNA leader sequence of ehxB to repress its expression in two different ways: CsrA either binds to the Shine–Dalgarno sequence of ehxB to block ribosome access or to ehxB transcript to promote its mRNA decay. The predicted CsrA-binding site 1 of ehxB is essential for its regulation. There is a single potential CsrA-binding site at the 5'-end of the hlyE transcript, and its mutation completely abolishes CsrA-dependent activation. CsrA can also stabilize hlyE mRNA by directly binding to its 5' UTR. Overall, our results indicate that CsrA acts as a hemolysis modulator to regulate pathogenicity under certain conditions.

Adaptation to various stresses during infection is important for Salmonella Typhimurium virulence, while the fitness determinants under infection-relevant stress conditions remain unknown. Here, we simulated conditions Salmonella encountered within the host or in the environment by 15 individual stresses as well as two model cell lines (epithelium and macrophage) to decipher the genes and pathways required for fitness. By high-resolution Tn-seq analysis, a total of 1242 genes were identified as essential for fitness under at least one stress condition. The comparative analysis of fitness determinants in 17 stress conditions indicated the essentiality of genes varied in different mimicking host niches. A total of 12 genes were identified as fitness determinants in all stress conditions, including recB, recC, and xseA (encode three exonuclease subunits necessary for DNA recombination repair) and a novel essential fitness gene yheM. YheM is a putative sulfurtransferase subunit that is responsible for tRNA modification, and our results showed that Salmonella lacking yheM accumulated more aggregates of endogenous protein than wild-type. Moreover, we established a scoring scheme for sRNA essentiality analysis and found STnc2080 of unknown function was essential for resistance to LL-37. In summary, we systematically dissected Salmonella gene essentiality profiling and demonstrated the general and specific adaptive requirements in infection-relevant niches. Our data not only provide valuable insights on how Salmonella responds to environmental stresses during infections but also highlight the potential clinical application of fitness determinants in vaccine development.

The recognition of the profound impact of the human gastrointestinal microbiome (GM) on human autoimmune diseases has gradually increased thanks to deeper research efforts. As a systemic autoimmune disease, primary Sjögren's syndrome (pSS) cannot be completely cured. Human studies have revealed that GM species and diversity are altered in patients with pSS compared with healthy individuals. Animal studies have provided possible mechanisms for the association between pSS and GM. The potential role of GM in pSS is exerted through several mechanisms. GM dysbiosis leads to increased intestinal permeability, which increases the risk of GM antigen exposure and activates specific autoreactive T lymphocytes via “molecular mimicry”. In addition, GM antigen exposure and intestinal immune tolerance loss caused by GM dysbiosis together induce chronic local gut mucosal inflammation, which deteriorates to systemic chronic non-specific inflammation with the circulation of pro-inflammatory lymphocytes and cytokines. These factors eventually activate autoreactive B lymphocytes and lead to pSS. If GM plays a key role in the pathogenesis of pSS, clarifying the underlying mechanisms will be helpful for the development of new therapies targeting GM for dry eye associated with pSS. This review summarizes the latest knowledge about the relationship between GM and pSS, with the aim of contributing to future research and to the development of new clinical applications.

Abstract We aimed to examine whether the efficacy of the risk of poor prognosis in patients with coronary artery disease is jointly affected by total cholesterol and baseline serum albumin in a secondary analysis of previous study. We analyzed the data of 204 patients from October 2014 to October 2017 for newly diagnosed stable CAD. The outcome was major adverse cardiac events (MACE; defined as all cause mortality, non fatal myocardial infarction, and non fatal stroke). The median duration of follow-up was 783 days. Multivariable COX model was performed to revalidate the relationship between the sALB and MACE and interaction tests were conducted to find the effects of total cholesterol on their association. A total of 28 MACE occurred among the 204 participants. The risk of MACE varied by baseline serum albumin and total cholesterol. Specifically, lower serum albumin indicated higher risk of MACE (HR 3.52, 95% CI 1.30–9.54), and a test for interaction between baseline serum albumin and total cholesterol on MACE was significant (P = 0.0005). We suggested that baseline serum albumin and total cholesterol could interactively affect the risk of poor prognosis of patients with coronary artery diseases. Our findings need to be confirmed by further randomized trials.

