Uninfected Tissue Research Articles

Single-Nucleus and Spatial Transcriptomics Revealing Host Response Differences Triggered by Mutated Virus in Severe Dengue.

Dengue virus (DENV) infection causes various disease manifestations ranging from an asymptomatic state to severe, life-threatening dengue. Despite intensive research, the molecular mechanisms underlying the abnormal host responses and severe disease symptoms caused by evolved DENV strains is not fully understood. First, the spatial structure of mutant DENV was compared via in silico molecular modeling analysis. Second, employing single-nucleus and spatial RNA sequencing, we analyzed and verified transcriptome samples in uninfected, mild (NGC group), and severe (N10 group) liver tissues from murine models. In this study, we obtained a cumulatively mutated DENV-2 N10 with enhanced capability of replication and pathogenicity post 10 serial passages in Ifnra-/- mice. This variant caused severe damage in the liver, as compared with other organs. Furthermore, mutated DENV infection elicited stronger responses in hepatocytes. The critical host factor Nrg4 was identified. It dominated mainly via the activation of the NRG/ErbB pathway in mice with severe symptoms. We report on evolved N10 viruses with changes observed in different organisms and tissue. This evolutionary variant results in high replicability, severe pathogenicity, and strong responses in murine. Moreover, the host responses may play a role by activating the NRG/ErbB signaling pathway. Our findings provide a realistic framework for defining disturbed host responses at the animal model level that might be one of the main causes of severe dengue and the potential application value.

Metabolic reprogramming tips vaccinia virus infection outcomes by stabilizing interferon-γ induced IRF1.

Interferon (IFN) induced activities are critical, early determinants of immune responses and infection outcomes. A key facet of IFN responses is the upregulation of hundreds of mRNAs termed interferon-stimulated genes (ISGs) that activate intrinsic and cell-mediated defenses. While primary interferon signaling is well-delineated, other layers of regulation are less explored but implied by aberrant ISG expression signatures in many diseases in the absence of infection. Consistently, our examination of tonic ISG levels across uninfected human tissues and individuals revealed three ISG subclasses. As tissue identity and many comorbidities with increased virus susceptibility are characterized by differences in metabolism, we characterized ISG responses in cells grown in media known to favor either aerobic glycolysis (glucose) or oxidative phosphorylation (galactose supplementation). While these conditions over time had a varying impact on the expression of ISG RNAs, the differences were typically greater between treatments than between glucose/galactose. Interestingly, extended interferon-priming led to divergent expression of two ISG proteins: upregulation of IRF1 in IFN-γ/glucose and increased IFITM3 in galactose by IFN-α and IFN-γ. In agreement with a hardwired response, glucose/galactose regulation of interferon-γ induced IRF1 is conserved in unrelated mouse and cat cell types. In galactose conditions, proteasome inhibition restored interferon-γ induced IRF1 levels to that of glucose/interferon-γ. Glucose/interferon-γ decreased replication of the model poxvirus vaccinia at low MOI and high MOIs. Vaccinia replication was restored by IRF1 KO. In contrast, but consistent with differential regulation of IRF1 protein by glucose/galactose, WT and IRF1 KO cells in galactose media supported similar levels of vaccinia replication regardless of IFN-γ priming. Also associated with glucose/galactose is a seemingly second block at a very late stage in viral replication which results in reductions in herpes- and poxvirus titers but not viral protein expression. Collectively, these data illustrate a novel layer of regulation for the key ISG protein, IRF1, mediated by glucose/galactose and imply unappreciated subprograms embedded in the interferon response. In principle, such cellular circuitry could rapidly adapt immune responses by sensing changing metabolite levels consumed during viral replication and cell proliferation.

