Epigenetic regulations in Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (Mtb) employs several sophisticated strategies to evade host immunity and facilitate its intracellular survival. One of them is the epigenetic manipulation of host chromatin by three strategies i.e., DNA methylation, histone modifications and miRNA involvement. A host-directed therapeutic can be an attractive approach that targets these host epigenetics or gene regulations and circumvent manipulation of host cell machinery by Mtb. Given the complexity of the nature of intracellular infection by Mtb, there are challenges in identifying the important host proteins, non-coding RNA or the secretory proteins of Mtb itself that directly or indirectly bring upon the epigenetic modifications in the host chromatin. Equally challenging is developing the methods of targeting these epigenetic factors through chemical or non-chemical approaches as host-directed therapeutics. The current review article briefly summarizes several of the epigenetic factors that serve to bring upon potential changes in the host transcriptional machinery and targets the immune system for immunosuppression and disease progression in Mtb infection.

Anti-glycating and anti-cytotoxic effect of silibinin on albumin at early glycation: A physiochemical study.

During persistent hyperglycaemia, albumin, one of the major blood proteins, can undergo fast glycation. It can be expected that timely inhibition of protein glycation might be add quality years to diabetic patients' life. Therefore, this study was designed to analyse the role of silibinin to reduced or delay amadori adduct formation at early glycation and its beneficial effect to improve the glycated albumin structure and conformation. We also analysed cytotoxic effect of amadori-albumin in the presence of silibinin on murine macrophage cell line RAW cells by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. Formation of early glycated product (furosine) in all samples was confirmed by LCMS. Albumin incubated with glucose only showed presence of furosine like structure. Albumin treated with silibinin in the presence of glucose did not show such furosine like peak. This LCMS result showed the silibinin play a protective role in the formation of early glycated product. HMF contents were also reduced in the presence of silibinin, when albumin was incubated with increasing concentrations of silibinin (100 and 200 µM) in the presence of glucose. ANS binding fluorescence decrease by increasing silibinin concentrations with amadori-albumin. SDS-PAGE was also showed that no significant difference in the band mobility of albumin treated with silibinin as compared to native albumin. The secondary conformational alteration in amadori-albumin due to silibinin were confirmed by FTIR. This spectrum showed slight shift in amide I and Amide II band in albumin co-incubated with glucose and silibinin as compared to albumin incubated with glucose only. We further discussed about cytotoxic effect of amadori albumin and its prevention by silibinin. MTT assay results demonstrated that amadori-albumin showed cytotoxic effect on RAW cells but silibinin showed protective role and increased the cell viability. Moreover, the results showed that silibinin has anti-glycating potential and playing a role to prevent the formation of Amadori-albumin in-vitro. Silibinin possesses strong anti-glycating capacity and can improve albumin structure and function at early stage. It might be useful in delaying the progression of diabetes mellitus and its secondary complications at early stage.

Design and characterization of a binary CT complex of imidazole-oxyresveratrol: exploring its pharmacological and computational aspects.

A new binary charge transfer (CT) complex between imidazole (IMZ) and oxyresveratrol (OXA) was synthesized and characterized experimentally and theoretically. The experimental work was carried out in solution and solid state in selected solvents such as chloroform (CHL), methanol (Me-OH), ethanol (Et-OH), and acetonitrile (AN). The newly synthesized CT complex (D1) has been characterized by various techniques such as UV-visible spectroscopy, FTIR, 1H-NMR, and powder-XRD. The 1:1 composition of D1 is confirmed by Jobs' method of continuous variation and spectrophotometric (at λmax 554 nm) methods at 298 K. The infrared spectra of D1 confirmed the existence of proton transfer hydrogen bond beside charge transfer interaction. These findings indicate that the cation and anion are joined together by the weak hydrogen bonding as N+-H-O-. Reactivity parameters strongly recommended that IMZ behaves as a good electron donor and OXA an efficient electron acceptor. Density functional theory (DFT) computations with basis set B3LYP/6-31G (d,p) was applied to support the experimental results. TD-DFT calculations gives HOMO (-5.12 eV) → LUMO (-1.14 eV) electronic energy gap (ΔE) to be 3.80 eV. The bioorganic chemistry of D1 was well established after antioxidant, antimicrobial, and toxicity screening in Wistar rat. The type of interactions between HSA and D1 at the molecular level was studied through fluorescence spectroscopy. Binding constant along with the type of quenching mechanism, was investigated through the Stern-Volmer equation. Molecular docking demonstrated that D1 binds perfectly with human serum albumin and EGFR (1M17) and exposes free energy of binding (FEB) values of -295.2 and -283.3 kcal/mol, respectively. The D1 fits successfully into the minor groove of HAS and 1M17, the results of molecular docking show that the D1 binds perfectly with the HAS and 1M17, the higher value of binding energy shows stronger interaction between HAS and 1M17 with D1. Our synthesized complex shows good binding results with HAS compared to 1M17.Communicated by Ramaswamy H. Sarma.