Increasing evidence highlights the role of bacteria in promoting tumorigenesis. The underlying mechanisms may be diverse and remain poorly understood. Here, we report that Salmonella infection leads to extensive de/acetylation changes in host cell proteins. The acetylation of mammalian cell division cycle 42 (CDC42), a member of the Rho family of GTPases involved in many crucial signaling pathways in cancer cells, is drastically reduced after bacterial infection. CDC42 is deacetylated by SIRT2 and acetylated by p300/CBP. Non-acetylated CDC42 at lysine 153 shows an impaired binding of its downstream effector PAK4 and an attenuated phosphorylation of p38 and JNK, consequently reduces cell apoptosis. The reduction in K153 acetylation also enhances the migration and invasion ability of colon cancer cells. The low level of K153 acetylation in patients with colorectal cancer (CRC) predicts a poor prognosis. Taken together, our findings suggest a new mechanism of bacterial infection-induced promotion of colorectal tumorigenesis by modulation of the CDC42-PAK axis through manipulation of CDC42 acetylation.

In order to deepen the understanding of the role and regulation mechanisms of prokaryotic global transcription regulators in complex processes, including virulence, the associations between the affinity and binding sequences of Mycobacterium tuberculosis MtrA have been explored extensively. Analysis of MtrA 294 diversified 26 bp binding sequences revealed that the sequence similarity of fragments was not simply associated with affinity. The unique variation patterns of GC content and periodical and sequential fluctuation of affinity contribution curves were observed along the sequence in this study. Furthermore, docking analysis demonstrated that the structure of the dimer MtrA-DNA (high affinity) was generally consistent with other OmpR family members, while Arg 219 and Gly 220 of the wing domain interacted with the minor groove. The results of the binding box replacement experiment proved that box 2 was essential for binding, which implied the differential roles of the two boxes in the binding process. Furthermore, the results of the substitution of the nucleotide at the 20th and/or 21st positions indicated that the affinity was negatively associated with the value of minor groove width precisely at the 21st position. The dimerization of the unphosphorylated MtrA facilitated by a low-affinity DNA fragment was observed for the first time. However, the proportion of the dimer was associated with the affinity of substrate DNA, which further suggested that the affinity was actually one characteristic of the stability of dimers. Based on the finding of 17 inter-molecule hydrogen bonds identified in the interface of the MtrA dimer, including 8 symmetric complementary ones in the conserved α4-β5-α5 face, we propose that hydrogen bonds should be considered just as important as salt bridges and the hydrophobic patch in the dimerization. Our comprehensive study on a large number of binding fragments with quantitative affinity values provided new insight into the molecular mechanism of dimerization, binding specificity and affinity determination of MtrA and clues for solving the puzzle of how global transcription factors regulate a large quantity of target genes.

Abstract Background To report on corneal endothelial regeneration, graft clarity, and vision recovery when using endothelium-free grafts. Methods We evaluated the donor’s cell viability using trypan blue staining and dual staining with calcein acetoxy methyl ester and ethidium homodimer-1. To preserve eyeball integrity, we performed therapeutic penetrating keratoplasty using cryopreserved donor tissue without endothelium on 195 consecutive patients who suffered from corneal perforation due to progressive primary corneal disease such as herpes simplex keratitis, fungal keratitis, ocular thermal burns, keratoconus, and phlyctenular keratoconjunctivitis. Of these, 18 eyes recovered corneal graft clarity and underwent periodic slit-lamp microscopy, A-scan pachymetry, and in vivo confocal microscopy to observe the clinical manifestations, variations in corneal thickness, and repopulation of the corneal endothelial cells on the donor grafts. Results No viable cells were detected in the cryopreserved corneas. After the therapeutic penetrating keratoplasty, notable corneal graft edema was observed in all 18 eyes for 1–4 months, and no corneal endothelial cells were detected on the grafts during this period. Thereafter, we observed gradual and progressive regression and final resolution of the stromal edema, with complete recovery of corneal graft clarity. Through periodic confocal microscopy, we observed the corneal endothelium’s regenerating process, along with single cells bearing multiple nuclei and cell division-like morphology. The regenerated endothelium on the grafts reached a mean cell density of 991 cells/mm2. Remarkable vision rehabilitation was achieved in all 18 patients. Conclusions We obtained conclusive evidence that host-derived endothelial cells can regenerate a new endothelium over the endothelium-free graft, which possesses normal functions for corneal clarity and vision recovery.

