Mechanism of Heterogenization of Dirhodium Catalysts: Insights from DFT Calculations.

Dirhodium(II) complexes such as [Rh2(TFA)4] bound to a functionalized mesoporous SBA-15 carrier material have proven to be valuable candidates for heterogeneous catalysis in the field of pharmaceutical synthesis. However, the mechanistic steps of immobilization by linker molecules containing carboxyl or amine functionalities remain the subject of discussion. Here we present a theoretical study of possible mechanistic binding pathways for the [Rh2(TFA)4] complex through model representations of synthetically investigated linkers, namely n-butylamine and n-butyric acid. Experimentally proposed intermediates of the immobilization process are investigated and analyzed by density functional theory calculations to gain insights into structural properties and the influence of solvation. An evaluation of the thermodynamic data for all identified intermediates allowed distinguishing between two possible reaction pathways that are characterized by a first axial complexation of either n-butyric acid or n-butylamine. In agreement with results from NMR spectroscopy, singly or doubly n-butylamine-fixated complexes were found to present possible immobilization products. Initial binding through a carboxy-functionalized linker is proposed as the most favorable reaction pathway for the formation of the mixed linker pattern [Rh2(TFA)3]·(n-butylamine)·(n-butyrate). The linkers n-butyric acid and n-butyrate, respectively, are found to exhibit an unaltered binding affinity to the dirhodium complex despite their protonation states, indicating invariance to the acidic environment unlike an immobilization by n-butylamine. These results present a theoretical framework for the rationalization of observed product distributions while also providing inspiration and guidance for the preparation of functionalized heterogeneous SBA-15/dirhodium catalyst systems.

Japanese Encephalitis Virus-induced let-7a/b interacted with the NOTCH-TLR7 pathway in microglia and facilitated neuronal death via caspase activation.

MicroRNAs (miRNAs) released from the activated microglia upon neurotropic virus infection may exacerbate the neuronal damage. Here, we identified let-7a and let-7b (let-7a/b) as one of the essential miRNAs over-expressed upon Japanese Encephalitis virus (JEV) infection and released in the culture supernatant of the JEV-infected microglial cells through extracellular vesicles. The let-7a/b was previously reported to modulate inflammation in microglial cells through Toll-like receptor 7 (TLR7) pathways; although their role in accelerating JEV pathogenesis remain unexplored. Therefore, we studied the role of let-7a/b in modulating microglia-mediated inflammation during JEV infection and investigated the effect of let-7a/b-containing exosomes on primary neurons. To this end, we examined let-7a/b and NOTCH signaling pathway in TLR7 knockdown (KD) mice. We observed that TLR7 KD or inhibition of let-7a/b suppressed the JEV-induced NOTCH activation possibly via NF-κB dependent manner and subsequently, attenuated JEV-induced TNFα production in microglial cells. Furthermore, exosomes secreted from let-7a/b over-expressed microglia when transferred to uninfected mice brain induced caspase activation. Exosomes secreted from virus-infected or let-7a/b over-expressed microglia when co-incubated with mouse neuronal (Neuro2a) cells or primary cortical neurons also facilitated caspase activation leading to neuronal death. Thus, our results provide evidence for the multifaceted role of let-7a/b miRNAs in JEV pathogenesis. Let-7a/b can interact with TLR7 and NOTCH signaling pathway and enhance TNFα release from microglia. On the other hand, the exosomes secreted by JEV-infected microglia can activate caspases in uninfected neuronal cells which possibly contribute to bystander neuronal death. Cover Image for this issue: doi: 10.1111/jnc.14506.

Structural Insights into Peptides Bound to the Surface of Silica Nanopores.

The structure and surface functionalization of biologically relevant silica-based hybrid materials was investigated by 2D solid-state NMR techniques combined with dynamic nuclear polarization (DNP). This approach was applied to a model system of mesoporous silica, which was modified through in-pore grafting of small peptides by solid-phase peptide synthesis (SPPS). To prove the covalent binding of the peptides on the surface, DNP-enhanced solid-state NMR was used for the detection of 15 N NMR signals in natural abundance. DNP-enhanced heterocorrelation experiments with frequency switched Lee-Goldburg homonuclear proton decoupling (1 H-13 C and 1 H-15 N CP MAS FSLG HETCOR) were performed to verify the primary structure and configuration of the synthesized peptides. 1 H FSLG spectra and 1 H-29 Si FSLG HETCOR correlation spectra were recorded to investigate the orientation of the amino acid residues with respect to the silica surface. The combination of these NMR techniques provides detailed insights into the structure of amino acid functionalized hybrid compounds and allows for the understanding for each synthesis step during the in-pore SPPS.

MiR-155 induction in microglial cells suppresses Japanese encephalitis virus replication and negatively modulates innate immune responses.

