c-Maf-dependent Treg cell control of intestinal TH17 cells and IgA establishes host-microbiota homeostasis.

Foxp3+ regulatory T cells (Treg cells) are crucial for the maintenance of immune homeostasis both in lymphoid tissues and in non-lymphoid tissues. Here we demonstrate that the ability of intestinal Treg cells to constrain microbiota-dependent interleukin (IL)-17-producing helper T cell (TH17 cell) and immunoglobulin A responses critically required expression of the transcription factor c-Maf. The terminal differentiation and function of several intestinal Treg cell populations, including RORγt+ Treg cells and follicular regulatory T cells, were c-Maf dependent. c-Maf controlled Treg cell-derived IL-10 production and prevented excessive signaling via the kinases PI(3)K (phosphatidylinositol-3-OH kinase) and Akt and the metabolic checkpoint kinase complex mTORC1 (mammalian target of rapamycin) and expression of inflammatory cytokines in intestinal Treg cells. c-Maf deficiency in Treg cells led to profound dysbiosis of the intestinal microbiota, which when transferred to germ-free mice was sufficient to induce exacerbated intestinal TH17 responses, even in a c-Maf-competent environment. Thus, c-Maf acts to preserve the identity and function of intestinal Treg cells, which is essential for the establishment of host-microbe symbiosis.

Implications of SARS-CoV-2 spike protein interactions with Zn-bound form of ACE2: a computational structural study.

The COVID-19 pandemic has generated a major interest in designing inhibitors to prevent SARS-CoV-2 binding on host cells to protect against infection. One promising approach to such research utilizes molecular dynamics simulation to identify potential inhibitors that can prevent the interaction between spike (S) protein on the virus and angiotensin converting enzyme 2 (ACE2) receptor on the host cells. In these studies, many groups have chosen to exclude the ACE2-bound zinc (Zn) ion, which is critical for its enzymatic activity. While the relatively distant location of Zn ion from the S protein binding site (S1 domain), combined with the difficulties in modeling this ion has motivated the decision of exclusion, Zn can potentially contribute to the structural stability of the entire protein, and thus, may have implications on S protein-ACE2 interaction. In this study, the authors model both the ACE2-S1 and ACE2-inhibitor (mAb) system to investigate if there are variations in structure and the readouts due to the presence of Zn ion. Although distant from the S1 or inhibitor binding region, inclusion/exclusion of Zn has statistically significant effects on the structural stability and binding free energy in these systems. In particular, the binding free energy of the ACE2-S1 and ACE2-inhibitor structures is - 3.26 and - 14.8 kcal/mol stronger, respectively, in the Zn-bound structure than in the Zn-free structures. This finding suggests that including Zn may be important in screening potentially inhibitors and may be particularly important in modeling monoclonal antibodies, which may be more sensitive to changes in antigen structure.

Regulating T-cell differentiation through the polyamine spermidine.

BACKGROUND: The cross-talk between the host and its microbiota plays a key role in the promotion of health. The production of metabolites such as polyamines by intestinal-resident bacteria is part of this symbiosis shaping host immunity. The polyamines putrescine, spermine, and spermidine are abundant within the gastrointestinal tract and might substantially contribute to gut immunity. OBJECTIVE: We aimed to characterize the polyamine spermidine as a modulator of T-cell differentiation and function. METHODS: Naive T cells were isolated from wild-type mice or cord blood from healthy donors and submitted to polarizing cytokines, with and without spermidine treatment, to evaluate CD4+ T-cell differentiation in vitro. Moreover, mice were subjected to oral supplementation of spermidine, or its precursor l-arginine, to assess the frequency and total numbers of regulatory T (Treg) cells in vivo. RESULTS: Spermidine modulates CD4+ T-cell differentiation in vitro, preferentially committing naive T cells to a regulatory phenotype. After spermidine treatment, activated T cells lacking the autophagy gene Atg5 fail to upregulate Foxp3 to the same extent as wild-type cells. These results indicate that spermidine's polarizing effect requires an intact autophagic machinery. Furthermore, dietary supplementation with spermidine promotes homeostatic differentiation of Treg cells within the gut and reduces pathology in a model of T-cell transfer-induced colitis. CONCLUSION: Altogether, our results highlight the beneficial effects of spermidine, or l-arginine, on gut immunity by promoting Treg cell development.