Infections are highly orchestrated and dynamic processes, which involve both pathogen and host. Transcriptional profiling at the single-cell level enables the analysis of cell diversity, heterogeneity of the immune response, and detailed molecular mechanisms underlying infectious diseases caused by bacteria, viruses, fungi, and parasites. Herein, we highlight recent remarkable advances in single-cell RNA sequencing (scRNA-seq) technologies and their applications in the investigation of host-pathogen interactions, current challenges and potential prospects for disease treatment are discussed as well. We propose that with the aid of scRNA-seq, the mechanism of infectious diseases will be further revealed thus inspiring the development of novel interventions and therapies.

Herpes simplex virus type 1 (HSV-1) is human specific virus. The intercellular transmission of HSV-1 is essential in its pathogenesis. The tunneling nanotube (TNT), a new mode connecting distant cells, has been found to play an important role in the spread of various viruses like human immunodeficiency virus (HIV) and influenza virus. However, whether HSV-1 can be transmitted through TNTs has not been confirmed. The purpose of this study was to clarify this, and further to determine the effect of inhibiting the actin-related protein 2/3 (Arp2/3) complex on the intercellular transmission of HSV-1. A scanning electron microscope and fluorescence microscope detected the formation of TNTs between HSV-1 infected cells. Envelope glycoprotein D (gD) and envelope glycoprotein E (gE) of HSV-1 and viral particles were observed in TNTs. Treatment with CK666, an inhibitor of the Arp2/3 complex, reduced the number of TNTs by approximately 40–80%. At the same time, the DNA level of HSV-1 in cells and the number of plaque formation units (PFU) were also reduced by nearly 30%. These findings indicated that TNT contributes to HSV-1 transmission and that the inhibition of the Arp2/3 complex could impair HSV-1 transmission, which not only provides a novel insight into the transmission mode of HSV-1, but also a putative new antiviral target.

Infectious keratitis is the most common condition of corneal diseases in which a pathogen grows in the cornea leading to inflammation and destruction of the corneal tissues. Infectious keratitis is a medical emergency for which a rapid and accurate diagnosis is needed to ensure prompt and precise treatment to halt the disease progression and to limit the extent of corneal damage; otherwise, it may develop a sight-threatening and even eye-globe-threatening condition. In this paper, we propose a sequential-level deep model to effectively discriminate infectious corneal disease via the classification of clinical images. In this approach, we devise an appropriate mechanism to preserve the spatial structures of clinical images and disentangle the informative features for clinical image classification of infectious keratitis. In a comparison, the performance of the proposed sequential-level deep model achieved 80% diagnostic accuracy, far better than the 49.27% ± 11.5% diagnostic accuracy achieved by 421 ophthalmologists over 120 test images.

Salmonella are a type of bacteria known to cause food-borne illness. Their host range varies widely, and their susceptibility to the host determines its pathogenicity. Salmonella enterica serovar Rissen (S. Rissen) is a widely distributed serotype; however, its virulence and pathogenicity are poorly understood. In this study, the pathogenicity and antibiotic resistance of a representative S. Rissen isolate were investigated. The cell model results showed that S. Rissen preferred to replicate in human macrophage cells U937 compared to murine macrophage cells RAW264.7, suggesting that it has a level of host adaptability. Genome sequencing and comparison analysis revealed that the distribution and nonsynonymous single nucleotide polymorphisms of virulence factors in S. Rissen were similar to those in S. Typhi rather than to those in S. Typhimurium. Taken together, our results suggest that although S. Rissen is a common serotype distributed in swine herds, pork and chicken products, it has strong ability to infect humans.