BACKGROUND: Microglial cells, which are resident macrophages of the central nervous system, play important roles in immune responses and pathogenesis. Japanese encephalitis virus (JEV) is a neurotropic virus that infects microglial cells in brain. Several microRNAs including miR-155 and miR-146a play an important role in defining the microglia inflammatory profile. In this study, we have investigated the effect of miR-155 and miR-146a modulation on JEV infection as well as innate immune responses in human microglial cells. METHODS: In vitro studies were performed in JEV-infected human microglial CHME3 cells. miR-155 or miR-146a were overexpressed and total RNA and protein were extracted following JEV-infection. Expression of genes involved in innate immune responses was studied by PCR array, quantitative real-time PCR (qPCR), western blot and Fluorescence activated cell sorter (FACS). JEV replication was monitored by studying the viral RNA by qPCR, protein by western blot, and titres by plaque assay. RESULTS: Overexpression of miR-155 in CHME3 cells resulted in significantly reduced JEV replication whereas miR-146a overexpression had an insignificant effect. Additionally, interferon regulatory factor 8 (IRF8) and complement factor H (CFH) were induced during JEV infection; however, this induction was attenuated in miR-155 overexpressing cells following JEV infection. Further, JEV-induced NF-κB regulated downstream gene expression was attenuated. Interestingly, an increased level of CD45, a negative regulator of microglia activation and a reduced phosphorylated-Signal Transducers and Activators of Transcription (p-STAT1) expression was observed in miR-155 overexpressing cells upon JEV infection. CONCLUSION: Induction of miR-155 in human microglial cells may negatively modulate JEV-induced innate immune gene expression and may have a beneficial role in limiting JEV replication in human microglial cells.

Diagnostic, prognostic, and predictive importance of neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and CA125 in endometrial hyperplasia and carcinoma.

BACKGROUND: Inflammation has an important role in the progression of endometrial carcinoma. AIMS: The aim of this study is to find the association between neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and CA125 in endometrial hyperplasia and endometrial carcinoma. The study also focuses on the association of CA125, NLR, and PLR with histopathological parameters in endometrial carcinoma that are of prognostic importance. MATERIALS AND METHODS: This is a prospective cross-sectional study on 57 biopsy-proven cases of endometrial hyperplasia and carcinoma conducted over a period of two years. The NLR, PLR, and CA125 were calculated and recorded in all the 57 cases. RESULTS: The 57 cases were divided into three groups: endometrial hyperplasia without atypia group which included 36 cases, endometrial atypical hyperplasia group which included 10 cases, and the endometrial carcinoma group which included 11 cases. Comparison was done between the groups, and parity, NLR, PLR, and CA125 were found to be significant, but patient age and postmenopausal status were not significant. NLR, PLR, and CA125 were found to increase with higher grade, pT-stage, and nodal stage for the endometrial carcinoma cases. CONCLUSION: NLR, PLR, and CA125 were marginally increased or normal in the case of endometrial hyperplasia without atypia and endometrial atypical hyperplasia, while they were significantly increased in endometrial carcinoma, and also correlated with an increase in grade, pT-stage, and nodal stage. Hence, these can be considered for additional screening as diagnostic, prognostic, and predictive markers in case of abnormal uterine bleeding with endometrial pathology.

The evaluation of frequency and predictors of delirium and its short-term and long-term outcomes in hospitalized older adults'.

BACKGROUND: Delirium is a common complication in hospitalized older adults with multifactorial etiology and poor health outcomes. AIM: To determine the frequency and predictors of delirium and its short-term and long-term outcomes in hospitalized older adults. METHODS: A prospective observational study was performed in patients aged ≥60 years consecutively admitted to geriatric ward. Potential risk factors were assessed within 24 hours of hospital admission. Delirium screening was performed on admission and daily thereafter throughout the hospital stay using Confusion Assessment Method (CAM). Patients were followed up at 1-year post-discharge. RESULTS: The study included 200 patients with mean age 73.1 ± 8.83 years. Incidence and prevalence rate of delirium were 5% and 20% respectively. Multivariable regression analysis revealed emergency admission (OR= 5.12 (1.94-13.57), p=0.001), functional dependency (Katz index of Independence in Activities of Daily Living (Katz-ADL) score <5) 2 weeks before admission (OR= 3.08 (1.30-7.33), p=0.011) and more psychopathological symptoms (higher Brief Psychiatric Rating Scale (BPRS) total score) (OR=1.12 (1.06-1.18), p=0.001) to be independently associated with delirium. Patients in delirium group had significantly high in-hospital mortality (OR= 5.02 (2.12-11.8), p=0.001) and post-discharge mortality (HR= 2.02 (1.13-3.61), p=0.017) and functional dependency (Katz-ADL score <5) (OR= 5.45 (1.49-19.31), p=0.01) at 1-year follow up. CONCLUSION: Delirium is quite frequent in geriatric inpatients and is associated with high in-hospital and post-discharge mortality risk and long-term functional dependency. Emergency admission, pre-hospitalization functional dependency, and more general psychopathological symptoms are independently associated factors. Hence, earliest identification and treatment with early implementation of rehabilitation services is warranted.