Inorganic nitrogen inhibits symbiotic nitrogen fixation through blocking NRAMP2-mediated iron delivery in soybean nodules

Symbiotic nitrogen fixation (SNF) in legume-rhizobia serves as a sustainable source of nitrogen (N) in agriculture. However, the addition of inorganic N fertilizers significantly inhibits SNF, and the underlying mechanisms remain not-well understood. Here, we report that inorganic N disrupts iron (Fe) homeostasis in soybean nodules, leading to a decrease in SNF efficiency. This disruption is attributed to the inhibition of the Fe transporter genes Natural Resistance-Associated Macrophage Protein 2a and 2b (GmNRAMP2a&2b) by inorganic N. GmNRAMP2a&2b are predominantly localized at the tonoplast of uninfected nodule tissues, affecting Fe transfer to infected cells and consequently, modulating SNF efficiency. In addition, we identified a pair of N-signal regulators, nitrogen-regulated GARP-type transcription factors 1a and 1b (GmNIGT1a&1b), that negatively regulate the expression of GmNRAMP2a&2b, which establishes a link between N signaling and Fe homeostasis in nodules. Our findings reveal a plausible mechanism by which soybean adjusts SNF efficiency through Fe allocation in response to fluctuating inorganic N conditions, offering valuable insights for optimizing N and Fe management in legume-based agricultural systems.

Metabolic reprogramming tips vaccinia virus infection outcomes by stabilizing interferon-γ induced IRF1.

Interferon (IFN) induced activities are critical, early determinants of immune responses and infection outcomes. A key facet of IFN responses is the upregulation of hundreds of mRNAs termed interferon-stimulated genes (ISGs) that activate intrinsic and cell-mediated defenses. While primary interferon signaling is well-delineated, other layers of regulation are less explored but implied by aberrant ISG expression signatures in many diseases in the absence of infection. Consistently, our examination of tonic ISG levels across uninfected human tissues and individuals revealed three ISG subclasses. As tissue identity and many comorbidities with increased virus susceptibility are characterized by differences in metabolism, we characterized ISG responses in cells grown in media known to favor either aerobic glycolysis (glucose) or oxidative phosphorylation (galactose supplementation). While these conditions over time had a varying impact on the expression of ISG RNAs, the differences were typically greater between treatments than between glucose/galactose. Interestingly, extended interferon-priming led to divergent expression of two ISG proteins: upregulation of IRF1 in IFN-γ/glucose and increased IFITM3 in galactose by IFN-α and IFN-γ. In agreement with a hardwired response, glucose/galactose regulation of interferon-γ induced IRF1 is conserved in unrelated mouse and cat cell types. In galactose conditions, proteasome inhibition restored interferon-γ induced IRF1 levels to that of glucose/interferon-γ. Glucose/interferon-γ decreased replication of the model poxvirus vaccinia at low MOI and high MOIs. Vaccinia replication was restored by IRF1 KO. In contrast, but consistent with differential regulation of IRF1 protein by glucose/galactose, WT and IRF1 KO cells in galactose media supported similar levels of vaccinia replication regardless of IFN-γ priming. Also associated with glucose/galactose is a seemingly second block at a very late stage in viral replication which results in reductions in herpes- and poxvirus titers but not viral protein expression. Collectively, these data illustrate a novel layer of regulation for the key ISG protein, IRF1, mediated by glucose/galactose and imply unappreciated subprograms embedded in the interferon response. In principle, such cellular circuitry could rapidly adapt immune responses by sensing changing metabolite levels consumed during viral replication and cell proliferation.

Understanding the Pathogenesis of Endothelial Injury Induced by Bluetongue Virus in Experimentally Infected Sheep

Bluetongue (BT) is an economically important contagious viral disease transmitted by insect vectors of Culicoides species affecting both domestic and wild ruminants caused by Genus Orbivirus of Reoviridae family. Present in vivo study for the first time conducted to understand the role of oxidative stress and acute phase proteins in the pathogenesis of endothelial injury in target blood vessels of BTV-4 infected sheep. Twelve sheep of either sex were divided into two groups of six animals each and infected intradermally at multiple sites in the neck region with 6×105.9 TCID50/ml of BTV-4 and animals of control group were inoculated with uninfected tissue culture fluid. Animals were sacrificed at 3, 7, 15, 28, 60, and 90 days post infection (dpi) and postmortem examination was conducted. Oxidative stress parameters like LPO, SOD, GPO and catalase in the blood vessels, hemolysate and serum were estimated. Oxidative stress parameter like LPO was upregulated and anti-oxidant parameters like SOD and GPO were downregulated in blood vessels homogenates, hemolysate and serum of BTV-4 infected animals than uninfected control. This shows the role of oxidative stress in causing endothelial cell damage during BTV infection. Acute phase proteins like CRP and fibrinogen in serum and plasma were estimated and found upregulated in BTV-4 infected animals, which shows that inflammatory changes during the disease causes vascular damage. Prothrombin time was estimated in plasma of BTV-4 infected animals and was prolonged in BTV infected animals indicating coagulation disorder. Increased oxidative stress and fibrinogen, and prolonged prothrombin time indicated the coagulation disorders induced by BTV infection by causing endothelial cell damage and severe vascular disease results in congestion, haemorrhages and edema in the blood vessels and other target organs in sheep.

