Thermodynamics of ferredoxin binding to cyanobacterial nitrate reductase.

The role of the seven negatively charged amino acids of Synechocystis sp. PCC 6803 ferredoxin (Fd), i.e., Glu29, Glu30, Asp60, Asp65, Asp66, Glu92, and Glu93, predicted to form complex with nitrate reductase (NR), was investigated using site-directed mutagenesis and isothermal titration calorimetry (ITC). These experiments identified four Fd amino acids, i.e., Glu29, Asp60, Glu92, and Glu93, that are essential for the Fd binding and efficient electron transfer to the NR. ITC measurements showed that the most likely stoichiometry for the wild-type NR/wild-type Fd complex is 1:1, a Kd value 4.7 µM for the complex at low ionic strength residues and both the enthalpic and entropic components are associated with complex formation. ITC titrations of wild-type NR with four Fd variants, E29N, D60N, E92Q, and E93N demonstrated that the complex formation, although favorable, was less energetically favorable when compared to complex formation between the two wild-type proteins, suggesting that these negatively charged Fd residues at these positions are important for the effective and productive interaction with wild-type enzyme.

Corrigendum to "Role of UHRF1 in malignancy and its function as a therapeutic target for molecular docking towards the SRA domain" [Int. J. Biochem. Cell Biol. 114 (September 2019) 105558].

Cancer pathogenesis has been attributed to the minor and major disruptions in the cell cycle, with a key role being played by several of the recently discovered epigenetic factors. Lately, UHRF1 (Ubiquitin-like with containing PHD and RING Finger domains 1), an epigenetic regulator has been shown to be evidently over expressed in numerous malignancies through an in-depth review of literature. Molecular docking studies have found that existing drugs such as propranolol, naphthazarin and thymoquinone have favourable interactions with specific domains of UHRF1. However, these findings would need large scale clinical trials to confirm their potency and safety during chemotherapy. UHRF1 (Ubiquitin-like with containing PHD and RING Finger domains 1), an epigenetic factor, plays a crucial role as an important checkpoint in the cell machinery. Basic science continues to unravel multiple facets of this five domain protein which includes a detailed elucidation of its roles and mechanisms of interaction with various enzymes during DNA replication. The gene has recently begun to be also termed as the "Universal Oncogene" in response to the results of research conducted in heterogenous populations and in over 17 cancers displaying heightened mRNA and protein expression in breast, liver, lung, head and neck cancers and many more. This gene could therefore, be a potential biomarker for diagnosis and for the prediction of the prognosis and survival of the diseased. A scientifically established solution in the form of targeted treatment must follow such a discovery and therefore, several natural and synthetic compounds such as thymoquinone and the well-known antihypertensive, propranolol have been docked and reported to have favourable interactions with the SRA (Set and Ring Associated) domain of UHRF1 in this review. This comprehensive review is thus, a brief synopsis of details regarding the structure and heightened levels of UHRF1 in several malignancies. Furthermore, pharmacogenomic research revolving around this oncogene is a potential sphere for clinical studies to be conducted in much larger and heterogenous populations to not only validate these therapeutic docking results but to also to bring personalised medicine to the bedside for the benefit of the patients.

Role of UHRF1 in malignancy and its function as a therapeutic target for molecular docking towards the SRA domain.

The figure describes the location of UHRF1 (Ubiquitin-like with containing PHD and RING Finger domains 1) gene, mRNA and protein synthesis in the tumor cell and its structural domains with a focus on the docking of Naphthazarin on the SRA domain of UHRF1, resulting in reduction of tumor size.

Photobiomodulation therapy in neuroischaemic diabetic foot ulcers: a novel method of limb salvage.

