Fecal microbiota transplantation stimulates type 2 and tolerogenic immune responses in a mouse model.

OBJECTIVES: Clostridioides difficile infection (CDI) is the leading hospital-acquired infection in North America. While previous work on fecal microbiota transplantation (FMT), a highly effective treatment for CDI, has focused on colonization resistance mounted against C. difficile by FMT-delivered commensals, the effects of FMT on host gene expression are relatively unexplored. This study aims to identify transcriptional changes associated with FMT, particularly changes associated with protective immune responses. METHODS: Gene expression was assessed on day 2 and day 7 after FMT in mice after antibiotic-induced dysbiosis. Flow cytometry was also performed on colon and mesenteric lymph nodes at day 7 to investigate changes in immune cell populations. RESULTS: FMT administration after antibiotic-induced dysbiosis successfully restored microbial alpha diversity to levels of donor mice by day 7 post-FMT. Bulk RNA sequencing of cecal tissue at day 2 identified immune genes, including both pro-inflammatory and Type 2 immune pathways as upregulated after FMT. RNA sequencing was repeated on day 7 post-FMT, and expression of these immune genes was decreased along with upregulation of genes associated with restoration of intestinal homeostasis. Immunoprofiling on day 7 identified increased colonic CD45+ immune cells that exhibited dampened Type 1 and heightened regulatory and Type 2 responses. These include an increased abundance of eosinophils, alternatively activated macrophages, Th2, and T regulatory cell populations. CONCLUSION: These results highlight the impact of FMT on host gene expression, providing evidence that FMT restores intestinal homeostasis after antibiotic treatment and facilitates tolerogenic and Type 2 immune responses.

The impact of existing total anti-toxin B IgG immunity in outcomes of recurrent Clostridioides difficile infection.

Late anti-toxin-B humoral immunity acquired after treatment is important for preventing recurrent Clostridioides difficile infection. We prospectively-measured anti-toxin-B IgG and neutralization titers at diagnosis as potential early predictors of recurrence. High anti-toxin-B-IgG/neutralizing antibodies were associated with short-lasting protection within 6-weeks, however, no difference in recurrence risk was observed by 90-days post-infection.

Host Immunity and Immunization Strategies for Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) represents a significant challenge to public health. C. difficile-associated mortality and morbidity have led the U.S. CDC to designate it as an urgent threat. Moreover, recurrence or relapses can occur in up to a third of CDI patients, due in part to antibiotics being the primary treatment for CDI and the major cause of the disease. In this review, we summarize the current knowledge of innate immune responses, adaptive immune responses, and the link between innate and adaptive immune responses of the host against CDI. The other major determinants of CDI, such as C. difficile toxins, the host microbiota, and related treatments, are also described. Finally, we discuss the known therapeutic approaches and the current status of immunization strategies for CDI, which might help to bridge the knowledge gap in the generation of therapy against CDI.

Structural, Theoretical Investigations, Hirshfeld Surface Analysis, and Cytotoxicity Profile of a Neocuproine-Co(II)-Based Discrete Homodinuclear Complex.

In this work, we aimed to synthesize a new cobalt(II) complex, namely [Co2(µ-HIPA)(NC)2(H2O)3(NO3)]·(NO3)(C2H5OH)(1) (where H3IPA = 5-hydroxy isophthalic acid and NC = 2,9-dimethyl-1,10-phenanthroline or neocuproine), as a promising chemotherapeutic agent. The diffraction (single crystal-XRD and powder-XRD), spectroscopic (FTIR and UV-visible), molar conductance, and thermal techniques were used to characterize complex 1. Single-crystal X-ray diffraction analysis reveals that Co(II) exists in an octahedral geometry, with the ligation of four oxygen atoms, and two nitrogen atoms. Topological analysis of complex 1 reveals 2,6C6 topological type as an underlying net. The plausible intermolecular interactions within complex 1 that control the crystal packing were analyzed by Hirshfeld surface analysis. In vitro cytotoxicity of complex 1 was evaluated against acute myeloid leukemia (THP-1), colorectal (SW480), and prostate (PC-3) cancer cell lines by utilizing an MTT assay. The result shows that complex 1 can inhibit the growth of cancer cells (THP-1, SW480, and PC-3) at lower inhibitory concentration (IC50) values of > 100, 43.6, and 95.1 µM respectively. The morphological changes induced by complex 1 on THP-1 and SW480 cancer cell lines were carried out with acridine orange/ethidium bromide staining methods. Additionally, comprehensive molecular docking studies were performed to understand the potential binding interactions of complex 1 with different bio-macromolecules.