Teratocarcinosarcoma of the head and neck: Clinicopathologic review of a rare entity.

Teratocarcinosarcoma is a rare, highly aggressive malignancy of the head and neck, characterized by multiphenotypic and triphasic growth of epithelial, mesenchymal, and primitive neuroepithelial elements. Owing to its rarity and morphological heterogeneity, as well as the lack of experience with this neoplasm, teratocarcinosarcoma is often misdiagnosed, particularly in small biopsy samples when only some of the elements are identified, thus leading to delayed management. Aggressive clinical behavior and poor survival outcomes, necessitate an accurate diagnosis and appropriate treatment. This review describes the main demographic and clinicopathological features of teratocarcinosarcoma, with an emphasis on the recent advances that have attempted to identify the molecular signature of this neoplasm.

A strip-based assay for detection of CrfA enzyme activity to differentiate Mycobacterium tuberculosis and non-tuberculous mycobacteria.

There is an unmet need for tools that permit diagnosis of Tuberculosis (TB) that are affordable, low-tech, and can differentiate Mycobacterium tuberculosis (M.tb) from non-tuberculous mycobacteria (NTM). In this study, we have developed a strip-based assay to detect the activity of a unique Carbapenem Resistance Factor A (CrfA) enzyme present only in M.tb. The strip comprises of PVDF (Polyvinylidene fluoride) membrane that has an immobilized anti-CrfA antibody to capture the CrfA enzyme from M.tb lysate. Lysate of mycobacteria is applied to the strip, washed, and incubated in the presence of chromogenic reporter dye which is a substrate for CrfA. A change in the color of the dye that is readily visible to the naked eye is the readout. We evaluated lysates from M.tb and various NTMs namely, M. abscessus, M. chelonae, M. avium, M. obuense, M. paraintracellulare, M. kansasi, including the patient-derived sputum samples. The strip assay selectively identified only those samples containing M.tb. Based on this evidence, this new assay enables the identification and differentiation of M.tb from NTMs in patient sputum samples. As this tool can be simple to use, therefore has the potential to serve the unmet need for diagnosis of TB and NTM infections in resource-limited settings.

The Spectrum of Digestive Tract Histopathologic Findings in the Setting of Severe Acute Respiratory Syndrome Coronavirus-2 Infection: What Pathologists Need to Know.

Although the novel severe acute respiratory syndrome coronavirus-2 is known primarily to affect the respiratory system, current evidence supports its capability to infect and induce gastrointestinal tract injury. Data describing the histopathologic alterations of the digestive system in patients infected with severe acute respiratory syndrome coronavirus-2 are becoming more detailed, as the number of studies is increasing and the quality of our insight into the infection and the histopathologic findings is improving. This review highlights the range of pathologic findings that could be observed in gastrointestinal specimens from patients infected with coronavirus disease 2019 and the potential underlying pathogenetic mechanisms of this disease.

Biochemical and histopathological analysis after sub-chronic administration of oxyresveratrol in Wistar rats.

Oxyresveratrol (OXY) is a naturally occurring phenolic compound; however, there are no toxicity studies reported on its long term use. The aim of our work was to demonstrate the evaluation of acute and sub-chronic toxicity of oxyresveratrol in rats to assess its safety profile. To evaluate the LD50 value, 2000 mg/kg of oxyresveratrol was administered to Wistar rats by oral gavage. For sub-chronic toxicity assessment, 80 Wistar rats were randomly divided into four groups (10 animal/sex/group) and oxyresveratrol administered at a dose of 50, 100, 150 mg/kg/day by oral gavage. Bodyweight, food, and water consumption were monitored every week. At the end of the experiments, biochemical and hematological parameters were analyzed. Gross and microscopic organ analyses were also carried out. LD50 of oxyresveratrol was greater than 2000 mg/kg sub-chronic administration of oxyresveratrol did not influence any mortality. Doses of 50 and 100 mg/kg of oxyresveratrol did not produce any sign of toxicity. However, the 150 mg/kg oxyresveratrol group depicted changes in multiple biochemical and hematological parameters with changes in the pathology of cardiac, hepatic, and renal tissues when compared with control. Therefore, no observed adverse effect level (NOAEL) of oxyresveratrol was observed to be 100 mg/kg per day for both male and female rats.

