Search

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered an ongoing global pandemic, necessitating rapid and accurate diagnostic tools to monitor emerging variants and preparedness for the next outbreak. This study introduces a multidisciplinary approach combining Fourier Transform Infrared (FTIR) microspectroscopy and Machine learning to comprehensively characterize and strain-type SARS-CoV-2 variants. FTIR analysis of pharyngeal swabs from different pandemic waves revealed distinct vibrational profiles, particularly in nucleic acid and protein vibrations. The spectral wavenumber range between 1150 and 1240 cm -1 was identified as the classification marker, distinguishing Healthy (noninfected) and infected samples. Machine learning algorithms, with neural networks exhibiting superior performance, successfully classified SARS-CoV-2 variants with a remarkable accuracy of 98.6%. Neural networks were also able to identify and differentiate a small cohort infected with influenza A variants, H1N1 and H3N2, from SARS-CoV-2-infected and Healthy samples. FTIR measurements further show distinct red shifts in vibrational energy and secondary structural alterations in the spike proteins of more transmissible forms of SARS-CoV-2 variants, providing experimental validation of the computational data. This integrated approach presents a promising avenue for rapid and reliable SARS-CoV-2 variant identification, enhancing our understanding of viral evolution and aiding in diagnostic advancements, particularly for an infectious disease with unknown etiology.

Extracellular matrix diseases like fibrosis are elusive to diagnose early on, to avoid complete loss of organ function or even cancer progression, making early diagnosis crucial. Imaging the matrix densities of proteins like collagen in fixed tissue sections with suitable stains and labels is a standard for diagnosis and staging. However, fine changes in matrix density are difficult to realize by conventional histological staining and microscopy as the matrix fibrils are finer than the resolving capacity of these microscopes. The dyes further blur the outline of the matrix and add a background that bottlenecks high-precision early diagnosis of matrix diseases. Here we demonstrate the multiple signal classification method-MUSICAL-otherwise a computational super-resolution microscopy technique to precisely estimate matrix density in fixed tissue sections using fibril autofluorescence with image stacks acquired on a conventional epifluorescence microscope. We validated the diagnostic and staging performance of the method in extracted collagen fibrils, mouse skin during repair, and pre-cancers in human oral mucosa. The method enables early high-precision label-free diagnosis of matrix-associated fibrotic diseases without needing additional infrastructure or rigorous clinical training., (© 2024. The Author(s).)

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes., (© 2024. The Author(s).)

Digital dermatitis (DD) is a skin disease in cattle characterized by painful inflammatory ulcerative lesions in the feet, mostly associated with local colonization by Treponema spp., including Treponema phagedenis . The reason why most DD lesions remain actively inflamed and progress to chronic conditions despite antibiotic treatment remains unknown. Herein, we show an abundant infiltration of proinflammatory (CD14 high CD16 low ) monocytes/macrophages in active DD lesions, a skin response that was not mitigated by topical treatment with oxytetracycline. The associated bacterium, T. phagedenis , isolated from DD lesions in cattle, when injected subcutaneously into mice, induced abscesses with a local recruitment of Ly6G + neutrophils and proinflammatory (Ly6C high CCR2 + ) monocytes/macrophages, which appeared at infection onset (4 days post challenge) and persisted for at least 7 days post challenge. When exploring the ability of macrophages to regulate inflammation, we showed that bovine blood-derived macrophages challenged with live T. phagedenis or its structural components secreted IL-1β via a mechanism dependent on the NLRP3 inflammasome. This study shows that proinflammatory characteristics of monocytes/macrophages and neutrophils dominate active non-healing ulcerative lesions in active DD, thus likely impeding wound healing after antibiotic treatment., Competing Interests: The authors declare no conflict of interest.

Ex vivo gene correction with CRISPR-Cas9 and a recombinant adeno-associated virus serotype 6 (rAAV6) in autologous hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD) has now entered early-phase clinical investigation. To facilitate the progress of CRISPR-Cas9/rAAV6 genome editing technology, we analyzed the molecular changes in key reagents and cellular responses during and after the genome editing procedure in human HSPCs. We demonstrated the high stability of rAAV6 to serve as the donor DNA template. We assessed the benefit of longer HSPC pre-stimulation in terms of increased numbers of edited cells. We observed that the p53 pathway was transiently activated, peaking at 6 h, and resolved over time. Notably, we revealed a strong correlation between p21 mRNA level and rAAV6 genome number in cells and beneficial effects of transient inhibition of p53 with siRNA on genome editing, cell proliferation, and cell survival. In terms of potential immunogenicity, we found that rAAV6 capsid protein was not detectable, while a trace amount of residual Cas9 protein was still detected at 48 h post-genome editing. We believe this information will provide important insights for future improvements of gene correction protocols in HSPCs., Competing Interests: M.H.P. holds equity in CRISPR Tx, holds equity and serves on the SAB of Allogene Tx and Ziopharm Therapeutics, on the board of directors of Graphite Bio, and as an advisor to Versant Ventures., (© 2023 The Author(s).)

Incorporation of more than one noncanonical amino acid (ncAA) within a single protein endows the resulting construct with multiple useful features such as augmented molecular recognition or covalent cross-linking capabilities. Herein, for the first time, we demonstrate the incorporation of two chemically distinct ncAAs into proteins biosynthesized in Saccharomyces cerevisiae . To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Readthrough efficiency at TGA was modulated by factors including the local nucleotide environment, gene deletions related to the translation process, and the identity of the suppressor tRNA. These observations facilitated systematic investigation of dual ncAA incorporation in both intracellular and yeast-displayed protein constructs, where we observed efficiencies up to 6% of wild-type protein controls. The successful display of doubly substituted proteins enabled the exploration of two critical applications on the yeast surface─(A) antigen binding functionality and (B) chemoselective modification with two distinct chemical probes through sequential application of two bioorthogonal click chemistry reactions. Lastly, by utilizing a soluble form of a doubly substituted construct, we validated the dual incorporation system using mass spectrometry and demonstrated the feasibility of conducting selective labeling of the two ncAAs sequentially using a "single-pot" approach. Overall, our work facilitates the addition of a 22nd amino acid to the genetic code of yeast and expands the scope of applications of ncAAs for basic biological research and drug discovery.

The efficient monitoring and early detection of viruses may provide essential information about diseases. In this work, we have highlighted the interaction between DNA and a two-dimensional (2D) metal oxide for developing biosensors for further detection of viral infections. Spectroscopic measurements have been used to probe the efficient interactions between single-stranded DNA (ssDNA) and the 2D metal oxide and make them ideal candidates for detecting viral infections. We have also used fully atomistic molecular dynamics (MD) simulation to give a microscopic understanding of the experimentally observed ssDNA-metal oxide interaction. The adsorption of ssDNA on the inorganic surface was found to be driven by favourable enthalpy change, and 5'-guanine was identified as the interacting nucleotide base. Additionally, the in silico assessment of the conformational changes of the ssDNA chain during the adsorption process was also performed in a quantitative manner. Finally, we comment on the practical implications of these developments for sensing that could help design advanced systems for preventing virus-related pandemics.