Differential microRNA Expression Analysis in Patients with HPV-Infected Ovarian Neoplasms.

This study aimed to identify microRNAs (miRNAs) whose expression levels are altered by high-risk human papillomavirus (HR-HPV) infection in women with epithelial ovarian neoplasms. MiRNA expression was quantified by real-time polymerase chain reaction, while HR-HPV DNA was quantified using digital-droplet PCR. Analysis of 11 miRNAs demonstrated significantly lower hsa-miR-25-5p expression in HPV-infected compared to uninfected ovarian tissues (p = 0.0405), while differences in miRNA expression in corresponding serum were statistically insignificant. The expression of hsa-miR-218-5p in ovarian tumors was significantly higher in high-grade serous ovarian carcinoma (HGSOC) cases than in other neoplasms (p = 0.0166). In addition, hsa-miR-218-5p was significantly upregulated, whereas hsa-miR-191-5p was significantly downregulated in tissues with stage III/IV FIGO (p = 0.0009 and p = 0.0305, respectively). Using unsupervised clustering, we identified three unique patient groups with significantly varied frequencies of HPV16/18-positive samples and varied miRNA expression profiles. In multivariate analysis, high expression of hsa-miR-16-5p was an independent prognostic factor for poor overall survival (p = 0.0068). This preliminary analysis showed the changes in miRNA expression in ovarian neoplasms during HPV infection and those collected from HGSOCs or patients with advanced disease. This prospective study can provide new insights into the pathogenesis of ovarian neoplasms and host-virus interactions.

Splenic proteome profiling in response to Marek’s disease virus strain GX0101 infection

Marek’s disease virus (MDV) strain GX0101 was the first reported field strain of recombinant gallid herpesvirus type 2 (GaHV-2). However, the splenic proteome of MDV-infected chickens remains unclear. In this study, a total of 28 1-day-old SPF chickens were intraperitoneally injected with chicken embryo fibroblast (CEF) containing 2000 PFU GX0101. Additionally, a control group, consisting of four one-day-old SPF chickens, received intraperitoneal equal doses of CEF. Blood and various tissue samples were collected at different intervals (7, 14, 21, 30, 45, 60, and 90 days post-infection; dpi) for histopathological, real-time PCR, and label-free quantitative analyses. The results showed that the serum expressions of MDV-related genes, meq and gB, peaked at 45 dpi. The heart, liver, and spleen were dissected at 30 and 45 dpi, and their hematoxylin-eosin staining indicated that virus infection compromised the normal organizational structure at 45 dpi. Particularly, the spleen structure was severely damaged, and the lymphocytes in the white medulla were significantly reduced. Furthermore, liquid chromatography-mass spectrometry (LC-MS) and label-free techniques were used to analyze the difference in splenic proteome profiles of the experimental and control groups at 30 and 45 dpi. Proteomic analysis identified 1660 and 1244 differentially expressed proteins (DEPs) at 30 and 40 dpi, respectively, compared with the uninfected spleen tissues. According to GO analysis, these DEPs were involved in processes such as organelle organization, cellular component biogenesis, cellular component assembly, anion binding, small molecule binding, metal ion binding, cation binding, cytosol, nuclear part, etc. Additionally, KEGG analysis indicated that the following pathways were linked to MDV-induced inflammation, apoptosis, and tumor: Wnt, Hippo, AMPK, cAMP, Notch, TGF-β, PI3K-Akt, Rap1, Ras, Calcium, NF-κB, PPAR, cGMP-PKG, Apoptosis, VEGF, mTOR, FoxO, TNF, JAK-STAT, MAPK, Prion disease, T cell receptor, and B cell receptor. We finally screened 674 DEPs that were linked to MDV infection in spleen tissue. This study improves our understanding of the MDV response mechanism in the spleen.