Correlation Between BMI, Caries Prevalence, and Sugar-containing Beverage Intake in 6-10 Year Old Children.

Background: The beverages containing sugar are proven risk factors for obesity and dental caries. Therefore, owing to the shared risk factors, an interrelationship is suspected between BMI, sugar beverage consumption, and dental caries in children. Aims: The present trial was carried out to assess the interrelationship between BMI, sugar beverage consumption, and dental caries in children aged 6-10 years. Materials and Methods: Eighty-six children within the age range of 6-10 years answered the health questionnaire. The BMI was calculated, intra-oral assessment was done, the frequency of sweetened beverage consumption was recorded, and the collected data were subjected to the statistical evaluation to formulate results. Results: On evaluation, a non-significant difference was observed in BMI levels in the four groups (P = 0.12). Whole-milk intake also showed an inverse correlation with dental caries and BMI, but this correlation was statistically non-significant with the respective values of P = 0.57 and 0.55. A similar inverse relationship was seen for low-fat milk for caries and BMI with P = 0.65 and 0.45, respectively. Regarding soft drinks, 44.1% (n = 38) took soft drinks, and a non-significant relation between caries and intake as well as BMI and intake with P = 0.86 and 0.55, respectively. Conclusion: Within its limitations, the present study concludes that no correlation exists between BMI and dental caries as well as between sugar-containing beverage consumption and dental caries. Also, BMI and sugar-containing beverage consumption showed no correlation in children aged 6-10 years.

Sphingosine-1-phosphate receptors 2 and 3 reprogram resting human macrophages into M1 phenotype following mycobacteria infection.

Mycobacteria tuberculosis (M.tb) the causative agent for tuberculosis has been accredited for a high rate of morbidity and mortality worldwide. The rise in MDR and XDR cases has further created new obstacles in achieving the "End TB Strategy", which is aimed for 2035. In this article, we have demonstrated the potential of sphingosine-1-phosphate (S1P) analogs in providing an anti-mycobacterial effector response by altering macrophage polarity into M1. Among S1PR1 and S1PR3 analogs, S1PR2 analogs proficiently favor selective polarization of infected human macrophages into M1 phenotypes, marked by increased expression of M1 markers and decreased M2 markers. Furthermore, S1PR1-3 analogs treated macrophages were also able to decrease the secretion of anti-inflammatory cytokine IL-10 and can induce NO secretion in infected macrophages. Lastly, only S1PR2-3 analogs were able to restrict the growth of mycobacteria in human macrophages. Taken together our study reflects the potential of S1PR2-3 analogs in providing host defenses following mycobacterial infection by favoring M1 macrophage polarization.

Macrophage plasticity as a therapeutic target in tuberculosis.

The macrophages contribute to host defense against intracellular pathogens such as mycobacteria. Mycobacteria interact with macrophages altering their polarization state, which propagates establishment of infection. Thus, molecular macrophage properties in mycobacterial infections are critical both for understanding the biology of the infections as well as identifying therapeutic targets. Here, we review recent advances in the understanding how altered macrophage polarization in mycobacterial infections may lead to the design of targeted therapies that may reprogram these macrophages for enhanced mycobactericidal function.

GPCRs as an emerging host-directed therapeutic target against mycobacterial infection: From notion to reality.

Mycobacterium tuberculosis (M. tb) is one of the successful pathogens and claim millions of deaths across the globe. The emergence of drug resistance in M. tb has created new hurdles in the tuberculosis elimination programme worldwide. Hence, there is an unmet medical need for alternative therapy, which could be achieved by targeting the host's critical signalling pathways that are compromised during M. tb infection. In this review, we have summarized some of the findings involving the modulation of host GPCRs in the regulation of the mycobacterial infection. Understanding the role of these GPCRs not only unravels signalling pathways during infection but also provides clues for targeting critical signalling intermediates for the development of GPCR-based host-directive therapy. LINKED ARTICLES: This article is part of GPCR Review Series. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/toc/10.1111/(ISSN)1476-5381.GPCRReviews.

Personalized therapy: can it tame the COVID-19 monster?