Colitis Promotes a Pathological Condition of the Liver in the Absence of Foxp3+ Regulatory T Cells.

Inflammatory bowel disease is associated with extraintestinal diseases such as primary sclerosing cholangitis in the liver. Interestingly, it is known that an imbalance between Foxp3+ regulatory T cells (Treg) and Th17 cells is involved in inflammatory bowel disease and also in primary sclerosing cholangitis. To explain these associations, one hypothesis is that intestinal inflammation and barrier defects promote liver disease because of the influx of bacteria and inflammatory cells to the liver. However, whether and how this is linked to the Treg and Th17 cell imbalance is unclear. To address this, we used dextran sodium sulfate (DSS) and T cell transfer colitis mouse models. We analyzed the pathological conditions of the intestine and liver on histological, cellular, and molecular levels. We observed bacterial translocation and an influx of inflammatory cells, in particular Th17 cells, to the liver during colitis. In the DSS colitis model, in which Treg were concomitantly increased in the liver, we did not observe an overt pathological condition of the liver. In contrast, the T cell-mediated colitis model, in which Treg are not abundant, was associated with marked liver inflammation and a pathological condition. Of note, upon depletion of Treg in DEREG mice, DSS colitis promotes accumulation of Th17 cells and a pathological condition of the liver. Finally, we studied immune cell migration using KAEDE mice and found that some of these cells had migrated directly from the inflamed intestine into the liver. Overall, these data indicate that colitis can promote a pathological condition of the liver and highlight an important role of Treg in controlling colitis-associated liver inflammation.

A 8- year Bangladeshi girl with disseminated histoplasmosis, presented as chronic liver disease with portal hypertension: a rare case report.

BACKGROUND: Histoplasmosis is a rare infectious condition with mainly pulmonary involvement. Disseminated histoplasmosis may occur in immunocompromised condition. It can present in different ways but jaundice and ascites is very uncommon. CASE PRESENTATION: A 8- year old girl visited to department of pediatric gastroenterology & nutrition, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh. Child presented with fever, jaundice and abdominal distension for 2 ½ months. There was no history of contact with tuberculosis patient and travelling to kala-azar, malaria endemic zone and no history of previous jaundice, blood or blood product transfusion, history of sib death, family history of jaundice or neuropsychiatric disorder, significant weight loss. On general examination she was fretful, febrile, moderately icteric, mildly pale, vitally stable, severely wasted and moderately stunted, skin survey revealed infected scabies, BCG vaccine mark was absent, generalized lymphadenopathy, hepato-splenomegaly and ascites present. After evaluating the physical findings, several investigations was done including lymphnode biopsy, then the case was finally diagnosed as Disseminated histoplasmosis with portal hypertension. Child was treated with injectable Deoxycholate Amphotericin B for 28 days and improved on follow up. CONCLUSION: We suggest that children presenting with fever, jaundice, lymphadenopathy and hepatosplenomegaly and portal hypertension, disseminated histoplasmosis can be one differential.

Mass Spectrometry- and Computational Structural Biology-Based Investigation of Proteins and Peptides.

Recent developments of mass spectrometry (MS) allow us to identify, estimate, and characterize proteins and protein complexes. At the same time, structural biology helps to determine the protein structure and its structure-function relationship. Together, they aid to understand the protein structure, property, function, protein-complex assembly, protein-protein interaction, and dynamics. The present chapter is organized with illustrative results to demonstrate how experimental mass spectrometry can be combined with computational structural biology for detailed studies of protein's structures. We have used tumor differentiation factor protein/peptide as ligand and Hsp70/Hsp90 as receptor protein as examples to study ligand-protein interaction. To investigate possible protein conformation, we will describe two proteins-lysozyme and myoglobin. As an application of MS-based assignment of disulfide bridges, the case of the spider venom polypeptide Phα1ß will also be discussed.