Objective To evaluate changes in retinal nerve fiber layer (RNFL) and macular thicknesses, included ganglion cell-inner plexiform layer (GCIPL) thickness, in patients with spontaneous intracranial hypotension (SIH). Methods This was a retrospective, nonrandom, observational case series study. Comprehensive ophthalmic examinations and systemic examinations were performed. Spectral domain optical coherence tomography angiography scanning was used to measure peripapillary RNFL thickness and macular volume. Results In total, 108 eyes in 54 patients with SIH were evaluated; these were compared with 108 eyes in 54 healthy controls. The mean ages were 38.2 ± 9.4 years (patients with SIH) and 38.9 ± 9.4 years (healthy controls). In both groups, 33 patients were women (61.1%). The peripapillary RNFL and GCIPL were thinner in patients with SIH than in healthy controls (100.08 ± 9.94 µm vs 104.83 ± 8.35 µm and 81.46 ± 5.67 µm vs 85.67 ± 4.57 µm, respectively). Among patients with SIH, the GCIPL was thinner in patients with visual field defects (79.81 ± 5.62 µm vs 82.39 ± 5.12 µm). Conclusions The RNFL and GCIPL were thinner in patients with SIH than in healthy controls. The GCIPL was thinner in eyes with visual field defects among patients with SIH.

Posttranslational modifications (PTMs) play an important role in regulating enzyme activities, protein-protein interactions, or DNA-protein recognition and, consequently, modulate many biological functions. We demonstrated that PhoP, the response regulator of PhoP/PhoQ two-component system, could be methylated on several evolutionally conserved amino acid residues.

Abstract A surface Ag nano-network pattern is formed by first depositing Ag nanoparticles (NPs) on a conductive template, which has a certain defect structure, and then illuminating the Ag NPs with ultraviolet (UV) light in a moist environment. Such an Ag nano-network pattern consists of multiple connected Brownian trees (BTs), which are produced through the diffusion-limited aggregation (DLA) process. In the DLA process, diffuse Ag+ ions, which are generated by UV light illumination and dissolved by a thin adsorbed water layer on the surfaces of the Ag NPs and used GaN template, settle to form a BT through the combination with excited hot electrons migrating into the template from the Ag NPs. The lateral transport of hot electrons in the template is regulated by the distributions of threading dislocation and point defect cluster in the template, which eventually become the centers of BTs. The structure of a surface Ag nano-network can potentially serve as a transparent conductor.

ABSTRACTAccumulating evidence indicates that bacterial metabolism plays an important role in virulence. Acetyl phosphate (AcP), the high-energy intermediate of the phosphotransacetylase-acetate kinase pathway, is the major acetyl donor in E. coli. PhoP is an essential transcription factor for bacterial virulence. Here, we show in Salmonella typhimurium that PhoP is non-enzymatically acetylated by AcP, which modifies its transcriptional activity, demonstrating that the acetylation of Lysine 102 (K102) is dependent on the intracellular AcP. The acetylation level of K102 decreases under PhoP-activating conditions including low magnesium, acid stress or following phagocytosis. Notably, in vitro assays show that K102 acetylation affects PhoP phosphorylation and inhibits its transcriptional activity. Both cell and mouse models show that K102 is critical to Salmonella virulence, and suggest acetylation is involved in regulating PhoP activity. Together, the current study highlights the importance of the metabolism in bacterial virulence, and shows AcP might be a key mediator.