SARS-CoV-2, a recently emerged zoonotic virus, has resulted in unstoppable high morbidity and mortality rates worldwide. However, due to a limited knowledge of the dynamics of the SARS-CoV-2 infection, it has been observed that the current COVID-19 therapy has led to some clinical repercussions. We discuss the adverse effects of drugs for COVID-19 primarily based on some clinical trials. As therapeutic efficacy and toxicity of therapy may vary due to different, genetic determinants, sex, age and the ethnic background of test subjects, hence biomarker-based personalized therapy could be more appropriate. We will share our thoughts on the current landscape of personalized therapy as a roadmap to fight against SARS-CoV-2 or another emerging pathogen.

Battling COVID-19 Pandemic: Sphingosine-1-Phosphate Analogs as an Adjunctive Therapy?

With the sudden outbreak of COVID-19 patient worldwide and associated mortality, it is critical to come up with an effective treatment against SARS-CoV-2. Studies suggest that mortality due to COVID 19 is mainly attributed to the hyper inflammatory response leading to cytokine storm and ARDS in infected patients. Sphingosine-1-phosphate receptor 1 (S1PR1) analogs, AAL-R and RP-002, have earlier provided in-vivo protection from the pathophysiological response during H1N1 influenza infection and improved mortality. Recently, it was shown that the treatment with sphingosine-1-phosphate receptor 1 analog, CYM5442, resulted in the significant dampening of the immune response upon H1N1 challenge in mice and improved survival of H1N1 infected mice in combination with an antiviral drug, oseltamivir. Hence, here we suggest to investigate the possible utility of using S1P analogs to treat COVID-19.

Sphingosine-1-phosphate signaling in Leishmania donovani infection in macrophages.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a crucial regulator of a wide array of cellular processes, such as apoptosis, cell proliferation, migration, and differentiation, but its role in Leishmania donovani infection is unknown. METHODOLOGY/ PRINCIPAL FINDINGS: In the present study, we observed that L. donovani infection in THP-1 derived macrophages (TDM) leads to decrease in the expression of S1pr2 and S1pr3 at mRNA level. We further observed that Leishmania infection inhibits the phosphorylation of sphingosine kinase 1 (sphK1) in a time-dependent manner. Exogenous S1P supplementation decreases L. donovani induced ERK1/2 phosphorylation and increases p38 phosphorylation in TDM, resulting in a decrease in the intracellular parasite burden in a dose-dependent manner. On the other hand, sphK inhibition by DMS increases ERK1/2 phosphorylation leading to increased IL-10 and parasite load. To gain further insight, cytokines expression were checked in S1P supplemented TDM and we observed increase in IL-12, while decrease IL-10 expression at mRNA and protein levels. In addition, treatment of antagonist of S1PR2 and S1PR3 such as JTE-013 and CAY10444 respectively enhanced Leishmania-induced ERK1/2 phosphorylation and parasite load. CONCLUSIONS: Our overall study not only reports the significant role of S1P signaling during L. donovani infection but also provides a novel platform for the development of new drugs against Leishmaniasis.

Investigation of molecular mechanism of recognition between citral and MARK4: A newer therapeutic approach to attenuate cancer cell progression.

Microtubule affinity regulating kinase 4 (MARK4) is a member of AMP-activated protein kinase, found to be involved in apoptosis, inflammation and many other regulatory pathways. Since, its aberrant expression is directly associated with the cell cycle and thus cancer. Therefore, MARK4 is being considered as a potential drug target for cancer therapy. Here, we investigated the mechanism of inhibition of MARK4 activity by citral. Docking studies suggested that citral effectively binds to the active site cavity, and complex is stabilized by several interactions. We further performed molecular dynamics simulation of MARK4-citral complex under explicit water condition for 100ns and observed that binding of citral to MARK4 was quite stable. Fluorescence binding studies suggested that citral strongly binds to MARK4 and thereby inhibits its enzyme activity which was measured by the kinase inhibition assay. We further performed MTT assay and observed that citral inhibits proliferation of breast cancer cell line MCF-7. This work provides a newer insight into the use of citral as novel cancer therapeutics through the MARK4 inhibition. Results may be employed to design novel therapeutic molecule using citral as a scaffold for MARK4 inhibition to fight related diseases.

Elucidation of Dietary Polyphenolics as Potential Inhibitor of Microtubule Affinity Regulating Kinase 4: In silico and In vitro Studies.