The standard diagnosis of prostate cancer is accomplished by the identification of cytomorphological deviations in biopsied tissues while immunohistochemistry is used to resolve the equivocal cases. Accumulating evidence favors the concept that epithelial-to-mesenchymal transition (EMT) is a stochastic process composed of multiple intermediate states instead of a single binary switch. Despite its significant role in promoting cancer aggressiveness, the current tissue-based risk stratification tools do not include any of the EMT phenotypes as a metric. As a proof-of-concept, the present study analyzes the temporal progression of EMT in transforming growth factor-beta (TGF-β) treated PC3 cells encompassing multifarious characteristics such as morphology, migration and invasion, gene expression, biochemical fingerprint, and metabolic activity. Our multimodal approach reinstates EMT plasticity in TGF-β treated PC3 cells. Further, it highlights that mesenchymal transition is accompanied by discernible changes in cellular morphometry and molecular signatures particularly in the range of 1800-1600 cm -1 and 3100-2800 cm -1 of Fourier-transformed infrared (FTIR) spectra signifying Amide III and lipid, respectively. Investigation of attenuated total reflectance (ATR)-FTIR spectra of extracted lipids from PC3 cell populations undergoing EMT identifies changes in stretching vibration at FTIR peaks at 2852, 2870, 2920, 2931, 2954, and 3010 cm -1 characteristics of fatty acids and cholesterol. Chemometric analysis of these spectra indicates that the level of unsaturation and acyl chain length of fatty acid coregister with differential epithelial/mesenchymal states of TGF-β treated PC3 cells. Observed changes in lipids also correlate with cellular nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide dihydrogen (FADH2) levels and mitochondrial oxygen consumption rate. In summary, our study establishes that morphological and phenotypic traits of epithelial/mesenchymal variants of PC3 cells concur with their respective biochemical and metabolic properties. It also underscores that spectroscopic histopathology has a definitive potential to refine the diagnosis of prostate cancer reckoning its molecular and biochemical heterogeneities., (© 2023 Wiley Periodicals LLC.)

Introduction: Calves are highly susceptible to gastrointestinal infection with Cryptosporidium parvum ( C. parvum ), which can result in watery diarrhea and eventually death or impaired development. With little to no effective therapeutics, understanding the host's microbiota and pathogen interaction at the mucosal immune system has been critical to identify and test novel control strategies., Methods: Herein, we used an experimental model of C. parvum challenge in neonatal calves to describe the clinical signs and histological and proteomic profiling of the mucosal innate immunity and microbiota shifts by metagenomics in the ileum and colon during cryptosporidiosis. Also, we investigated the impact of supplemental colostrum feeding on C. parvum infection., Results: We showed that C. parvum challenged calves experienced clinical signs including pyrexia and diarrhea 5 days post challenge. These calves showed ulcerative neutrophil ileitis with a proteomic signature driven by inflammatory effectors, including reactive oxygen species and myeloperoxidases. Colitis was also noticed with an aggravated mucin barrier depletion and incompletely filled goblet cells. The C. parvum challenged calves also displayed a pronounced dysbiosis with a high prevalence of Clostridium species (spp.) and number of exotoxins, adherence factors, and secretion systems related to Clostridium spp. and other enteropathogens, including Campylobacter spp., Escherichia sp., Shigella spp., and Listeria spp. Daily supplementation with a high-quality bovine colostrum product mitigated some of the clinical signs and modulated the gut immune response and concomitant microbiota to a pattern more similar to that of healthy unchallenged calves., Discussion: C. parvum infection in neonatal calves provoked severe diarrheic neutrophilic enterocolitis, perhaps augmented due to the lack of fully developed innate gut defenses. Colostrum supplementation showed limited effect mitigating diarrhea but demonstrated some clinical alleviation and specific modulatory influence on host gut immune responses and concomitant microbiota., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gamsjäger, Cirone, Schluessel, Campsall, Herik, Lahiri, Young, Dufour, Sapountzis, Otani, Gomez, Windeyer and Cobo.)

Cathelicidin peptides secreted by leukocytes and epithelial cells are microbicidal but also regulate pathogen sensing via toll-like receptors (TLRs) in the colon by mechanisms that are not fully understood. Herein, analyses with the attaching/effacing pathogen Citrobacter rodentium model of colitis in cathelicidin-deficient (Camp-/-) mice, and colonic epithelia demonstrate that cathelicidins prevent apoptosis by sustaining post-transcriptional synthesis of a TLR adapter, toll-interacting protein (TOLLIP). Cathelicidins induced phosphorylation-activation of epidermal growth factor receptor (EGFR)-kinase, which phosphorylated-inactivated miRNA-activating enzyme Argonaute 2 (AGO2), thus reducing availability of the TOLLIP repressor miRNA-31. Cathelicidins promoted stability of TOLLIP protein via a proteosome-dependent pathway. This cathelicidin-induced TOLLIP upregulation prevented apoptosis in the colonic epithelium by reducing levels of caspase-3 and poly (ADP-ribose) polymerase (PARP)-1 in response to the proinflammatory cytokines, interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα). Further, Camp-/- colonic epithelial cells were more susceptible to apoptosis during C. rodentium infection than wild-type cells. This antiapoptotic effect of cathelicidins, maintaining epithelial TOLLIP protein in the gut, provides insight into cathelicidin's ability to regulate TLR signaling and prevent exacerbated inflammation., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Outer membrane vesicles (OMVs), also called as bacterial membrane vesicles (BMVs), are secreted by many Gram-negative bacterial pathogens. These nanoscale vesicles traffic discrete arrays of virulence factors that can often induce complex pathologies far from the infection sites. The OMVs of P. aeruginosa, often regarded as the gold standard of BMVs are known to traffic a battery of specific small MW alkyl-quinolones (AQs). These AQs function like primordial hormones by modulating intra-species and inter-species bacterial interactions. They can also perform cross-kingdom signaling with the human host and directly exacerbate pathogenesis. The discrete isotopic signatures of AQs enjoy potential in the mass spectrometry-based diagnosis P. aeruginosa infections. Matrix-free laser desorption/ionization mass spectrometry (LDI-MS) presents a robust, cost-effective platform to fit this demand. We describe a LDI-MS system using inert ceramic filters that performs dual role of single-step enrichment of OMVs and matrix-free ionization/identification of AQs in situ., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background In 2020 WHO declared the coronavirus disease 2019 (COVID-19) outbreak as a global pandemic. To flatten the curve of infection, a nationwide lockdown was declared by the Indian government. All the schools and colleges were shut for an indefinite period. Like all other streams, medical education also got severely hampered. Adapting themselves to the changing environment, teachers started using different teaching-learning methods and media to get across to the students. The objective of the research was to study the perception of medical students towards online teaching during the COVID pandemic. Methods A descriptive cross-sectional study was conducted by the distribution of a pre-validated online questionnaire to medical students of West Bengal. From the collected data, relevant statistical averages and census domains were calculated. The chi-square test was done and assessed with a p≤0.05 significance level. Results A significant increase was noted in the time spent by students on various online teaching activities (p<0.05). Video tutorials, e.g., YouTube, were ranked as the most effective mode (17.2%), followed by live tutorials via Microsoft Teams, etc. (8.9%). A significant number (30.2%) of students strongly favoured online teaching over face-to-face teaching. Major challenges of online learning cited by students were internet connectivity issues (79.8%) followed by family distraction (37.9 %) and inconvenient timing of the classes (20.1%). Conclusion Our study highlighted the benefits, disadvantages and barriers to online learning from the perspective of undergraduate medical education in India. Even though the online mode of teaching was found to be beneficial in the context of the COVID-19 pandemic, it cannot be used as an absolute substitution for face-to-face teaching in the given context., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Rao(Lahiri) et al.)

Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in Escherichia coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remain limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNA CUA MaPyl support the incorporation of ncAAs into proteins produced in Saccharomyces cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the activity of previously reported Methanosarcina mazei , Methanosarcina barkeri , and chimeric M. mazei and M. barkeri PylRSs. Using S. cerevisiae RJY100 and pairing these PylRSs with the M. mazei tRNA CUA , we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS/tRNA CUA MaPyl to the orthogonal translation machinery toolkit in S. cerevisiae potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the β-globin gene ( HBB ). Ex vivo β-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34 + cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The choice of tissue fixation is critical for preserving the morphology and biochemical information of tissues. Fragile oral tissues with lower tensile strength are challenging to process for histological applications as they are prone to processing damage, such as tissue tear, wrinkling, and tissue fall-off from slides. This leads to loss of morphological information and unnecessary delay in experimentation. In this study, we have characterized the new PAXgene tissue fixation system on oral buccal mucosal tissue of cancerous and normal pathology for routine histological and immunohistochemical applications. We aimed to minimize the processing damage of tissues and improve the quality of histological experiments. We also examined the preservation of biomolecules by PAXgene fixation using FTIR microspectroscopy. Our results demonstrate that the PAXgene-fixed tissues showed significantly less tissue fall-off from slides. Hematoxylin and Eosin staining showed comparable morphology between formalin-fixed and PAXgene-fixed tissues. Good quality and slightly superior immunostaining for cancer-associated proteins p53 and CK5/6 were observed in PAXgene-fixed tissues without antigen retrieval than formalin-fixed tissues. Further, FTIR measurements revealed superior preservation of glycogen, fatty acids, and amide III protein secondary structures in PAXgene-fixed tissues. Overall, we present the first comprehensive evaluation of the PAXgene tissue fixation system in oral tissues. This study concludes that the PAXgene tissue fixation system can be applied to oral tissues to perform diagnostic molecular pathology experiments without compromising the quality of the morphology or biochemistry of biomolecules.

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory-Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62 - total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-β-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.

Cathelicidins are small, cationic peptides produced by macrophages with protective effects against infection although their involvement in phagocytosis is not fully understood. This study demonstrates that fewer macrophages were recruited in mice genetically deficient in cathelicidin (Camp -/- ) during acute Escherichia coli-induced peritonitis and those macrophages had impaired phagocytosis. These defects seem due to endogenous functions of murine cathelicidin (CRAMP) as phagocytosis was not improved by synthetic human cathelicidin (LL-37) in a murine phagocytic cell line. This knowledge contributes to understanding the function of cathelicidins in the recruitment and function of phagocytic cells and differential roles between endogenous and exogenous cathelicidins.

Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of aβ T cells and B cells has been shown to reduce the risk of acute and chronic graft-versus-host disease, thus allowing the use of haploidentical donors which expands the population of recipients in whom allogeneic hematopoietic stem cell transplantation can be used. Leukemic relapse, however, remains a challenge. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including those in the central nervous system. We hypothesized that by engineering the donor aβ T cells that are removed from the graft by genome editing to express a CD19-specific chimeric antigen receptor, while simultaneously inactivating the T-cell receptor, we could create a therapy that enhances the anti-leukemic efficacy of the stem cell transplant without increasing the risk of graft-versus-host disease. Using genome editing with Cas9 ribonucleoprotein and adeno-associated virus serotype 6, we integrated a CD19-specific chimeric antigen receptor inframe into the TRAC locus. More than 90% of cells lost T-cell receptor expression, while >75% expressed the chimeric antigen receptor. The initial product was further purified with less than 0.05% T-cell receptorpositive cells remaining. In vitro, the chimeric antigen receptor T cells efficiently eliminated target cells and produced high cytokine levels when challenged with CD19+ leukemia cells. In vivo, the gene-modified T cells eliminated leukemia without causing graft-versus-host disease in a xenograft model. Gene editing was highly specific with no evidence of off-target effects. These data support the concept that the addition of aβ T-cell-derived, genome-edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of aβ T-celldepleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.

SARS-CoV-2, a novel coronavirus, was first identified in Wuhan, China in December 2019. The rapid spread of the virus worldwide prompted the World Health Organization (WHO) to declare COVID-19 a pandemic in March 2020. COVID-19 discontinuing's a global health crisis. Approximately 80% of the patients infected with SARS-CoV-2 display undetectable to mild inflammation confined in the upper respiratory tract. In remaining patients, the disease turns into a severe form affecting almost all major organs predominantly due to an imbalance of innate and adaptive arms of host immunity. The purpose of the present review is to narrate the virus's invasion through the system and the host's reaction. A thorough discussion on disease severity is also presented regarding the behavior of the host's immune system, which gives rise to the cytokine storm particularly in elderly patients and those with comorbidities. A multifaceted yet concise description of molecular aspects of disease progression and its repercussion on biochemical and immunological features in infected patients is tabulated. The summary of pathological, clinical, immunological, and molecular accounts discussed in this review is of theranostic importance to clinicians for early diagnosis of COVID-19 and its management.

Digital dermatitis (DD) causes lameness in cattle with substantial negative impact on sustainability and animal welfare. Although several species of Treponema bacteria have been isolated from various DD stages, their individual or synergistic roles in the initiation or development of lesions remain largely unknown. The objective of this study was to compare effects of the three most common Treponema species isolated from DD lesions in cattle ( T. phagedenis, T. medium and T. pedis ), both as individual and as mixed inoculations, in a murine abscess model. A total of 10 9 or 5 × 10 8 Treponema spp. were inoculated subcutaneously, and produced abscess was studied after 7 days post infection. There were no synergistic effects when two or three species were inoculated together; however, T. medium produced the largest abscesses, whereas those produced by T. phagedenis were the smallest and least severe. Treponema species were cultured from skin lesions at 7 days post infection and, additionally, from the kidneys of some mice (2/5), confirming systemic infection may occur. Taken together, these findings suggest that T. medium and T. pedis may have more important roles in DD lesion initiation and development than T. phagedenis .