Cyclic dinucleotides modulate induced type I IFN responses in innate immune cells by degradation of STING.

The cyclic dinucleotides, GMP-AMP (cGAMP) and c-di-AMP [bis-(3',5')-cyclic dimeric AMP], are potent type I IFN inducers via STING-TBK1-IRF3 cascade. They are promising adjuvants that promote antigen-specific humoral and cellular immune responses in different preclinical models; however, an optimal outcome of vaccination depends on a balanced immune activation. Here, we characterize the process of IFN-ß induction by c-di-AMP and cGAMP in an in vitro model on the basis of primary mouse dendritic cells. Results obtained show decreased IFN-ß production upon prolonged cell stimulation. We demonstrate that this effect depends on c-di-AMP/cGAMP-mediated down-regulation of stimulator of IFN gene (STING) protein levels. These results were confirmed by using human peripheral blood mononuclear cell-derived dendritic cells. Studies performed to explore the potential mechanism of STING modulation suggested proteolytic degradation to be a contributing factor to the observed decrease in cellular STING levels. Our work contributes to the elucidation of the molecular mode of action of vaccine constituents, which, in turn, is a prerequisite for the rational design of vaccines with predictable efficacy and safety profiles-Rueckert, C., Rand, U., Roy, U., Kasmapour, B., Strowig, T., Guzmán, C. A. Cyclic dinucleotides modulate induced type I IFN responses in innate immune cells by degradation of STING.

Structural modeling of tumor necrosis factor: A protein of immunological importance.

Tumor necrosis factor (TNF) is a multifunctional pro-inflammatory cytokine responsible for various immunoregulatory activities. Upon binding with its receptor, TNF triggers multiple complex signaling pathways such as the activations of nuclear factor kappa B and caspase cascade, which are the leading determining factors for cell survival or cell death. The present work studies certain modeling aspects of the TNF, with comparative structural analyses of the wild and mutant types of this protein. Additionally, nanoscale molecular dynamics simulations are performed to assess the structure-property relationships of proteins as functions of time.

Protein-protein interactions: switch from classical methods to proteomics and bioinformatics-based approaches.

Following the sequencing of the human genome and many other organisms, research on protein-coding genes and their functions (functional genomics) has intensified. Subsequently, with the observation that proteins are indeed the molecular effectors of most cellular processes, the discipline of proteomics was born. Clearly, proteins do not function as single entities but rather as a dynamic network of team players that have to communicate. Though genetic (yeast two-hybrid Y2H) and biochemical methods (co-immunoprecipitation Co-IP, affinity purification AP) were the methods of choice at the beginning of the study of protein-protein interactions (PPI), in more recent years there has been a shift towards proteomics-based methods and bioinformatics-based approaches. In this review, we first describe in depth PPIs and we make a strong case as to why unraveling the interactome is the next challenge in the field of proteomics. Furthermore, classical methods of investigation of PPIs and structure-based bioinformatics approaches are presented. The greatest emphasis is placed on proteomic methods, especially native techniques that were recently developed and that have been shown to be reliable. Finally, we point out the limitations of these methods and the need to set up a standard for the validation of PPI experiments.

Mass spectrometry investigation of glycosylation on the NXS/T sites in recombinant glycoproteins.

We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Fc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed.

Characterization of tumor differentiation factor (TDF) and its receptor (TDF-R).