Microtubule affinity regulating kinase 4 (MARK4) is a Ser/Thr kinase belonging to AMPK-like family, has recently become an important drug target against cancer and neurodegenerative disorders. In this study, we have evaluated different natural dietary polyphenolics including rutin, quercetin, ferulic acid, hesperidin, gallic acid and vanillin as MARK4 inhibitors. All compounds are primarily binds to the active site cavity of MARK4. In silico observations were further complemented by the fluorescence-binding studies and isothermal titration calorimetry (ITC) measurements. We found that rutin and vanillin bind to MARK4 with a reasonably high affinity. ATPase and tau-phosphorylation assay further suggesting that rutin and vanillin inhibit the enzyme activity of MARK4 to a great extent. Cell proliferation, ROS quantification and Annexin-V staining studies are clearly providing sufficient evidences for the apoptotic potential of rutin and vanillin. In conclusion, rutin and vanillin may be considered as potential inhibitors for MARK4 and further exploited to design novel therapeutic molecules against MARK4 associated diseases.

Evaluation of human microtubule affinity-regulating kinase 4 inhibitors: fluorescence binding studies, enzyme, and cell assays.

Human microtubule affinity-regulating kinase 4 (MARK4) is considered as an encouraging drug target for the design and development of inhibitors to cure several life-threatening diseases such as Alzheimer disease, cancer, obesity, and type-II diabetes. Recently, we have reported four ligands namely, BX-912, BX-795, PKR-inhibitor, and OTSSP167 (hydrochloride) which bind preferentially to the two different constructs of human MARK4 containing kinase domain. To ensure the role of ubiquitin-associated (UBA) domain in the ligand binding, we made a newer construct of MARK4 which contains both kinase and UBA domains, named as MARK4-F3. We observed that OTSSP167 (hydrochloride) binds to the MARK4-F3 with a binding constant (K) of 3.16 × 106, M-1 (±.21). However, UBA-domain of MARK4-F3 doesn't show any interaction with ligands directly as predicted by the molecular docking. To validate further, ATPase inhibition assays of all three constructs of MARK4 in the presence of mentioned ligands were carried out. An appreciable correlation between the binding experiments and ATPase inhibition assays of MARK4 was observed. In addition, cell-proliferation inhibition activity for all four ligands on the Human embryonic kidney (HEK-293) and breast cancer cell lines (MCF-7) was performed using MTT assay. IC50 values of OTSSP167 for HEK-293 and MCF-7 were found to be 58.88 (±1.5), and 48.2 (±1.6), respectively. OTSSP167 among all four inhibitors, showed very good enzyme inhibition activity against three constructs of MARK4. Moreover, all four inhibitors showed anti-neuroblastoma activity and anticancer properties. In conclusion, OTSSP167 may be considered as a promising scaffold to discover novel inhibitors of MARK4.

Ubiquitin-associated domain of MARK4 provides stability at physiological pH.

Microtubule affinity regulating Kinase 4 (MARK4) belongs to the family of AMP-activated protein kinase. It phosphorylates microtubule associated proteins at specific sites (Serine in KXGS motifs) in the microtubule-binding repeats. In our previous studies, two constructs, namely, kinase domain with 59 N-terminal residues (residues 1-310) and only kinase domain (residues 59-310) of MARK4 show aggregation at physiological pH. However, these two constructs were stable at extremes of pH conditions. Now the question arises: how is MARK4 stable at physiological pH in-vivo? To answer this question, we have successfully cloned, expressed, and purified UBA-domain along with the kinase domain of MARK4 and performed spectroscopic measurements and activity assays. We observed a pronounced secondary and tertiary structure and ATPase activity in the MARK4 at physiological pH. In conclusion, UBA domain may be important to maintain the structure, stability and activity of MARK4 under physiological conditions.

Human microtubule affinity-regulating kinase 4 is stable at extremes of pH.