The 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02 (GtGBE, EC 2.4.1.18) does not possess the thermostability required by modified starch industry. To increase its thermostability, a rational design strategy was used to introduce additional salt bridges into GtGBE. The strategy involved in mutation of individual residues to form "local" two-residue salt bridges. Accordingly, five of local salt bridges (Q231R-D227, Q231K-D227, T339E-K335, T339D-K335, and I571D-R569 mutants) were separately introduced into GtGBE. The half-times of these mutants at 60 °C were 17% to 51% longer than that of wild-type. Subsequently, these two-residue salt bridges were extended to form salt bridge networks (Q231R/K-D227-D131H, T339D/E-K335-I291H, and I571D-R569-R617H mutants). Among these mutants, except I571D-R569-R617H, the half-times of Q231R/K-D227-D131H, T339D/E-K335-I291H mutants at 60 °C were 15%, 17%, 21% and 17% longer than those of the corresponding two-residue salt bridges, respectively. The results showed that design and introduction of salt bridges improves enzyme thermostability in GtGBE., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The supplementation of 50 ppm dosed silver nanoparticles (AgNPs) as a disinfectant in broilers drinking water was investigated to examine their growth performance, blood serum biochemistry, and organ histology in the case group, compared to the control. The growth performance parameters, such as water intake, feed intake, and body weight were recorded 6 times, each in an interval of 7 days, over a period of 42 days. At the end of each 42 days, the blood and major organs of the 1 case boiler out of 75 and 1 control broiler out of 75 were collected in random. The procedure was repeated 3 sets one after another, each consisting 42 day intervening period. The liver enzyme, lipid profile, glucose level, organ histology, and concentration of AgNPs in liver, spleen, heart, and small intestine were determined. The obtained results show that the growth performance of the case broilers was significantly higher than the control section ( p  < 0.05). However, in all the three sets the changes in lipid profile, liver enzyme, and glucose level of the case broilers were not statistically significantly different compared to the control ( p  > 0.05). The histology of liver, kidney, heart, spleen, and small intestine of broilers has not shown any damages to the cells as compared to the control samples. In the case samples, the highest concentration of AgNPs was observed in the small intestine (5.44 µg/g) followed by liver (4.32 µg/g), kidney (3.94 µg/g), heart (3.82 µg/g), and spleen (3.49 µg/g). The present study concludes that the administering 50 ppm AgNPs of average 15 nm size in the poultry drinking water was found safe for consumption as well as for growth enhancing, due to better bird comfort., Competing Interests: Conflict of interestThe authors do not have conflict of interest in this present work., (© King Abdulaziz City for Science and Technology 2020.)

Digital dermatitis (DD), a common ulcerative disease of the bovine foot causing lameness and reducing productivity and animal welfare, is associated with infection by spirochete Treponema bacteria. Topical tetracycline, the most common treatment, has inconsistent cure rates; therefore, new therapeutic options are needed. We compared effects of topical oxytetracycline and vitamin D 3 on innate immunity in DD-affected skin. Cows with active DD lesions were treated topically with oxytetracycline or vitamin D 3 and skin biopsies were collected from lesions. Tissue samples were examined histologically, transcriptional expression of pro-inflammatory cytokines, Toll-like receptors (TLRs), and host defense peptides assessed, and the presence of specific treponeme species determined. Effects of treatments at a mechanistic level were studied in a human keratinocyte model of treponeme infection. Oxytetracycline promoted hyperplastic scab formation in ulcerated DD lesions and decreased transcriptional expression of Cxcl-8 (neutrophil chemoattractant). Oxytetracycline also reduced numbers of Treponema phagedenis and T. pedis and enhanced Tlr2 mRNA expression. Vitamin D 3 did not modify expression of cytokines or Tlrs, or bacterial loads, but enhanced transcription of tracheal antimicrobial peptide (Tap), a key bovine β-defensin. Combing oxytetracycline and vitamin D 3 provides complementary clinical benefits in controlling DD through a combination of antimicrobial, immunomodulatory, and pro-healing activities.

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic diarrheic intestinal infections in domestic and wild ruminants (paratuberculosis or Johne's disease) for which there is no effective treatment. Critical in the pathogenesis of MAP infection is the invasion and survival into macrophages, immune cells with ability to carry on phagocytosis of microbes. In a search for effective therapeutics, our objective was to determine whether human cathelicidin LL-37, a small peptide secreted by leuckocytes and epithelial cells, enhances the macrophage ability to clear MAP infection. In murine (J774A.1) macrophages, MAP was quickly internalized, as determined by confocal microscopy using green fluorescence protein expressing MAPs. Macrophages infected with MAP had increased transcriptional gene expression of pro-inflammatory TNF-α, IFN-γ, and IL-1β cytokines and the leukocyte chemoattractant IL-8. Pretreatment of macrophages with synthetic LL-37 reduced MAP load and diminished the transcriptional expression of TNF-α and IFN-γ whereas increased IL-8. Synthetic LL-37 also reduced the gene expression of Toll-like receptor (TLR)-2, key for mycobacterial invasion into macrophages. We concluded that cathelicidin LL-37 enhances MAP clearance into macrophages and suppressed production of tissue-damaging inflammatory cytokines. This cathelicidin peptide could represent a foundational molecule to develop therapeutics for controlling MAP infection.

Cell types differentiated from induced pluripotent stem cells (iPSCs) are frequently arrested in their development program, more closely resembling a fetal rather than an adult phenotype, potentially limiting their utility for downstream clinical applications. The fetal phenotype of iPSC-derived dendritic cells (ipDCs) is evidenced by their low expression of MHC class II and costimulatory molecules, impaired secretion of IL-12, and poor responsiveness to conventional maturation stimuli, undermining their use for applications such as immune-oncology. Given that iPSCs display an epigenetic memory of the cell type from which they were originally derived, we investigated the feasibility of reprogramming adult DCs to pluripotency to determine the impact on the phenotype and function of ipDCs differentiated from them. Using murine bone marrow-derived DCs (bmDCs) as proof of principle, we show here that immature DCs are tractable candidates for reprogramming using non-integrating Sendai virus for the delivery of Oct4, Sox2, Klf4, and c-Myc transcription factors. Reprogramming efficiency of DCs was lower than mouse embryonic fibroblasts (MEFs) and highly dependent on their maturation status. Although control iPSCs derived from conventional MEFs yielded DCs that displayed a predictable fetal phenotype and impaired immunostimulatory capacity in vitro and in vivo, DCs differentiated from DC-derived iPSCs exhibited a surface phenotype, immunostimulatory capacity, and responsiveness to maturation stimuli indistinguishable from the source DCs, a phenotype that was retained for 15 passages of the parent iPSCs. Our results suggest that the epigenetic memory of iPSCs may be productively exploited for the generation of potently immunogenic DCs for immunotherapeutic applications., (©2019 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2019.)

Background: In the context of early diagnosis and prevention of oral cancer, precise assessment of malignant potentiality of the oral potentially malignant disorders, particularly oral submucous fibrosis (OSF) is crucial. Till date, the assessment of malignant potentiality suffers from predictive ambiguity due to the lack of precision in the gold standard techniques. This can be addressed by integrating heuristic domain knowledge with quantitative analysis., Aim: The aim of this study is to propose an index for enhancing accuracy in malignant potentiality evaluation., Materials and Methods: The present study analyzes important histomorphometric attributes (epithelial thickness, basal cell nuclear size, nuclear-to-cytoplasmic area ratio of basal cells, chromaticity of basal cell nucleus, thickness of basement membrane, ratio of vasculature in juxta-epithelial connective tissue [i.e., area covered by blood vessels/total area], collagen density in the lamina propria) of oral mucosa in dysplastic and nondysplastic OSF in association with relevant oncopathological appreciations (weightage of different features as suggested by oral pathologists) toward proposing a "Malignant Potentiality Index" (MPI)., Results: Analysis of variance and notch box plot analysis depict statistically significant differences ( P < 0.0001) in the histopathological features among different study groups (normal oral mucosa, OSF without dysplasia, OSF with dysplasia). Histopathological observation of one OSF patient with calculated MPI is shown., Conclusion: This newly proposed diagnostic cum prognostic decision-making parameter, the "MPI" may bring a value addition to the conventional diagnostic gold standard., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Journal of Oral and Maxillofacial Pathology.)