Abstract Background Keratoconus normally presents as a sporadic disease. Although different studies have found sequence variants of the visual system homeobox 1 (VSX1) gene associated with keratoconus in humans, no research has detected such variants in sporadic keratoconus patients from China. To investigate the possibility of VSX1 being a candidate susceptibility gene for Chinese patients with sporadic keratoconus, we performed sequence screening of this gene in such patients. Methods Whole DNA was obtained from the leukocytes in the peripheral venous blood of 50 patients with sporadic keratoconus and 50 control subjects without this ocular disorder. Polymerase chain reaction single-strand conformation polymorphism analysis and direct DNA sequencing technology were used to detect sequence variation in the five exons and splicing regions of the introns of the VSX1 gene. The sequencing results were analyzed using DNAstar software. Results One novel missense heterozygous sequence variant (p.Arg131Pro) was found in the first exon of the VSX1 gene in one keratoconus patient. Another heterozygous sequence variant (p.Gly160Val) in the second exon was found in two keratoconus patients. These variants were not detected in the control subjects. In the third intron of the VSX1 gene, c.8326G > A nucleotide substitution (including heterozygous and homozygous change) was also discovered. The frequency of this variation did not differ significantly between patients and controls, it should belong to single-nucleotide polymorphism of the VSX1 gene. Bioinformatic analysis also predicted that one missense sequence variation (p.Arg131Pro) may not cause a pathogenic change. Conclusions In this study, we added one novel missense sequence variation (p.Arg131Pro) in the coding region of the VSX1 gene to the range of VSX1 coding region variations observed in patients with sporadic keratoconus from China. Our work suggests that VSX1 sequence variants might be involved in the pathogenesis of sporadic keratoconus, but their precise role in disease causation requires further investigation.

Multidrug-resistant tuberculosis (TB), defined as TB resistant to the two first-line drugs, isoniazid and rifampin, is a serious challenge to global TB eradication efforts. Although mutations in rpoA or rpoC have been proposed to compensate for this fitness cost due to rpoB mutation in rifampicin-resistant Mycobacterium tuberculosis mutants, whether the compensatory effect exists and the underlying mechanisms of compensation remain unclear. Here, we used RNA sequencing to investigate the global transcriptional profiles of 6 rifampin-resistant clinical isolates with either single mutation in rpoB or dual mutations in rpoB/rpoC, as well as 3 rifampin-susceptible clinical isolates, trying to prove the potential compensatory effect of rpoC by transcriptomic alteration. In rifampin-free conditions, rpoC mutation was associated with M. tuberculosis upregulation of ribosomal protein-coding genes, dysregulation of growth-related essential genes and balancing the expression of arginine and glutamate synthesis-associated genes. Upon rifampin exposure of M. tuberculosis isolates, rpoC mutations were associated with the upregulation of the oxidative phosphorylation machinery, which was inhibited in the rpoB single mutants, as well as stabilization of the expression of rifampin-regulated essential genes and balancing the expression of genes involved in metabolism of sulfur-containing amino acids. Taken together, our data suggest that rpoC mutation may compensate for the fitness defect of rifampicin-resistant M. tuberculosis by altering gene expression in response to rifampin exposure.

Streptococcus agalactiae and Candida albicans often co-colonize the female genital tract, and under certain conditions induce mucosal inflammation. The role of the interaction between the two organisms in candidal vaginitis is not known. In this study, we found that co-infection with S. agalactiae significantly attenuated the hyphal development of C. albicans, and that EFG1-Hwp1 signal pathway of C. albicans was involved in this process. In a mouse model of vulvovaginal candidiasis (VVC), the fungal burden and the levels of pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α showed a increase on co-infection with S. agalactiae, while the level of TH17 T cells and IL-17 in the cervicovaginal lavage fluid were significantly decreased. Our results indicate that S. agalactiae inhibits C. albicans hyphal development by downregulating the expression of EFG1-Hwp1. The interaction between S. agalactiae and C. albicans may attenuate host vaginal mucosal TH17 immunity and contribute to mucosal colonization by C. albicans.