Artificial MicroRNA-Mediated Inhibition of Japanese Encephalitis Virus Replication in Neuronal Cells.

Artificial microRNA (amiRNA)-mediated inhibition of viral replication has recently gained importance as a strategy for antiviral therapy. In this study, we evaluated the benefit of using the amiRNA vector against Japanese encephalitis virus (JEV). We designed three single amiRNA sequences against the consensus sequence of 3' untranslated region (3'UTR) of JEV and tested their efficacy against cell culture-grown JEV Vellore strain (P20778) in neuronal cells. The binding ability of three amiRNAs on 3'UTR region was tested in vitro in HEK293T cells using a JEV 3'UTR tagged with luciferase reporter vector. Transient transfection of amiRNAs was nontoxic to cells as evident from the MTT assay and caused minimal induction in interferon-stimulated gene expression. Furthermore, our result suggested that transient expression of two amiRNAs (amiRNA #1 and amiRNA #2) significantly reduced intracellular viral RNA and nonstructural 1 (NS1) protein, as well as diminished infectious viral particle release up to 95% in the culture supernatant as evident from viral plaque reduction assay. Overall, our results indicated that RNA interference based on amiRNAs targeting viral conserved regions at 3'UTR was a useful approach for improvements of nucleic acid inhibitors against JEV.

Differential Expression and Significance of Circulating microRNAs in Cerebrospinal Fluid of Acute Encephalitis Patients Infected with Japanese Encephalitis Virus.

Changes in circulating microRNAs (miRNAs) in the cerebrospinal fluid (CSF) have been associated with different neurological diseases. Here, we presented results of a pilot study aimed at determining the feasibility of detecting miRNAs in the CSF of Japanese Encephalitis virus (JEV) infected individuals with acute encephalitis syndrome (AES). We demonstrated the circulating miRNA profile in CSF of acute encephalitis patients infected with JEV. Using a quantitative real-time PCR-based miRNA array, we examined the level of 87 miRNAs expressed in human exosomes isolated from CSF. Subsequently, correlation between cytokine level and miRNAs expression in CSF samples was examined. In this study, we identified and validated the upregulated expression of three miRNAs, miR-21-5p, miR-150-5p, and miR-342-3p that were specifically circulated in CSF of acute encephalitis patients infected with JEV. CSF miR-21-5p, miR-150-5p, and miR-342-3p expressions were also elevated in infected mice brain. However, the expression pattern of these miRNAs differed in neuronal cells, microglial cells, and the exosome derived from JEV-infected cell culture supernatant. Interestingly, neuronal cells infected with vaccine strain (SA-14-14) did not lead to any upregulation of these three miRNAs. Further, miR-150-5p expression was found to be negatively correlated(r = -0.5279, p = 0.016) with TNFα level. Pathway analysis of putative target genes of these miRNAs indicated involvement of TGF-ß, NGF, axon guidance, and MAPK signaling pathways in JEV/AES patients. This study for the first time represents the circulating miRNA in CSF of AES patients and identified the upregulated miRNAs in JEV-infected patients and offers the basis for future investigation.

Dynamic changes in global microRNAome and transcriptome reveal complex miRNA-mRNA regulated host response to Japanese Encephalitis Virus in microglial cells.

Microglia cells in the brain play essential role during Japanese Encephalitis Virus (JEV) infection and may lead to change in microRNA (miRNA) and mRNA profile. These changes may together control disease outcome. Using Affymetrix microarray platform, we profiled cellular miRNA and mRNA expression at multiple time points during viral infection in human microglial (CHME3) cells. In silico analysis of microarray data revealed a phased pattern of miRNAs expression, associated with JEV replication and provided unique signatures of infection. Target prediction and pathway enrichment analysis identified anti correlation between differentially expressed miRNA and the gene expression at multiple time point which ultimately affected diverse signaling pathways including Notch signaling pathways in microglia. Activation of Notch pathway during JEV infection was demonstrated in vitro and in vivo. The expression of a subset of miRNAs that target multiple genes in Notch signaling pathways were suppressed and their overexpression could affect JEV induced immune response. Further analysis provided evidence for the possible presence of cellular competing endogenous RNA (ceRNA) associated with innate immune response. Collectively, our data provide a uniquely comprehensive view of the changes in the host miRNAs induced by JEV during cellular infection and identify Notch pathway in modulating microglia mediated inflammation.