Evaluating the Interaction between Arbuscular Mycorrhizal Fungi (AMF) and Plant Pathogens

The presence of arbuscular mycorrhizal fungi (AMF) in the rhizosphere has been shown to exert a substantial influence on plant disease resistance. The colonization of AMF induces a cascade of defense responses in plants, resulting in heightened resistance to pathogen invasion. The current study unveils the essential role of AMF in initiating systemic defense responses in plants. When plants undergo colonization by AMF, they initiate a variety of molecular and biochemical mechanisms that enhance their immune system. These systemic defense responses equip the plant with the ability to both withstand invading pathogens and ready neighboring uninfected tissues for potential future attacks. In addition, the establishment of AMF in the roots can induce physical alterations in the root architecture, thereby increasing the difficulty for pathogens to infiltrate and infect plants. While there have been notable advancements in understanding the overall impact of AMF colonization on plant disease resistance, further investigation is needed. Future investigations should delve into the mechanisms of AMF colonization on plant disease resistance, with a particular emphasis on elucidating the signaling pathways and biochemical factors that trigger systemic defense responses. This will offer valuable insights into the molecular foundation of plant immunity and aid in the development of precise disease management strategies. AMF can also serve as biocontrol agents for sustainable management of plant diseases, thereby decreasing dependency on chemical pesticides and encouraging environmentally-conscious practices. The efficiency of AMF colonization and the expression of plant defense responses can be significantly altered by environmental factors, such as soil conditions and nutrient availability. Comprehending these factors will facilitate the formulation of efficient management tactics to enhance biotic interactions in agricultural systems.

Cellular inflammatory response in the bovine uterus by Leptospira infection may be related to embryo death and subfertility

Bovine leptospirosis is a chronic disease that causes various reproductive disorders and consequent economic losses worldwide, particularly embryo death. Although Leptospira spp. has already been detected in the genital tract of cows, little is known about the uterine cellular immune response or the intrinsic factors that could contribute to that reproductive failure. In this context, the aim of this study was to assess the uterine cellular inflammatory response after the quantification of cytokine IL-6 in bovine uteri naturally infected with leptospires compared to uninfected. Our results demonstrated that uterine tissues infected with leptospires have higher levels of IL-6 compared to uninfected tissues (p < 0.001). It suggests that the presence of leptospires in the bovine uterus can induce a cellular inflammatory response, which may be related to embryo death and consequent subfertility.

Evaluating the Performance of Pathogen-Targeted Positron Emission Tomography Radiotracers in a Rat Model of Vertebral Discitis-Osteomyelitis.

Vertebral discitis-osteomyelitis (VDO) is a devastating infection of the spine that is challenging to distinguish from noninfectious mimics using computed tomography and magnetic resonance imaging. We and others have developed novel metabolism-targeted positron emission tomography (PET) radiotracers for detecting living Staphylococcus aureus and other bacteria in vivo, but their head-to-head performance in a well-validated VDO animal model has not been reported. We compared the performance of several PET radiotracers in a rat model of VDO. [11C]PABA and [18F]FDS were assessed for their ability to distinguish S aureus, the most common non-tuberculous pathogen VDO, from Escherichia coli. In the rat S aureus VDO model, [11C]PABA could detect as few as 103 bacteria and exhibited the highest signal-to-background ratio, with a 20-fold increased signal in VDO compared to uninfected tissues. In a proof-of-concept experiment, detection of bacterial infection and discrimination between S aureus and E coli was possible using a combination of [11C]PABA and [18F]FDS. Our work reveals that several bacteria-targeted PET radiotracers had sufficient signal to background in a rat model of S aureus VDO to be potentially clinically useful. [11C]PABA was the most promising tracer investigated and warrants further investigation in human VDO.

Wolbachia-mediated resistance to Zika virus infection in Aedes aegypti is dominated by diverse transcriptional regulation and weak evolutionary pressures.

A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the wMel strain of Wolbachia that are being released by the World Mosquito Program in Medellín, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of wMel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia-infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia. Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia-mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system.

Evaluation of a human mucosal tissue explant model for SARS-CoV-2 replication.