Background: Currently, exosomes that act as mediators of intercellular communication are being researched extensively. Our previous studies confirmed that these exosomes contain microRNAs (miRNAs) that could alter chemo-susceptibility, which is partly attributed to the successful intercellular transfer of multidrug resistance (MDR)-specific miRNAs. We also confirmed that β-elemene could influence MDR-related miRNA expression and regulate the expression of the target genes PTEN and Pgp, which may lead to the reversal of the chemoresistant breast cancer (BCA) cells. We are the first to report these findings, and we propose the following logical hypothesis: β-elemene can mediate MDR-related miRNA expression in cells, thereby affecting the exosome contents, reducing chemoresistance transmission via exosomes, and reversing the drug resistance of breast cancer cells. Methods: MTT-cytotoxic, miRNA microarray, real-time quantitative PCR, Dual Luciferase Activity Assay, and Western blot analysis were performed to investigate the impact of β-elemene on the expression of chemoresistance specific miRNA and PTEN as well as Pgp in chemoresistant BCA exosomes. Results: Drug resistance can be reversed by β-elemene related to exosomes. There were 104 differentially expressed miRNAs in the exosomes of two chemoresistant BCA cells: adriacin (Adr) - resistant MCF-7 cells (MCF-7/Adr) and docetaxel (Doc) - resistant MCF-7 cells (MCF-7/Doc) that underwent treatment. Of these, 31 miRNAs were correlated with the constant changes in the MDR. The expression of miR-34a and miR-452 can lead to changes in the characteristics of two chemoresistant BCA exosomes: MCF-7/Adr exosomes (A/exo) and MCF-7/Doc exosomes (D/exo). The PTEN expression affected by β-elemene was significantly increased, and the Pgp expression affected by β-elemene was significantly decreased in both cells and exosomes. β-elemene induced a significant increase in the apoptosis rate in both MCF-7/Doc and MCF-7/Adr cells. Conclusions: Drug resistance can be reversed by β-elemene, which can alter the expression of some MDR-related miRNAs, including PTEN and Pgp in MCF-7/Adr and MCF-7/Doc in cells. It can therefore affect the exosome contents and induce the reduction of resistance transmission via exosomes.

The type VI secretion system (T6SS) is a class of sophisticated cell contact-dependent apparatus with anti-eukaryotic or anti-bacterial function. Klebsiella pneumoniae is one of the most common bacterial pathogens with resistance to the carbapenem antibiotics. However, little is known about the antibacterial T6SS in K. pneumoniae. Using core-component protein searches, we identified a putative T6SS gene cluster on the chromosome of the carbapenemase-producing K. pneumoniae (CRKP) strain HS11286. Intraspecies and interspecies competition assays revealed an antibacterial function of the HS11286 T6SS. The phospholipase Tle1KP was found to be an effector protein that is transferred by T6SS. The overexpression of this effector gene in the periplasm caused severe growth inhibition of Escherichia coli. A sub-inhibitory concentration of β-lactam antibiotics stimulated the expression and secretion of the HS11286 T6SS and enhanced T6SS-dependent killing. It suggested that the antibiotics might be an impact factor for the T6SS secretion and antibacterial activity.

DNA replication initiation is a central event in the cell cycle, and it is strictly controlled by multiple regulatory mechanisms. Our previous work showed that acetylation of residue lysine (K) 178 prevents DnaA from binding to ATP, which leads to the inhibition of DNA replication initiation. Here, we show that another residue, K243, is critical for DnaA full activity in vivo. K243 can be acetylated, and its acetylation level varies with cell growth. A homogeneous, recombinant DnaA that contains N𝜀-acetyllysine at K243 (K243Ac) retained its ATP/ADP binding ability, but showed decreased binding activity to the oriC region. A DNase I footprinting assay showed that DnaA K243Ac failed to recognize DnaA boxes I3, C1, and C3, and, thus, it formed an incomplete initiation complex with oriC. Finally, we found that acetyl phosphate and the deacetylase CobB can regulate the acetylation level of K243 in vivo. These findings suggest that DnaA K243 acetylation disturbs its binding to low-affinity DnaA boxes, and they provide new insights into the regulatory mechanisms of DNA replication initiation.