Tumor differentiation factor (TDF) is an under-investigated protein produced by the pituitary with no definitive function. TDF is secreted into the bloodstream and targets the breast and prostate, suggesting that it has an endocrine function. Initially, TDF was indirectly discovered based on the differentiation effect of alkaline pituitary extracts of the mammosomatotropic tumor MtTWlO on MTW9/PI rat mammary tumor cells. Years later, the cDNA clone responsible for this differentiation activity was isolated from a human pituitary cDNA library using expression cloning. The cDNA encoded a 108-amino-acid polypeptide that had differentiation activity on MCF7 breast cancer cells and on DU145 prostate cancer cells in vitro and in vivo. Recently, our group focused on identification of the TDF receptor (TDF-R). As potential TDF-R candidates, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cells fibroblasts or fibroblast-like cells. Here we review the current advances on TDF, with particular focus on the structural investigation of its receptor and on its functional effects on breast and prostate cells.

Bactericidal activity of nukacin ISK-1: an alternative mode of action.

We previously reported bacteriostatic action of nukacin ISK-1 against Bacillus subtilis JCM 1465(T). Here, we found its bactericidal activity against Micrococcus luteus DSM 1790 and Staphylococcus simulans 22, showing decrease in cell viability, cell lysis, and dissipation of the membrane potential. Moreover, leakage of small molecules such as K(+), suggested the formation of small-sized or specific K(+)-conducting-pores by nukacin ISK-1.

Utility of computational structural biology in mass spectrometry.

Recent developments of mass spectrometry (MS) allow us to identify, estimate, and characterize proteins and protein complexes. At the same time, structural biology helps to determine the protein structure and its structure-function relationship. Together, they aid to understand the protein structure, property, function, protein-complex assembly, protein-protein interaction and dynamics. The present chapter is organized with illustrative results to demonstrate how experimental mass spectrometry can be combined with computational structural biology for detailed studies of protein's structures. We have used tumor differentiation factor protein/peptide as ligand and Hsp70/Hsp90 as receptor protein as examples to study ligand-protein interaction. To investigate possible protein conformation we will describe two proteins, lysozyme and myoglobin.

Prediction of severity of dengue infection in children based on hepatic involvement.

Background and Aim: To assess the spectrum of hepatic involvement in children with dengue fever (DF) and prediction of severity of dengue infection by early detection of elevated liver enzymes. Methods: This prospective observational study was conducted at a tertiary care hospital from June 2019 to September 2019. Children admitted with DF were included. Severity of DF was graded as dengue without warning sign (DNWS), with warning sign (DWS), and severe dengue fever (SDF) according to WHO criteria. Liver injury (LI) was defined as alanine aminotransferase (ALT) more than upper limit of normal irrespective of sex. Results: Of 190 children (male, 109) with DF, 60 had DNWS, 49 had DWS, and 81 had SDF. A total of 100 children (52.6%) had LI. The distribution of hepatic involvement spectrum involves hepatomegaly (26.3%), hepatic tenderness (25.2%), features of acute liver failure (1.5%), raised level of ALT (52.6%), raised level of aspartateaminotransferase (AST) (65.8%), prolonged prothrombin time (7.3%), and reduced level of serum albumin (44.7%) in children. Of them, 5.8% and 6.8% of children had >tenfold increase in ALT and AST values. The degree of liver function derangement significantly (P < 0.05) increased with DF severity. In our study, ALT at 422 IU/L (10 times upper limit of normal [ULN]) and AST 689 IU/L (17 times ULN) had similar sensitivity and specificity as WHO recommended cutoff of 1000 IU/L (25 times of ULN) to detect SDF. Conclusion: ALT ≥10 times and AST ≥17 times of ULN are as sensitive as ≥25 times (as recommended by WHO) to detect SDF.

Investigation of stable and transient protein-protein interactions: Past, present, and future.