With the onset of COVID-19, the development of ex vivo laboratory models became an urgent priority to study host-pathogen interactions in response to the pandemic. In this study, we aimed to establish an ex vivo mucosal tissue explant challenge model for studying SARS-CoV-2 infection and replication. Nasal or oral tissue samples were collected from eligible participants and explants generated from the tissue were infected with various SARS-CoV-2 strains, including IC19 (lineage B.1.13), Beta (lineage B.1.351) and Delta (lineage B.1.617.2). A qRT-PCR assay used to measure viral replication in the tissue explants over a 15-day period, demonstrated no replication for any viral strains tested. Based on this, the ex vivo challenge protocol was modified by reducing the viral infection time and duration of sampling. Despite these changes, viral infectivity of the nasal and oral mucosa was not improved. Since 67% of the enrolled participants were already vaccinated against SARS-CoV-2, it is possible that neutralizing antibodies in explant tissue may have prevented the establishment of infection. However, we were unable to optimize plaque assays aimed at titrating the virus in supernatants from both infected and uninfected tissue, due to limited volume of culture supernatant available at the various collection time points. Currently, the reasons for the inability of these mucosal tissue samples to support replication of SARS-CoV-2 ex vivo remains unclear and requires further investigation.

Successful surgical treatment of infected aorto-femoral­ bifurcation prosthesis by its explantation and bifurcation bypass grafting outside the area of previous reconstruction. Clinical case description

Vascular graft infections are associated with a significant risk of mortality and morbidity and cost approximately $640 million per year in the United States. Clinical pattern depends on the time elapsed since the implantation and surgery location. A thorough collection of medical history and physical examination in combination with various imaging methods are often necessary to confirm a diagnosis. There are several treatment options for infected aortic grafts, including graft excision with extra-anatomic bypass grafting, in situ reconstruction or neo-aortoiliac system reconstruction. The treatment of infected endovascular aortic grafts is similar. In cases of infected peripheral shunts, graft preservation techniques may be used, but if these are not feasible, graft removal and revascularization through uninfected tissues are necessary. The authors discuss the diagnosis, general management and surgical approaches to the treatment of infected vascular grafts, as well as a clinical case of successful surgical management of an infected aortofemoral bifurcation prosthesis by its explantation and bifurcation bypass grafting outside the area of previous reconstruction. Keywords: graft infection, reconstructive surgery, re-intervention

A robust platform for integrative spatial multi-omics analysis to map immune responses to SARS-CoV-2 infection in lung tissues.

The SARS-CoV-2 (COVID-19) virus has caused a devastating global pandemic of respiratory illness. To understand viral pathogenesis, methods are available for studying dissociated cells in blood, nasal samples, bronchoalveolar lavage fluid and similar, but a robust platform for deep tissue characterization of molecular and cellular responses to virus infection in the lungs is still lacking. We developed an innovative spatial multi-omics platform to investigate COVID-19-infected lung tissues. Five tissue-profiling technologies were combined by a novel computational mapping methodology to comprehensively characterize and compare the transcriptome and targeted proteome of virus infected and uninfected tissues. By integrating spatial transcriptomics data (Visium, GeoMx and RNAScope) and proteomics data (CODEX and PhenoImager HT) at different cellular resolutions across lung tissues, we found strong evidence for macrophage infiltration and defined the broader microenvironment surrounding these cells. By comparing infected and uninfected samples, we found an increase in cytokine signalling and interferon responses at different sites in the lung and showed spatial heterogeneity in the expression level of these pathways. These data demonstrate that integrative spatial multi-omics platforms can be broadly applied to gain a deeper understanding of viral effects on cellular environments at the site of infection and to increase our understanding of the impact of SARS-CoV-2 on the lungs.

Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues.