Enterohemorrhagic Escherichia coli (EHEC) is one major type of contagious and foodborne pathogens. The type VI secretion system (T6SS) has been shown to be involved in the bacterial pathogenicity and bacteria-bacteria competition. Here, we show that EHEC could secrete a novel effector KatN, a Mn-containing catalase, in a T6SS-dependent manner. Expression of katN is promoted by RpoS and OxyR and repressed by H-NS, and katN contributes to bacterial growth under oxidative stress in vitro. KatN could be secreted into host cell cytosol after EHEC is phagocytized by macrophage, which leads to decreased level of intracellular reactive oxygen species (ROS) and facilitates the intramacrophage survival of EHEC. Finally, animal model results show that the deletion mutant of T6SS was attenuated in virulence compared with the wild type strain, while the deletion mutant of katN had comparable virulence to the wild type strain. Taken together, our findings suggest that EHEC could sense oxidative stress in phagosome and decrease the host cell ROS by secreting catalase KatN to facilitate its survival in the host cells.

The metabolic and pharmacokinetic studies on complanatuside, a quality marker of a Chinese materia medicatonic, Semen Astragali Complanati, were carried out. The UHPLC-Q-TOF/MS (ultra-high performance liquid chromatography coupled with electrospray ionization tandem quadrupole-time-of-flight mass spectrometry) method was applied to identify the metabolites of complanatuside in rat plasma, bile, stool, and urine after oral administration at the dosage of 72 mg/kg. Up to 34 metabolites (parent, 2 metabolites of the parent drug, and 31 metabolites of the degradation products) were observed, including processes of demethylation, hydroxylation, glucuronidation, sulfonation, and dehydration. The results indicated glucuronidation and sulfonation as major metabolic pathways of complanatuside in vivo. Meanwhile, a HPLC-MS method to quantify complanatuside and its two major metabolites—rhamnocitrin 3-O-β-glc and rhamnocitrin—in rat plasma for the pharmacokinetic analysis was developed and validated. The Tmax (time to reach the maximum drug concentration) of the above three compounds were 1 h, 3 h, and 5.3 h, respectively, while the Cmax (maximum plasma concentrations)were 119.15 ng/mL, 111.64 ng/mL, and 1122.18 ng/mL, and AUC(0-t) (area under the plasma concentration-time curve) was 143.52 µg/L·h, 381.73 µg/L·h, and 6540.14 µg/L·h, accordingly. The pharmacokinetic characteristics of complanatuside and its two metabolites suggested that complanatuside rapidly metabolized in vivo, while its metabolites—rhamnocitrin—was the main existent form in rat plasma after oral administration. The results of intracorporal processes, existing forms, and pharmacokinetic characteristics of complanatuside in rats supported its low bioavailability.

The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix-turn-helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms.

Quality control of Chinese herbal tea remains a challenge due to our poor knowledge of their complex chemical profile. This study aims to investigate the chemical composition of one of the best-selling and famous brand of beverage in China, Wanglaoji Herbal Tea (WLJHT), via a full component quantitative analysis. In this paper, a total of thirty-four representative constituents were identified or tentatively characterized using ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Moreover, the quantitative analyses of fourteen constituents were performed by high performance liquid chromatography with a triple quadruple tandem mass spectrometry (HPLC-MS/MS) method and saccharide compositions of WLJHT were also quantitatively determined by high performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) on a Hilic column, separately. Using multiple chromatographic techniques presented a good precision, sensitivity, repeatability and stability, and was successfully applied to analyze 16 batches of WLJHT samples. Therefore, it would be a reliable and useful approach for the quality control of WLJHT.