The contribution of newly designed salt bridges to protein stabilization remains controversial even today. In order to solve this problem, we investigated salt bridges from two aspects: spatial distribution and evolutionary characteristics of salt bridges. Firstly, we analyzed spatial distribution of salt bridges in proteins, elucidating the basic requirements of forming salt bridges. Then, from an evolutionary point of view, the evolutionary characteristics of salt bridges as well as their neighboring residues were investigated in our study. The results demonstrate that charged residues appear more frequently than other neutral residues at certain positions of sequence even under evolutionary pressure, which are able to form electrostatic interactions that could increase the evolutionary stability of corresponding amino acid regions, enhancing their importance to stability of proteins. As a corollary, we conjectured that the newly designed salt bridges with more contribution to proteins, not only, are qualified spatial distribution of salt bridges, but also, are needed to further increase the evolutionary stability of corresponding amino acid regions. Based on analysis, the 8 mutations were accordingly constructed in the 1,4-α-glucan branching enzyme (EC 2.4.1.18, GBE) from Geobacillus thermoglucosidans STB02, of which 7 mutations improved thermostability of GBE. The enhanced thermostability of 7 mutations might be a result of additional salt bridges on residue positions that at least one of amino acids positions is conservative, improving their contribution of stabilization to proteins.

The metabolites of the mammalian kynurenine (KYN) pathway are generated from a branch of tryptophan metabolic pathway. The latter generates 3-hydroxykynurenine (3-HK), kynurenic acid (KYNA), quinolinic acid (QUIN), and picolinic acid (PIC) which are all strongly neuroactive, often with dramatically contrasting functional outcomes. Whereas KYNA and PIC are neuroprotective, 3-HK and QUIN are potently neurotoxic and attributed in major neurodegenerative diseases like schizophrenia, Alzheimer's disease, Huntington's disease, bipolar disorder, and depression. It is increasingly evident that the ratio(s) between the neurotoxic and neuroprotective metabolites may help predict the manifestations of disease vs. health. Therefore high-throughput platforms for determining the relative levels of these kynurenine metabolites in biofluids offer considerable potential. Current analytical tools for studying KYN pathway include assays of branching enzymes, PCR, immunoanalysis, and LCMS. None of these offer high-throughput, cost-effective analyses suited for clinical or drug-screening applications. In this report a laser desorption ionization mass spectrometry (LDI-MS) method is described using SBA-15 mesoporous silica. The system allows fast, high-resolution quantitation of neurotoxic kynurenines using targeted metabolomics on conventional MALDI platforms.

Background: Flow cytometry, with its high throughput nature, combined with the ability to measure an increasing number of cell parameters at once can surpass the throughput of prevalent genomic and metagenomic approaches in the study of microbiomes. Novel computational approaches to analyze flow cytometry data will result in greater insights and actionability as compared to traditional tools used in the analysis of microbiomes. This paper is a demonstration of the fruitfulness of machine learning in analyzing microbial flow cytometry data generated in anaerobic microbiome perturbation experiments., Results: Autoencoders were found to be powerful in detecting anomalies in flow cytometry data from nanoparticles and carbon sources perturbed anaerobic microbiomes but was marginal in predicting perturbations due to antibiotics. A comparison between different algorithms based on predictive capabilities suggested that gradient boosting (GB) and deep learning, i.e. feed forward artificial neural network with three hidden layers (DL) were marginally better under tested conditions at predicting overall community structure while distributed random forests (DRF) worked better for predicting the most important putative microbial group(s) in the anaerobic digesters viz. methanogens, and it can be optimized with better parameter tuning. Predictive classification patterns with DL (feed forward artificial neural network with three hidden layers) were found to be comparable to previously demonstrated multivariate analysis. The potential applications of this approach have been demonstrated for monitoring the syntrophic resilience of the anaerobic microbiomes perturbed by synthetic nanoparticles as well as antibiotics., Conclusion: Machine learning can benefit the microbial flow cytometry research community by providing rapid screening and characterization tools to discover patterns in the dynamic response of microbiomes to several stimuli., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Intratumor heterogeneity is increasingly recognized as a major factor impacting diagnosis and personalized treatment of cancer. We characterized stochastic phenotype switching as a mechanism contributing to intratumor heterogeneity and malignant potential of liver cancer. Clonal analysis of primary tumor cell cultures of a human sarcomatoid cholangiocarcinoma identified different types of self-propagating subclones characterized by stable (keratin-7-positive or keratin-7-negative) phenotypes and an unstable phenotype consisting of mixtures of keratin-7-positive and keratin-7-negative cells, which lack stem cell features but may reversibly switch their phenotypes. Transcriptome sequencing and immunohistochemical studies with the markers Zeb1 and CD146/MCAM demonstrated that switching between phenotypes is linked to changes in gene expression related but not identical to epithelial-mesenchymal transition. Stochastic phenotype switching occurred during mitosis and did not correlate with changes in DNA methylation. Xenotransplantation assays with different cellular subclones demonstrated increased tumorigenicity of cells showing phenotype switching, resulting in tumors morphologically resembling the invasive component of primary tumor and metastasis., Conclusion: Our data demonstrate that stochastic phenotype switching contributes to intratumor heterogeneity and that cells with a switching phenotype have increased malignant potential. (Hepatology 2017)., (© 2017 The Authors. Hepatology published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.)

Chronic intoxication of mice with the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to morphological and metabolic changes closely resembling steatohepatitis, a severe form of metabolic liver disease in humans. Since human steatohepatitis (both the alcoholic and non-alcoholic type) is characterized by reduced expression of PPARα and disturbed lipid metabolism we investigated the role of this ligand-activated receptor in the development of DDC-induced liver injury. Acute DDC-intoxication was accompanied by early significant downregulation of Pparα mRNA expression along with PPARα-controlled stress-response and lipid metabolism genes that persisted in the chronic stage. Administration of the specific PPARα agonist fenofibrate together with DDC prevented the downregulation of PPARα-associated genes and also improved the stress response of Nrf2-dependent redox-regulating genes. Moreover, oxidative stress and inflammation were strongly reduced by DDC/fenofibrate co-treatment. In addition, fenofibrate prevented the disruption of hepatocyte intermediate filament cytoskeleton and the formation of Mallory-Denk bodies at late stages of DDC intoxication. Our findings show that, like in human steatohepatitis, PPARα is downregulated in the DDC model of steatohepatitis-like hepatocellular damage. Its downregulation and the pathomorphologic features of steatohepatitis are prevented by co-administration of fenofibrate.