Eccrine Angiomatous Hamartoma With Arteriovenous Malformation: A Rare Entity Re-Explored.

Eccrine angiomatous hamartoma (EAH) is a rare, benign, slow-growing cutaneous lesion characterized by hamartomatous proliferation of the eccrine glands and vascular structures. It usually arises in early childhood; however, cases in adults have also been reported. It is diagnosed based on the clinical features of the lesion as well as the histopathological findings of the excised tissue. As the name indicates, EAH shows a close association with mature eccrine elements and capillary-sized blood vessels at the histopathological level. In rare instances, the vascular component can show the features of arteriovenous malformations. Here, we report a rare case of EAH with a component of arteriovenous malformation in a 39-year-old woman who presented with a foot lesion.

Allosteric cooperation in ß-lactam binding to a non-classical transpeptidase.

L,D-transpeptidase function predominates in atypical 3 → 3 transpeptide networking of peptidoglycan (PG) layer in Mycobacterium tuberculosis. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or ß-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by ß-lactams. Here, we report identification of an allosteric site at a distance of 21 Å from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second ß-lactam molecule and influences binding at the catalytic site. We provide evidence that two ß-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-pocket and one covalently at the catalytic site. This dual ß-lactam-binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new drugs against M. tuberculosis.

Deciphering the involvement of iron targets in colorectal cancer: a network biology approach.

Several studies suggested the role of heme iron, but not non-heme iron in colorectal cancer. A network and system biology-based approach was used to understand the role of heme and non-heme iron on colorectal cancer etiology. Heme and non-heme iron targets were screened in addition to CRC targets. The protein-protein interaction map of both iron targets was prepared with CRC targets. Moreover, functional enrichment analysis was performed in order to understand their role in cancer etiology. The heme iron is predicted to modulate several cancer-associated pathways. Our results indicate several targets and pathways, including IL-4/IL-13, ACE, and HIF-1 signaling, that may have an important role in heme iron-mediated CRC and must be given consideration for understanding their role in colorectal cancer.

Panniculitis Ossificans in Posterior Knee: An Unusual Presentation.

Panniculitis ossificans (PO) is a heterotopic ossification, benign recurring lesion, presenting in a variety of presentations anywhere in the body. The condition can sometimes be mistaken for a malignant bone tumor or long list of other differential diagnoses which have been mentioned in our report and that may lead to unnecessary wrong management. We report a case of a patient with subcutaneous ossification in the posterior aspect of the left knee. PO was confirmed histologically showing subcutaneous fat necrosis associated with osteoid material.

Anaplastic Large Cell Lymphoma, Giant Cell-Rich, Involving a Nonimplant Breast: A Case Report and Review of the Literature.

Primary breast lymphomas are uncommon tumors and account for <1% of all malignant neoplasms of the breast. They are almost always of non-Hodgkin type, with B-cell lymphomas being the most common subtype. Anaplastic large cell lymphoma (ALCL) is a rare T-cell lymphoma that can involve the breast. Most of the articles in the literature describe ALCL in association with breast implants. We present a 48-year-old woman with a left breast enlargement and no history of an implant. Microscopic sections showed a high-grade CD30-positive lymphoid neoplasm with frequent giant cells, which turned out to be a primary ALCL of the breast, giant cell-rich pattern. To our knowledge, no cases of primary ALCL, giant cell-rich variant, have been reported in the breast in the absence of an implant making our case unique.

Sporadic Cutaneous Keratocyst of the Scalp: A Report of an Extremely Rare Lesion.

Keratocysts, which are benign cystic lesions that usually arise in the jawbones, particularly the mandible, can rarely occur in the skin and soft tissues. Odontogenic keratocysts, which are keratocysts in the jaw, are considered hallmarks of nevoid basal cell carcinoma syndrome (NBCCS). In contrast, cutaneous keratocysts (CKCs), which can occur in association with NBCCS, are rare. To date, only a few studies have reported CKCs arising independently of NBCCS. Herein, we report a rare case of CKC in the scalp of a 49-year-old female patient without the clinical features of NBCCS, intending to increase awareness of this rare presentation among pathologists who encounter cutaneous cystic lesions in daily practice.

The safety and efficacy of BCG encapsulated alginate particle (BEAP) against M.tb H37Rv infection in Macaca mulatta : A pilot study.