MAP/microtubule affinity-regulating kinase 4 (MARK4) is a member of adenosine monophosphate-activated protein kinases, directly associated with cancer and neurodegenerative diseases. Here, we have cloned, expressed, and purified two variants of MARK4 [the kinase domain (MARK4-F2), and kinase domain along with 59 N-terminal residues (MARK4-F1)] and compared their stability at varying pH range. Structural and functional changes were observed by incubating both forms of MARK4 in buffers of different pH. We measured the secondary structure of MARK4 using circular dichroism and tertiary structure by measuring intrinsic fluorescence and absorbance properties along with the size of proteins by dynamic light scattering. We observed that at extremes of pH (below pH 3.5 and above pH 9.0), MARK4 is quite stable. However, a remarkable aggregate formation was observed at intermediate pH (between pH 3.5 and 9.0). To further validate this result, we have modeled both forms of MARK4 and performed molecular dynamics simulation for 15 ns. The spectroscopic observations are in excellent agreement with the findings of molecular dynamics simulation. We also performed ATPase activity at varying pH and found a significant correlation of structure of MARK4 with its enzyme activity. It is interesting to note that both forms of MARK4 are showing a similar pattern of structure changes with reference to pH.

Designing New Kinase Inhibitor Derivatives as Therapeutics Against Common Complex Diseases: Structural Basis of Microtubule Affinity-Regulating Kinase 4 (MARK4) Inhibition.

Drug development for common complex diseases is in need of new molecular entities and actionable drug targets. MAP/microtubule affinity-regulating kinase 4 (MARK4) is associated with numerous diseases such as neurodegenerative disorders, obesity, cancer, and type 2 diabetes. Understanding the structural basis of ligands' (inhibitors) and substrates' binding to MARK4 is crucial to design new kinase inhibitors for therapeutic purposes. This study reports new observations on docking three well-known kinase inhibitors in the kinase domain of MARK4 variants and the calculated binding affinity. These variants of MARK4 are named as MARK4-F1 (59 N-terminal residues along with kinase domain) and MARK4-F2 (kinase domain of MARK4). We additionally performed molecular dynamics (MD) simulation and fluorescence binding studies to calculate the actual binding affinity of kinase inhibitors, BX-912, BX-795, and OTSSP167 (hydrochloride) for the MARK4. Docking analyses revealed that ligands bind in the large hydrophobic cavity of the kinase domain of MARK4 through several hydrophobic and hydrogen-bonded interactions. Simulations suggested that OTSSP167 (hydrochloride) is forming a stable complex, and hence the best inhibitor of MARK4. Intrinsic fluorescence of MARK4 was significantly quenched by addition of ligands, indicating their potential binding to MARK4. A lower KD value of MARK4 with OTSSP167 (hydrochloride) suggested that it is a better interacting partner than BX-912 and BX-795. These data form a basis for designing novel and potent OTSSP167 (hydrochloride) derivatives as therapeutic candidates against common complex diseases. The inhibitors designed as such might possibly suppress the growth of tumor-forming cells and be potentially applied for treatment of a wide range of human cancers as well.

PKR-inhibitor binds efficiently with human microtubule affinity-regulating kinase 4.

MAP/microtubule affinity-regulating kinase 4 (MARK4) plays a central role in the cellular physiology, and it is inseparably linked with many human diseases including cancer, diet induced obesity, type2 diabetes and neurodegenerative disorders. Here, we studied the interaction of PKR-inhibitor with two variants of human MARK4. One variant is named as MARK4-F1 which has 59 N-terminal residues along with kinase domain while another variant is MARK4-F2 which has kinase domain only. Molecular-docking, molecular dynamics (MD) simulation and fluorescence-binding studies were undertaken to understand the role of N-terminal 59-residues in the binding of substrate/inhibitors. Molecular docking studies revealed that the PKR-inhibitor binds in the large hydrophobic cavity of the kinase domain of MARK4 through several hydrophobic and hydrogen-bonded interactions. Furthermore, MD simulation showed a stable parameters for the complexes of both MARK4-F1 and MARK4-F2 to PKR-inhibitor with marginal difference in their binding affinities. A significant decrease in the fluorescence intensity of MARK4 was observed on successive addition of the PKR-inhibitor. Using fluorescence data we have calculated the binding-affinity and the number of binding site of PKR-inhibitor to the MARK4. A significantly high binding affinity was observed for the PKR-inhibitor to the MARK4 variants. However, there is no any significant difference in the binding affinity of PKR-inhibitor to the MARK4 variants was observed, indicating that 59 N-terminal residues of MARK4-F1 are not playing a crucial role in the ligand binding. The present study will provide an insights into designing of new PKR-inhibitor derivative as potent and selective therapeutic agent against many life threatening diseases which are associated with MARK4.