Digital dermatitis (DD) commonly associated with Treponema spp. infection is a prevalent infectious bovine foot disease characterized by ulcerative and necrotic lesions. Lesions associated with DD are often classified using the M-stage scoring system, with M0 indicating healthy heel skin and M4 indicating chronic lesions. Current treatments utilizing antimicrobials or chemical footbaths are often ineffective and rarely cure DD lesions. Understanding the function of the innate immune response in the pathogenesis of DD will help to identify novel therapeutic approaches. In this study, the expression of the local innate host defense peptides cathelicidins and β-defensins was investigated in cows with DD and associated with the presence of treponemes and inflammatory reactions. Samples from active ulcerative DD lesions (M2) had considerable epidermal neutrophilic infiltration and increased gene expression of β-defensin tracheal antimicrobial peptides compared to control skin. Samples from acute lesions also had elevated local Cxcl-8 and TLR4 gene expression and abundant treponemes as identified by direct visualization, immunohistochemistry, and culture. Conversely, the anti-inflammatory peptide IL-10 was elevated in skin from chronic (M4) lesions, whereas bovine cathelicidin myeloid antimicrobial peptide 28 (Bmap-28) was increased in skin from oxytetracycline-treated M2 lesions. Experiments using cultured human keratinocytes challenged with Treponema spp. isolated from clinical cases of bovine DD showed that structural products from treponemes are able to initiate the innate immune response, in part through TLR2 signaling. These findings indicate that neutrophil influx, Cxcl-8, and β-defensin are key markers of active DD. Cathelicidins and IL-10 seem important in response to treatment or during the chronic proliferative stages of the disease.

Reverse turns are solvent-exposed motifs in proteins that are crucial in nucleating β-sheets and drive the protein folding. The solvent-exposed nature makes reverse turns more amenable to chemical modifications than α-helices or β-sheets towards modulating the stability of re-engineered proteins. Here, we utilize van der Waals repulsive forces in tuning the steric restraint at the reverse turn. The steric restraint induced upon N -methylation of the i +1- i +2 amide bond at the reverse turn results in well-folded and stable β-sheets in aqueous solution at room temperature. The developed superactive turn inducing motif is tolerant to a wide variety of functional groups present on coded amino acids making the designed turn fully compatible with bioactive loops in proteins. We demonstrate that the steric restraint and the functional groups at the reverse turn act in synergy to modulate the folding of re-engineered β-sheets. Introduction of the turn motifs onto a three-stranded β-sheet protein, Pin 1 WW domain, resulted in various analogs showing a cooperative two-state transition with thermal stability ( T M ) ranging from 62 °C to 82 °C. Despite modulating the stability of Pin 1 variants by ∼2.8 kcal mol -1 (ΔΔ G f ), the native fold in all the protein variants was found to be unperturbed. This structural stability is brought about by conformational preorganization at the engineered reverse turn that results in strong intramolecular hydrogen bonds along the three dimensional structure of the protein. Thus, this simple loop engineering strategy via two amino acid substitution provides us a "toolkit" to modulate the stability of β-sheet containing peptides and proteins in aqueous solution that will greatly expand the scope of de novo protein and foldamer design.

The dispersive optical activity of a homoconjugated bicyclic diene, (R)-methylene norbornene (R-MNB), was interrogated under complementary vapor-phase and solution-phase conditions to elucidate the structural/electronic provenance of its unusual chiroptical signatures and to explore the marked influence of environmental perturbations. The intrinsic (isolated-molecule) values of specific rotation measured at 355 and 633 nm (1623.5 ± 5.5 and 390.4 ± 3.7 deg dm -1 (g/mL) -1 ) were found to be factors of 3.9 and 2.1 smaller in magnitude than analogous quantities obtained for the kindred enone, (R)-norbornenone (R-NBO), reflecting, in part, the loss of prominent magnetic-dipole contributions from the C═O moiety and the exclusion of electron delocalization from the oxygen lone pairs. The wavelength-resolved rotatory powers of R-MNB were enhanced dramatically (by ∼40% on average) upon dissolution in any of the four common solvents targeted by the present study (acetonitrile, di-n-butylether, cyclohexane, and chloroform), yet displayed only a slight dependence on the exact nature of the surrounding liquid (±2.7% variation from the mean at 589.3 nm). Quantum-chemical calculations built upon the linear-response frameworks of density-functional theory and coupled-cluster theory were enlisted to interpret experimental results, with the substantial effects incurred by nonspecific solvation phenomena being explored through use of polarizable continuum models and bulk property-response relationships. Aside from enumerating the varied quality of agreement attained between computational predictions and polarimetric measurements, these efforts have found that refractive-index correlations, akin to those embodied in the venerable (albeit often discounted) Lorentz local-field correction, afford a viable means of linking the chiroptical behavior of R-MNB across phases.

Outer membrane vesicles (OMVs) are secreted by all Gram-ve pathogens. These nano-scale delivery vehicles contain discrete arrays of prokaryotic pathogenic determinants, including a family of low molecular weight (MW) lipidic quorum signaling alkyl-quinolones (AQs). These are synthesized from β-keto-fatty acids and function like primordial lipidic hormones, which regulate numerous pathogenic factors both inter-species and intra-species. Significantly, AQs can also directly exacerbate pathogenesis by cross-kingdom signaling with the host immune, metabolic, and other systems. In Pseudomonas aeruginosa more than 50 AQs are reported; many with pathogenic mechanisms that are largely unknown. Some of these AQs are exclusively associated with OMVs. Accurate characterization of these OMV-AQs may reveal novel mechanism of diseases and Pseudomonas aeruginosa presents an ideal model. Matrix-free laser desorption/ionization mass spectrometry (LDI-MS) technologies enjoy unique advantages in mass spectrometry (MS)-based imaging and low MW analysis. We report single-step isolation of Pseudomonas aeruginosa OMV on inert ceramic filters and high-resolution mass spectrometry (HRMS) analysis of AQs vesicle in situ.

Few topics in regenerative medicine have inspired such impassioned debate as the immunogenicity of cell types and tissues differentiated from pluripotent stem cells. While early predictions suggested that tissues derived from allogeneic sources may evade immune surveillance altogether, the pendulum has since swung to the opposite extreme, with reports that the ectopic expression of a few developmental antigens may prompt rejection, even of tissues differentiated from autologous cell lines. Here we review the evidence on which these contradictory claims are based in order to reach an objective assessment of the likely magnitude of the immunological challenges ahead. Furthermore, we discuss how the inherent properties of pluripotent stem cells may inform strategies for reducing the impact of immunogenicity on the future ambitions of regenerative medicine.