Rapid detection of microbial-induced cellular changes during the course of an infection is critical to understanding pathogenesis and immunological homeostasis. In the last two decades, fluorescence imaging has received significant attention for its ability to help characterize microbial induced cellular and tissue changes in in vitro and in vivo settings. However, most of these methods rely on the covalent conjugation of large exogenous probes and detection methods based on intensity-based imaging. Here, we report a quantitative, intrinsic, label-free, and minimally invasive method based on two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM) for imaging 1,4-dihydro-nicotinamide adenine dinucleotide (NADH) metabolism of virally infected cells and tissue sections. To better understand virally induced cellular and tissue changes in metabolism we have used 2p-FLIM to study differences in NADH intensity and fluorescence lifetimes in HIV-1 infected cells and tissues. Differences in NADH fluorescence lifetimes are associated with cellular changes in metabolism and changes in cellular metabolism are associated with HIV-1 infection. NADH is a critical co-enzyme and redox regulator and an essential biomarker in the metabolic processes. Label-free 2p-FLIM application and detection of NADH fluorescence using viral infection systems are in their infancy. In this study, the application of the 2p-FLIM assay and quantitative analyses of HIV-1 infected cells and tissue sections reveal increased fluorescence lifetime and higher enzyme-bound NADH fraction suggesting oxidative phosphorylation (OxPhos) compared to uninfected cells and tissues. 2p-FLIM measurements improve signal to background, fluorescence specificity, provide spatial and temporal resolution of intracellular structures, and thus, are suitable for quantitative studies of cellular functions and tissue morphology. Furthermore, 2p-FLIM allows distinguishing free and bound populations of NADH by their different fluorescence lifetimes within single infected cells. Accordingly, NADH fluorescence measurements of individual single cells should provide necessary insight into the heterogeneity of metabolic activity of infected cells. Implementing 2p-FLIM to viral infection systems measuring NADH fluorescence at the single or subcellular level within a tissue can provide visual evidence, localization, and information in a real-time diagnostic or therapeutic metabolic workflow.

Integration of Human and Viral miRNAs in Epstein-Barr Virus-Associated Tumors and Implications for Drug Repurposing.

Epstein-Barr virus (EBV) is associated with several tumors, and has substantial relevance for public health. Therapeutics innovation for EBV-related disorders is much needed. In this context, miRNAs are noncoding RNA molecules that play vital roles in EBV infection. miRNA-Seq and RNA-Seq data for EBV-associated clinical samples and cell lines have been generated, but their detailed integrative analyses, and exploitation for drug repurposing against EBV are lacking. Hence, we identified and analyzed the differentially expressed miRNAs (DEmiRs) in EBV-infected cell lines (28) and infected (28) and uninfected human tissue (20) samples using an in-house pipeline. We found significantly enriched host miRNAs like hsa-mir-3651, hsa-mir-1248, and hsa-mir-29c-3p in EBV-infected samples from EBV-associated nasopharyngeal carcinoma and Hodgkin's lymphoma, among others. Furthermore, we also identified significantly enriched novel miRNAs such as hsa-mir-29c-3p, hsa-mir-3651, and hsa-mir-98-3p, which were not previously reported in EBV-related tumors. Differentially expressed mRNAs (DEMs) were identified in EBV-infected cell lines (21) and uninfected human tissue (14) samples. We predicted and selected 1572 DEMs (upregulated) that are targeted by 547 DEmiRs (downregulated). These were further classified into essential (870) and nonessential (702) genes. Moreover, a miRNA-mRNA network was developed for the hub miRNAs. Importantly, we used the DEMs during EBV latent infection types I, II, and III to identify the candidate drugs for repurposing: Glyburide, Levodopa, Nateglinide, and Stiripentol, among others. To the best of our knowledge, this is the first integrative analyses that identified DEmiRs and DEMs as potential therapeutic targets and predicted drugs as potential candidates for repurposing against EBV-related tumors.

Candidatus Liberibacter asiaticus: An important factor affecting bacterial community composition and Wolbachia titers in Asian citrus psyllid.

Endosymbionts play crucial roles in various physiological activities within insect hosts. The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an important vector for Candidatus Liberibacter asiaticus (CLas), a fatal pathogenic bacterial agent causing the disease Huanglongbing in the citrus industry. This study combines high-throughput sequencing of 16S ribosomal RNA amplicons to explore how CLas affects the bacterial community in different color morphs (blue, gray), genders, and tissues (cuticle, gut, mycetome, Malpighian tubule, ovary, and testis) of ACP. We found that there was no significant differences in the bacterial community diversity and CLas acquired ratio between the different color morphs and genders of ACP adults. However, acquiring CLas could promote the adult bacterial community's diversity and richness more than in the uninfected condition. The presence of CLas could increase the Wolbachia and unclassified_Enterobacteriaceae proportions more than in the uninfected condition. The bacterial community diversity in the CLas infected tissues of ovary and cuticle, was lower than the uninfected condition, but the richness of all tissues was not different between the infected and uninfected conditions. CLas could also change the bacterial structure in different tissues and make the bacterial relationship network simpler than it is in an uninfected condition. Furthermore, we used quantitative real-time PCR to assess the dynamic changes of Wolbachia in CLas uninfected and infected color morphs and tissues of ACP. The results showed that Wolbachia titers were significantly higher in CLas infected adults than in uninfected adults. In different tissues, the Wolbachia titers in the testis, ovary, and Malpighian tubule were higher than their uninfected counterparts. Our results provide essential knowledge for understanding the symbionts of the ACP and how CLas affects the bacterial community of the ACP.