Due to the limited utility of Bacillus Calmette-Guérin (BCG), the only approved vaccine available for tuberculosis, there is a need to develop a more effective and safe vaccine. We evaluated the safety and efficacy of a dry powder aerosol (DPA) formulation of BCG encapsulated alginate particle (BEAP) and the conventional intradermal BCG immunization in infant rhesus macaques (Macaca mulatta). The infant macaques were immunized intratracheally with DPA of BEAP into the lungs. Animals were monitored for their growth, behaviour, any adverse and allergic response. The protective efficacy of BEAP was estimated by the ex-vivo H37Rv infection method. Post-immunization with BEAP, granulocytes count, weight gain, chest radiography, levels of liver secreted enzymes, cytokines associated with inflammation like TNF and IL-6 established that BEAP is non-toxic and it does not elicit an allergic response. The T cells isolated from BEAP immunized animals' blood, upon stimulation with M.tb antigen, secreted high levels of IFN-γ, TNF, IL-6 and IL-2. The activated T cells from BEAP group, when co-cultured with M.tb infected macrophages, eliminated largest number of infected macrophages compared to the BCG and control group. This study suggests the safety and efficacy of BEAP in Non-human primate model.

Development of a Lung Cancer Model in Wistar Rat and In Silico Screening of its Biomarkers.

BACKGROUND: Cancer is usually caused by three factors: Nutrition, inflammation and cigarette smoke. This study on rat experimental models would enable us to understand the mechanism of lung cancer caused by NNK to which humans are continuously exposed, help us understand possible molecular targets, and assist in designing drugs for humans against lung cancer. AIM: A lung cancer model was developed by administering tobacco-specific carcinogen: NNK [4- methylnitrosamino)-1-(3-pyridyl)-1-butanone] to male Wistar rats for 24 weeks. Furthermore, in silico approach was followed to screen the molecular targets. METHODS: A method was established in which subcutaneous and intraperitoneal injections of NNK were administered to male Wistar rats simultaneously. For authentication of lung cancer in vivo, we performed molecular docking simulations with protein biomarkers: Cox-2, p53, p38 MAPKs and EGFR using Hex-Discovery Studio, Schrödinger-maestro software. RESULTS: Lung morphology and histopathology indicated the initiation of bronchiolar epithelial hyperplasia and squamous dysplasia in the cancer 1 group after 16 weeks of NNK exposure. 66.66% incidence of squamous cell carcinoma (SCC) and 33.3% incidence of adenocarcinoma were observed in cancer 2 group after being exposed to NNK. Results indicated that the incidence of SCC and adenocarcinoma gradually increased from 66.66% to 85.71% in cancer 2 group and from 33.33% to 42.58% in cancer 3 group, respectively. Docking results indicate the total binding energy and glide energy of Cox-2, p53, p38 MAPKs, EGFR : 38.14, -211.58, -181.58, -213.05 Kcal/mol and -39.25, -32.16,-36.49, -40.19 Kcal/mol, respectively. CONCLUSION: Pulmonary adenocarcinoma model was developed by administering tobacco-specific carcinogen: NNK [4-methylnitrosamino)-1-(3-pyridyl)-1-butanone] to male Wistar rats in 24 weeks. In silico experiments confirmed EGFR to be the most potential target for NNK induced lung Cancer.

An inexpensive and easy-to-make customized antibiotics mix for mycobacterium culture.

The cultivation of mycobacteria often requires the use of several antibiotics to limit the growth of other rapidly growing micro-flora present in the growth medium. This antibiotic cocktail is one of the most expensive reagents required for mycobacterium culture. Here we present a customized antibiotics mix that is easy to prepare at a fraction of the cost of the commercially available antibiotic mixture that protects against transient flora, which are normally present in lungs, without affecting mycobacterial colony number.

s8ORF2 protein of infectious salmon anaemia virus is a RNA-silencing suppressor and interacts with Salmon salar Mov10 (SsMov10) of the host RNAi machinery.

The infectious salmon anaemia virus (ISAV) is a piscine virus, a member of Orthomyxoviridae family. It encodes at least 10 proteins from eight negative-strand RNA segments. Since ISAV belongs to the same virus family as Influenza A virus, with similarities in protein functions, they may hence be characterised by analogy. Like NS1 protein of Influenza A virus, s8ORF2 of ISAV is implicated in interferon antagonism and RNA-binding functions. In this study, we investigated the role of s8ORF2 in RNAi suppression in a well-established Agrobacterium transient suppression assay in stably silenced transgenic Nicotiana xanthi. In addition, s8ORF2 was identified as a novel interactor with SsMov10, a key molecule responsible for RISC assembly and maturation in the RNAi pathway. This study thus sheds light on a novel route undertaken by viral proteins in promoting viral growth, using the host RNAi machinery.