Ammonium assimilation is catalyzed by two enzymatic pathways, i.e., glutamine synthetase/glutamate synthase (GS/GOGAT) and alanine dehydrogenase (AlaDH) in Amycolatopsis mediterranei U32. Under nitrogen-rich conditions, the AlaDH pathway is the major route for ammonium assimilation, while the GS/GOGAT pathway takes over when the extracellular nitrogen supply is limited. The global nitrogen regulator GlnR was previously characterized to activate the transcription of the GS encoding gene glnA in response to nitrogen limitation and is demonstrated in this study as a repressor for the transcription of the AlaDH encoding gene ald, whose regulation is consistent with the switch of the ammonium assimilation pathways from AlaDH to GS/GOGAT responding to nitrogen limitation. Three transcription initiation sites (TISs) of ald were determined with primer extension assay, among which transcription from aldP2 contributed the major transcripts under nitrogen-rich conditions but was repressed to an undetectable level in response to nitrogen limitation. Through DNase I footprinting assay, two separate regions were found to be protected by GlnR within ald promoter, within which three GlnR binding sites (a1, b1 sites in region I and a2 site in region II) were defined. Interestingly, the major TIS aldP2 is located in the middle of a2 site within region II. Therefore, one may easily conclude that GlnR represses the transcription of ald via specific binding to the GlnR binding sites, which obviously blocks the transcription initiation from aldP2 and therefore reduces ald transcripts.

Background. Low expression or absence of dendritic cell (DC) surface B7 molecules can induce immune tolerance or hyporesponse. Whether DCs could induce indirect allogeneic-specific cross-tolerance or hyporesponse to recipient T cells remains unclear. Methods. Generated from C3H/He mice bone marrow cells pulsed with donor antigen from C57BL/6 mice, recipient DCs were incubated with B7 antisense peptide (B7AP). Immune regulatory activities were examined in vitro by a series of mixed lymphocyte reactions. Murine allogeneic carotid artery orthotopic transplantation was performed from C57BL/6 to C3H/He. Recipients were given B7AP-treated DCs 7 days before transplantation. Allograft pathological analysis was done 2 months after transplantation. Results. B7AP-pretreated DCs markedly inhibited T-cell proliferation compared with untreated group. Pretreated T cells exhibited markedly reduced response to alloantigen versus third-party antigen. Pathological analysis of arterial allografts demonstrated significant reduction of intimal hyperplasia in B7-AP pretreated group versus control. Conclusion. Blockade of B7 molecules by B7AP could induce indirect allogeneic-specific hyporesponse and inhibit arterial allograft intimal hyperplasia, which may be involved in future strategies for human allograft chronic rejection.

Background: Shenzao dripping pill (SZDP) is empirically prescribed for treating cardiac diseases. Nevertheless, there is a lack of comprehensive knowledge regarding the underlying mechanisms contributing to its therapeutic effects. The objective of this study is to investigate the underlying mechanism of SZDP against chronic myocardial ischemia (CMI) in a rat model. Methods: In this study, we utilized electrocardiographic and echocardiographic detection along with pathological tissue analysis to evaluate the efficacy of SZDP. The integration of network pharmacology and metabolomics was conducted to investigate the mechanisms. Molecular docking and molecular dynamics simulations were used to validate the binding energy between the compounds of SZDP and the associated targets. Results: The results showed that SZDP was able to improve T wave voltage, reverse CMI abnormalities in ejection fraction and fractional shortening, and restore histopathological heart damage. Metabolomics results indicated that disturbances of metabolic profile in CMI rats were partly corrected after SZDP administration, mainly affecting purine metabolism. 13-Docosenamide may be the potential metabolic biomarker of the therapeutic application of SZDP for CMI. Integrating network pharmacology and metabolomics, thiopurine S-methyltransferase (TPMT), xanthine dehydrogenase/oxidase (XDH), bifunctional purine biosynthesis protein ATIC (ATIC), and cytochrome p450 1A1 (CYP1A1) were identified as possible targets of SZDP to exert therapeutic effects by enhancing the metabolic levels of L-Tryptophan, Deoxyribose 1-phosphate and Phosphoribosyl formamidocarboxamide. Conclusion: SZDP has a therapeutic effect on CMI by regulating metabolite levels, acting on the targets of TMPT, XDH, ATIC, and CYP1A1, and reducing cardiomyocyte injury and myocardial fibrosis. [ABSTRACT FROM AUTHOR]