Transcriptional profiling of defense responses to Botrytis cinerea infection in leaves of Fragaria vesca plants soil-drenched with β-aminobutyric acid.

Grey mold caused by the necrotrophic fungal pathogen Botrytis cinerea can affect leaves, flowers, and berries of strawberry, causing severe pre- and postharvest damage. The defense elicitor β-aminobutyric acid (BABA) is reported to induce resistance against B. cinerea and many other pathogens in several crop plants. Surprisingly, BABA soil drench of woodland strawberry (Fragaria vesca) plants two days before B. cinerea inoculation caused increased infection in leaf tissues, suggesting that BABA induce systemic susceptibility in F. vesca. To understand the molecular mechanisms involved in B. cinerea susceptibility in leaves of F. vesca plants soil drenched with BABA, we used RNA sequencing to characterize the transcriptional reprogramming 24 h post-inoculation. The number of differentially expressed genes (DEGs) in infected vs. uninfected leaf tissue in BABA-treated plants was 5205 (2237 upregulated and 2968 downregulated). Upregulated genes were involved in pathogen recognition, defense response signaling, and biosynthesis of secondary metabolites (terpenoid and phenylpropanoid pathways), while downregulated genes were involved in photosynthesis and response to auxin. In control plants not treated with BABA, we found a total of 5300 DEGs (2461 upregulated and 2839 downregulated) after infection. Most of these corresponded to those in infected leaves of BABA-treated plants but a small subset of DEGs, including genes involved in 'response to biologic stimulus', 'photosynthesis' and 'chlorophyll biosynthesis and metabolism', differed significantly between treatments and could play a role in the induced susceptibility of BABA-treated plants.

Activity of a hypochlorous acid-producing electrochemical bandage as assessed with a porcine explant biofilm model.

The activity of a hypochlorous acid-producing electrochemical bandage (e-bandage) in preventing methicillin-resistant Staphylococcus aureus infection (MRSA) infection and removing biofilms formed by MRSA was assessed using a porcine explant biofilm model. e-Bandages inhibited S. aureus infection (p = 0.029) after 12 h (h) of exposure and reduced 3-day biofilm viable cell counts after 6, 12, and 24 h exposures (p = 0.029). Needle-type microelectrodes were used to assess HOCl concentrations in explant tissue as a result of e-bandage treatment; toxicity associated with e-bandage treatment was evaluated. HOCl concentrations in infected and uninfected explant tissue varied between 30 and 80 µM, decreasing with increasing distance from the e-bandage. Eukaryotic cell viability was reduced by an average of 71% and 65% in fresh and day 3-old explants, respectively, when compared to explants exposed to nonpolarized e-bandages. HOCl e-bandages are a promising technology that can be further developed as an antibiotic-free treatment for wound biofilm infections.

A case on novel treatment protocol for difficult to treat foot myiasis

Foot myiasis is a rare condition, with only a few reported cases and no treatment consensus. We proposed a conservative and unique treatment approach with VAC dressing method. The patient administered in the hospital with presentation of deep tissue injury from a sharp stone, from 3 months back which was maggot infested. The patient had approached local physicians before been admitted in our facility. The wound was severe with maggot infestation. The patient was non diabetic with no other comorbid conditions. We had the aim of infection free wound with faster rate of healing, which was fulfilled by the novel treatment protocol using vacuum-assisted closure (VAC®) dressing method. Culture sensitivity tests ruled out infection and maggot infestation. The wound was closed surgically after healthy uninfected granulation tissue confirmed with culture sensitivity report. No recurrence of maggot or any other infection were found. The protocol performed was safe and effective in critical case of myiasis.