Crystal structure of calcium binding protein-5 from Entamoeba histolytica and its involvement in initiation of phagocytosis of human erythrocytes.

Entamoeba histolytica is the etiological agent of human amoebic colitis and liver abscess, and causes a high level of morbidity and mortality worldwide, particularly in developing countries. There are a number of studies that have shown a crucial role for Ca2+ and its binding protein in amoebic biology. EhCaBP5 is one of the EF hand calcium-binding proteins of E. histolytica. We have determined the crystal structure of EhCaBP5 at 1.9 Šresolution in the Ca2+-bound state, which shows an unconventional mode of Ca2+ binding involving coordination to a closed yet canonical EF-hand motif. Structurally, EhCaBP5 is more similar to the essential light chain of myosin than to Calmodulin despite its somewhat greater sequence identity with Calmodulin. This structure-based analysis suggests that EhCaBP5 could be a light chain of myosin. Surface plasmon resonance studies confirmed this hypothesis, and in particular showed that EhCaBP5 interacts with the IQ motif of myosin 1B in calcium independent manner. It also appears from modelling of the EhCaBP5-IQ motif complex that EhCaBP5 undergoes a structural change in order to bind the IQ motif of myosin. This specific interaction was further confirmed by the observation that EhCaBP5 and myosin 1B are colocalized in E. histolytica during phagocytic cup formation. Immunoprecipitation of EhCaBP5 from total E. histolytica cellular extract also pulls out myosin 1B and this interaction was confirmed to be Ca2+ independent. Confocal imaging of E. histolytica showed that EhCaBP5 and myosin 1B are part of phagosomes. Overexpression of EhCaBP5 increases slight rate (∼20%) of phagosome formation, while suppression reduces the rate drastically (∼55%). Taken together, these experiments indicate that EhCaBP5 is likely to be the light chain of myosin 1B. Interestingly, EhCaBP5 is not present in the phagosome after its formation suggesting EhCaBP5 may be playing a regulatory role.

Transgenesis-mediated reproductive dysfunction and tumorigenesis: effects of immunological neutralization.

Human chorionic gonadotropin (hCG) was initially thought to be made only during pregnancy, but is now known to also be synthesized by a variety of cancers and is associated with poor patient prognosis. Transgenic expression of ßhCG in mice causes hyper-luteinized ovaries, a loss in estrous cyclicity and infertility, increased body weight, prolactinomas and mammary gland tumors. Strategies were devised to generate antibody responses against hCG to investigate whether reversal of the molecular processes driving tumorigenesis would follow. hCG-immunized transgenic mice did not exhibit increases in body weight or serum prolactin levels, and gross ovarian and pituitary morphology remained normal. While non-immunized transgenic animals demonstrated heightened levels of transcripts associated with pituitary tumorigenesis (HMG2A, E2F1, CCND1, PRL, GH, GAL, PTTG1, BMP4) and decreased levels of CDK inhibitors CDKN1B (p27), CDKN2A (p16) and CDKN2c (p18), immunization led to a reversal to levels found in non-transgenic animals. Serum derived from transgenic (but not non-transgenic) mice led to enhanced transcription as well as expression of VEGF, IL-8, KC (murine IL-8) and MMP-9 in tumor cells, effects not seen when sera derived from hCG-immunized transgenic mice was employed. As the definitive indication of the restoration of the reproductive axis, immunization led to the resumption of estrous cyclicity as well as fertility in transgenic mice. These results indicate that hCG may influence cancer pathogenesis and progression via several distinct mechanisms. Using a stringent in vivo system in which ßhCG acts both a "self" antigen and a tumor-promoting moiety (putatively akin to the situation in humans), the data builds a case for anti-gonadotropin vaccination strategies in the treatment of gonadotropin-dependent or secreting malignancies that frequently acquire resistance to conventional therapy.

Cloning, purification, crystallization and preliminary crystallographic study of calcium-binding protein 5 from Entamoeba histolytica.

Entamoeba histolytica is the causative agent of human amoebiasis. Phagocytosis is the major route of food intake by this parasite and is responsible for its virulence. Calcium and calcium-binding proteins play major roles in its phagocytosis. Calcium-binding protein 5 from E. histolytica (EhCaBP5) is a cytoplasmic protein; its expression is very sensitive to serum starvation and it seems to be involved in binding to myosin I. In this study, EhCaBP5 was cloned, expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. The purified protein crystallized in space group C222 and the crystals diffracted to 2 Šresolution. The Matthews coefficient indicated the presence of one molecule in the asymmetric unit, with a VM of 2.35 Å3 Da(-1) and a solvent content of 47.7%.