This article presents an overview of the literature and a review of recent advances in the analysis of stable and transient protein-protein interactions (PPIs) with a focus on their function within cells, organs, and organisms. The significance of PTMs within the PPIs is also discussed. We focus on methods to study PPIs and methods of detecting PPIs, with particular emphasis on electrophoresis-based and MS-based investigation of PPIs, including specific examples. The validation of PPIs is emphasized and the limitations of the current methods for studying stable and transient PPIs are discussed. Perspectives regarding PPIs, with focus on bioinformatics and transient PPIs are also provided.

Identification of potential tumor differentiation factor (TDF) receptor from steroid-responsive and steroid-resistant breast cancer cells.

Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.

Computational Investigation of Selected Spike Protein Mutations in SARS-CoV-2: Delta, Omicron, and Some Circulating Subvariants.

Among the multiple SARS-CoV-2 variants recently reported, the Delta variant has generated the most perilous and widespread effects. Another variant, Omicron, has been identified specifically for its high transmissibility. Omicron contains numerous spike (S) protein mutations and numbers much larger than those of its predecessor variants. In this report, the author has discussed some essential structural aspects and time-based structure changes of a selected set of spike protein mutations within the Delta and Omicron variants. The expected impact of multiple point mutations within the spike protein's receptor-binding domain (RBD) and S1 of these variants are examined. Additionally, the RBDs of the more recently emerged subvariants BA.4, BA.5, and BA.2.12.1 are discussed. Within the latter group, BA.5 represents the most prevalent form of SARS-CoV-2 globally until recently. This computational work also briefly explores the temporal mutation profile for the currently circulating variants of interest (VOIs), variants under monitoring (VUMs), and variants being monitored (VBMs) including XBB.1.5, BQ.1, BA.2.75, CH.1.1, XBB, XBF, EG.5 (or Eris), and BA.2.86 (or Pirola). It is expected that these structural data can facilitate the tasks of identifying drug targets and neutralizing antibodies for the evolving variants/subvariants of SARS-CoV-2.

Cross-reactivity of human monoclonal antibodies with canine peripheral blood mononuclear cells.

In drug development, the dog is often used as a model for non-rodent preclinical safety studies. In particular, immunophenotyping in dogs can be important to characterize the toxicological profile of a test item. A wide range of antibodies specific to surface antigens is needed, however, commercially available antibodies to dog are scarce. To date, numerous studies have reported the cross-reactivity of human monoclonal antibodies with canine peripheral blood mononuclear cells (PBMC). In this study, we aimed to increase the number of canine-specific antibodies and took a rather novel approach to further determine cross-reactivity of 378 human recombinant antibodies lacking Fc regions to surface antigens on canine PBMC. The screening resulted in 30 human monoclonal antibodies well reactive to canine PBMC. Sequence homology of the targeted human and canine antigens was analyzed with Basic Local Alignment Search Tool. Thirteen human cross-reactive antibodies of interest were analyzed with cells from canine whole blood in combination with lineage markers. Finally, ten antibodies were identified as useful markers for the application in dog. Except for CD27, the remaining nine antibodies are already commercially available human cross-reactive antibodies. This study provides a new source for all ten antibodies described here.

Structural investigation of tumor differentiation factor.

Tumor differentiation factor (TDF) is a 17 kDa protein produced by the pituitary and secreted into the bloodstream, with no definitive function and incomplete characterization. TDF has the following four cysteine (Cys) residues: Cys17, Cys70, Cys97, and Cys98. To understand the function of TDF, we (1) overexpressed and characterized recombinant TDF (rTDF); (2) investigated native, secreted TDF; and (3) assessed potential disulfide connectivities using molecular modeling. Our results from Western blotting (WB) experiments suggest that rTDF is mostly expressed as insoluble, monomeric, and dimeric forms. Mass spectrometry analysis of the overexpressed rTDF identified a peptide that is a part of TDF protein. WB of the native, secreted TDF detected it as a 50 kDa band. In addition, investigation of TDF by molecular modeling suggests that the Cys residues may form disulfide bridges between Cys17-Cys98 and Cys70-Cys17.