Xenobiotics exposure increases endoplasmic reticulum (ER) proliferation and cytochrome P-450 (CYP) induction to sustain metabolic requirements. Whether autophagy is essential for the removal of excess ER and CYP and whether an autophagy receptor is involved in this process in mammals remains elusive. In this study, we show that autophagy is induced in mouse livers after withdrawal of the hepatic mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Although isolated autophagosomes, autolysosomes, and lysosomes from mouse livers after withdrawal of TCPOBOP contained ER proteins, those in control mouse livers did not. Liver-specific Atg5 knockout mice had higher basal hepatic ER content that was further increased and sustained after withdrawal of TCPOBOP compared with wild-type mice. In addition to regulating ER degradation, our results also suggest that autophagy plays a role in regulating the homeostasis of hepatic CYP because blocking autophagy led to increased CYP2B10 accumulation either at the basal level or following TCPOBOP withdrawal. Furthermore, we found that the autophagy receptor protein sequestosome 1 (SQSTM1)/p62 is associated with the ER. After withdrawal of TCPOBOP, p62 knockout mice had increased ER content in the liver compared with wild-type mice. These results suggest that p62 may act as an autophagy receptor for the autophagic removal of excess ER in the mouse liver. Taken together, our results indicate that autophagy is important for the removal of excess ER and hepatic CYP enzymes in mouse livers, a process associated with the autophagy receptor protein p62., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background & Aims: Keratins (K) constitute the epithelial intermediate filaments. Among them, K7/K19 are widely used markers of the regenerative liver response termed ductular reaction (DR) that consists of activated biliary epithelial cells (BECs) and hepatic progenitor cells (HPCs) and correlates with liver disease severity. In the present study we aimed to characterize K23 in the liver., Methods: We analyzed the expression and localization of K23 in the digestive system under basal conditions as well as in various human and mouse liver diseases/stress models. Cell culture studies were used to study factors regulating K23 expression., Results: In untreated mice, K23 was restricted to biliary epithelia. It was (together with K7/K19) markedly upregulated in three different DR/cholestatic injury models, i.e., multidrug resistance protein 2 (Mdr2) knockouts, animals treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine or subjected to bile duct ligation. K23 levels correlated with the DR marker Fn14 and immunofluorescence staining showed a distinct co-localization with K7/K19. In chronic human liver disease, K23 expression increased in patients with a more prominent inflammation/fibrosis. A dramatic upregulation (>200times) was observed in patients with acute liver failure (ALF) and end-stage primary biliary cholangitis (PBC). Patients with alcoholic liver cirrhosis displayed increased K23 serum levels. In primary hepatocytes as well as hepatobiliary cell lines, treatment with TNF-related weak inducer of apoptosis (TWEAK), and the type I acute phase inducer interleukin (IL)-1β but not the type II inducer IL-6 elevated K23 expression., Conclusions: K23 represents a specific, stress-inducible DR marker, whose levels correlate with liver disease severity. K23 may represent a useful non-invasive DR marker., Lay Summary: Ductular reaction represents a basic response to liver injury and correlates with liver disease severity. Our study identifies K23 as a novel ductular reaction marker in mice and humans., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Mallory-Denk bodies (MDBs) are hepatocytic protein aggregates found in steatohepatitis and several other chronic liver diseases as well as hepatocellular carcinoma. MDBs are mainly composed of phosphorylated keratins and stress protein p62/Sequestosome-1 (p62), which is a common component of cytoplasmic aggregates in a variety of protein aggregation diseases. In contrast to the well-established role of keratins, the role of p62 in MDB pathogenesis is still elusive. We have generated total and hepatocyte-specific p62 knockout mice, fed them with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce MDBs and allowed the mice to recover from DDC intoxication on a standard diet to investigate the role of p62 in MDB formation and elimination. In the absence of p62, smaller, granular and less distinct MDBs appeared, which failed to mature to larger and compact inclusions. Moreover, p62 deficiency impaired the binding of other proteins such as NBR1 and Hsp25 to MDBs and altered the cellular defense mechanism by downregulation of Nrf2 target genes. Upon recovery from DDC intoxication on a standard diet, there was an enhanced reduction of p62-deficient MDBs, which was accompanied by a pronounced decrease in ubiquitinated proteins. Our data provide strong evidence that keratin aggregation is the initial step in MDB formation in steatohepatitis-related mouse models. Interaction of p62 with keratin aggregates then leads to maturation i.e., enlargement and stabilization of the MDBs as well as recruitment of other MDB-associated proteins.

There is a lack of functional group diversity in the reverse turn motifs nucleating a β-sheet conformation in designed peptides, proteins and foldamers. The majority of these sequences consist of d-Pro-l-Pro, d-Pro-Gly or Asn-Gly as the turn inducing motif restricting their biological application and physicochemical modulation. In this report, for the first time we elucidate that N -methylation of heterochiral amino acids in linear peptides nucleates β-sheet conformation without the necessity of having a ring or covalent constraint at the reverse turn. Our results show that d-Pro can be conveniently substituted by any other N -methylated d-amino acid followed by an N -methylated l-amino acid or sarcosine to adopt a βII' turn inducing the β-sheet folding. Furthermore, we reveal that a single amino acid either at the i + 1 or i + 2 site of the reverse turn can modulate the right-handed twist, which eventually dictates the extent of the foldedness of the β-hairpin.

Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The dispersive optical activity of (R)-(-)-glycidyl methyl ether (R-GME) has been interrogated under ambient vapor-phase and solution-phase conditions, with quantum-chemical analyses built on density functional (B3LYP and CAM-B3LYP) and coupled-cluster (CCSD) implementations of linear-response theory exploited to interpret experimental findings. Inherent flexibility of the heavy atom skeleton leads to nine low-lying structural isomers that possess distinct chiroptical and physicochemical properties, as evinced by marked changes in the magnitude and the sign of rotatory powers observed in various media. These species are interconverted by independent motion along two large-amplitude torsional coordinates and are stabilized differentially by interaction with the surroundings, thereby reapportioning their relative contributions to the collective response evoked from a thermally equilibrated ensemble. The intrinsic behavior exhibited by isolated (vapor-phase) R-GME molecules was calculated through use of both conformer-averaging and restricted vibrational-averaging procedures, the former affording moderately good agreement with measurements of optical rotatory dispersion (ORD) and the latter providing strong evidence for sizable effects arising from vibrational degrees of freedom. A similar conformer-averaging ansatz based on the polarizable-continuum model (PCM) for implicit solvation was deployed to elucidate R-GME specific-rotation parameters acquired for six dilute solutions. This approach gave reasonable predictions for sodium D-line (589.3 nm) experiments performed in the extremes of solvent polarity represented by cyclohexane and acetonitrile but failed to reproduce the overall shape of ORD profiles and suggested more complex processes might be involved in the case of an aromatic medium.

The National Health Interview Survey, conducted by the National Center for Health Statistics, is designed to provide reliable design-based estimates for a wide range of health-related variables for national and four major geographical regions of the USA. However, state-level or substate-level estimates are likely to be unreliable because they are based on small sample sizes. In this paper, we compare the efficiency of different area-level models in estimating smoking prevalence for the 50 US states and the District of Columbia. Our study is based on survey data from the 2008 National Health Interview Survey in conjunction with a number of potentially related auxiliary variables obtained from the American Community Survey, an ongoing large complex survey conducted by the US Census. A major portion of this study is devoted to the investigation of several methods for estimating survey sampling variances needed to implement an area-level hierarchical model. Based on our findings, a hierarchical Bayesian method that uses a survey-adjusted random sampling variance model to capture the complex survey sampling variability appears to be somewhat superior to the other considered area-level models in accounting for small sample behavior of estimated survey sampling variances. Several diagnostic procedures are presented to compare the proposed methods., (Copyright © 2014 John Wiley & Sons, Ltd.)

Measurements and calculations of specific rotation are indispensable for the characterization of chiral molecules and are now performed routinely. However, the factors that determine the magnitude of this property are still not well-understood. The anomalously large specific rotation of (1S,4S)-norbornenone, an outstanding puzzle for over three decades, offers the chance to examine these factors in detail. The present work provides an explanation for the unusual behavior of this molecule in terms of interactions between chemical groups and electronic excited-state transition properties by means of ab initio density functional theory and coupled cluster theory calculations. We show that one can focus on the first excited state and examine the relative orientation of its electric and magnetic transition dipole moments. The contribution of the two transition moments of this electronic state to the specific rotation in a sum-over-states formalism reveals a constructive interaction that is possible only when the two chromophores in norbornenone (C═O and C═C) are in-plane and pointing away from each other. This is due to a small but non-negligible charge transfer between the chromophores and is consistent with recent results from Autschbach's group [Moore et al., J. Chem. Theory Comput. 2012, 8, 4336-4346]. The analysis in this work improves our understanding of this fundamental property of chiral molecules and may help in the design of other molecules with large specific rotation.

The lack of knowledge about molecular pathology of uterine sarcomas with a representation of 3-7% of all malignant uterine tumors prevents the establishment of effective therapy protocols. Here, we explored advanced therapeutic options to the previously discovered antitumorigenic effects of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) by combined treatment with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L). In addition, we investigated the uterine sarcoma cell lines, MES-SA and ESS-1, regarding the underlying molecular mechanisms of SAHA and TRAIL-induced apoptosis and their resistance towards TRAIL. Compared to single SAHA or TRAIL treatment, the combination of SAHA with TRAIL led to complete cell death of both tumor cell lines after 24 to 48 hours. In contrast to single SAHA treatment, apoptosis occured faster and was more pronounced in ESS-1 cells than in MES-SA cells. Induction of SAHA- and TRAIL-induced apoptosis was accompanied by upregulation of the intrinsic apoptotic pathway via reduction of mitochondrial membrane potential, caspase-3, -6, and -7 activation, and PARP cleavage, but was also found to be partially caspase-independent. Apoptosis resistance was caused by reduced expression of caspase-8 and DR 4/TRAIL-R1 in ESS-1 and MES-SA cells, respectively, due to epigenetic silencing by DNA hypermethylation of gene promoter sequences. Treatment with the demethylating agent 5-Aza-2'-deoxycytidine or gene transfer therefore restored gene expression and increased the sensitivity of both cell lines against TRAIL-induced apoptosis. Our data provide evidence that deregulation of epigenetic silencing by histone acetylation and DNA hypermethylation might play a fundamental role in the origin of uterine sarcomas. Therefore, tumor growth might be efficiently overcome by a cytotoxic combinatorial treatment of HDAC inhibitors with TRAIL.

The anomalously large chiroptical response of (1R,4R)-norbornenone has been probed under complementary vapor-phase and solution-phase conditions to assess the putative roles of environmental perturbations. Measurements of the specific rotation for isolated (gas-phase) molecules could not be reproduced quantitatively by comprehensive quantum-chemical calculations based on density-functional or coupled-cluster levels of linear-response theory, which suggests that higher-order treatments may be needed to accurately predict such intrinsic behavior. A substantial, yet unexpected, dependence of the dispersive optical activity on the nature (phase) of the surrounding medium has been uncovered, with the venerable Lorentz local-field correction reproducing solvent-mediated trends in rotatory dispersion surprisingly well, whereas more modern polarizable continuum models for implicit solvation performed less satisfactorily., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Retrospective studies have demonstrated that nearly 50% of patients with ovarian cancer with normal cancer antigen 125 (CA125) levels have persistent disease; however, prospectively distinguishing between patients is currently impossible. Here, we demonstrate that for one patient, with the first reported fibroblast growth factor receptor 2 (FGFR2) fusion transcript in ovarian cancer, circulating tumor DNA (ctDNA) is a more sensitive and specific biomarker than CA125, and it can also inform on a candidate therapeutic. For a 4-year period, during which the patient underwent primary debulking surgery and chemotherapy, tumor recurrences, and multiple chemotherapeutic regimens, blood samples were longitudinally collected and stored. Whereas postsurgical CA125 levels were elevated only three times for 28 measurements, the FGFR2 fusion ctDNA biomarker was readily detectable by quantitative real-time reverse transcription-polymerase chain reaction (PCR) in all of these same blood samples and in the tumor recurrences. Given the persistence of the FGFR2 fusion, we treated tumor cells derived from this patient and others with the FGFR2 inhibitor BGJ398. Only tumor cells derived from this patient were sensitive to FGFR2 inhibitor treatment. Using the same methodologic approach, we demonstrate in a second patient with a different fusion that PCR and agarose gel electrophoresis can also be used to identify tumor-specific DNA in the circulation. Taken together, we demonstrate that a relatively inexpensive, PCR-based ctDNA surveillance assay can outperform CA125 in identifying occult disease.

The optical rotatory dispersion of two monocyclic ketones, (R)-3-methylcyclopentanone [R-3MCP] and (R)-3-methylcyclohexanone [R-3MCH], has been investigated under isolated and solvated conditions to explore the role of ring size/morphology and to elucidate the impact of environmental perturbations. Vapor-phase measurements of specific rotation, [α]λT, were performed at 355/633 nm by means of cavity ring-down polarimetry while complementary solution-phase work employed a canonical discrete-wavelength polarimeter to probe five distinct solvents. The magnitude of [α]λT was found to increase upon solvation, albeit to different extents for the two species of interest, with the attendant sign switching between the solution and vapor phases for λ ≥ 510.7 nm in the case of R-3MCH. Quantum-chemical analyses suggest two low-lying conformers to exist for each ketone, distinguished by an equatorial or axial arrangement of the methyl substituent. Linear-response calculations built upon density-functional [DFT(B3LYP)/aug-cc-pVTZ] and coupled-cluster [CCSD/aug-cc-pVDZ] frameworks gave antagonistic chiroptical parameters for these isomers, which were combined with various energy metrics in a conformer-averaging ansatz to simulate the response for a thermally equilibrated ensemble. The intrinsic behavior of R-3MCP was reproduced best by averaging DFT optical-activity predictions according to relative populations deduced from free-energy differences; however, less satisfactory agreement was realized for isolated R-3MCH molecules. The sizable circular birefringence of R-3MCP can be attributed to inherent chirality of its twisted carbon ring whereas the more modest response of R-3MCH stems mainly from the lone stereogenic center. The implicit polarizable continuum model treated solvation effects in R-3MCP with moderate success, but failed to replicate solvent-dependent trends in R-3MCH. The relationship of dispersive optical activity to bulk characteristics of the surrounding medium, including dielectric constant, refractive index, and polarizability, is discussed with the goal of bridging the gap between isolated and solvated chiroptical properties.