OBJECTIVE: Low-level laser therapy (also known as photobiomodulation therapy, PBMT) promotes accelerated healing of diabetic foot ulcers (DFUs), thereby preventing the risk of future complications and amputation. The aim of this study was to determine the effect of PBMT, with structured, graded mobilisation and foot care, on DFU healing dynamics. METHOD: Patients diagnosed with type 2 diabetes, diabetic peripheral neuropathy and presenting with a chronic neuroischaemic DFU, were treated with PBMT using scanning and non-contact probe methods. The DFU was clinically observed and the area measured every seven days until complete healing. Neuropathic parameters were also measured. The PBMT was administered until complete closure of the DFU and patients also undertook a programme of graded mobilisation. RESULTS: A total of 17 participants were recruited, with a mean age of 69±8 years, and a mean duration of diabetes of 13±5 years. Mean complete closure time was 26±11days. In addition, a mean reduction of the semi-quantitative vibration pressure threshold from 49±2 volts to 20±4 volts was observed in all participants. CONCLUSION: PBMT can be effectively used as a treatment mode for neuroischaemic DFUs in patients with type 2 diabetes. Graded mobilisation with focused foot care could improve the function of people living with type 2 diabetes with a chronic DFU.

Evaluation of efficacy of thermographic breast imaging in breast cancer: A pilot study.

BACKGROUND: Thermographic imaging is a non-invasive and radiation free imaging modality that measures the infrared radiation released by the body. Recently, there is a renewed interest regarding the scope of thermal imaging for breast cancer. OBJECTIVE: To evaluate the efficacy of thermographic breast imaging in detecting breast cancer. METHODS: A Prospective observational study was carried out from January 2014 to December 2014 at Kasturba Hospital, Manipal, India. Patients in whom breast cancer was confirmed on FNAC or biopsy, were included in the study and further evaluated with thermographic imaging of the breast. RESULTS: 65 patients with FNAC or biopsy proven breast carcinoma were included in the study. Using thermographic imaging, malignancy was accurately detected in 60 patients (92.31%). Mammography was able to detect malignancy in 62 out of the 65 patients (95.38%). Thermography was able to detect malignancy in all 3 cases in which conventional mammography missed it. CONCLUSION: Thermography may have a role in detection of breast cancer. However, it is too early to recommend thermographic imaging as a standard imaging modality for breast cancer. Larger studies are required to evaluate the usefulness of thermography in diagnosis and/or screening of breast cancer.

Semi-automated breast cancer tumor detection with thermographic video imaging.

Screening for breast cancer enables early detection by which curative treatment can be possible. While mammography is the current gold standard for screening, it has low sensitivity in younger women and its harmful X-rays in frequent screening can increase the risk of cancer. Incidence rates are rising in younger women, causing a relook at thermography for low cost and non-harmful screening. In this paper, thermography is compared to mammography correlated with sono-mammography in 65 FNAC/biopsy proven cancer subjects in India. Thermography is comparable to mammography correlated with sono-mammography, having 94% and 95% sensitivity, respectively. A novel semi-automated thermographic tumor detection and location algorithm used in this paper also provides 97% sensitivity. This shows the promise of automated thermographic screening for reaching large populations in a cost effective manner in low resource settings in countries like India. Further studies in a large scale need to be done to evaluate the specificity to enable such solutions.

Overexpression of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) in HepG2 cells leads to increased hyaluronan synthesis and cell proliferation by up-regulation of cyclin D1 in AKT-dependent pathway.

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.

Photobiomodulation by helium neon and diode lasers in an excisional wound model: A single blinded trial.

BACKGROUND: Application of different kinds of lasers in clinical and experimental studies causes photobiomodulation that works at localized cellular and humoral level on various biological systems. Increased numbers of fibroblasts, myofibroblast, and degranulation of mast cells have been the observed benefits post-irradiation. OBJECTIVE: Was to find out the effect of irradiation with energy densities of 3.38 J/cm(2), 8 J/cm(2), and 18 J/cm(2) on animal tissue (albino wistar rats) in an excisional wound model and to assess changes in biochemical (hydroxyproline) and histopathological levels in excisional wound model. MATERIALS AND METHODS: The animals were divided into 4 groups, which were labeled as L1, diode laser (18 J/cm(2)), L2 Helium-neon (He-Ne, 8 J/cm(2)), L3 diode laser (3.38 J/cm(2)), and sham treatment for control was depicted by C, respectively. Histological and hydroxyproline analysis was performed on 7, 14, 21 days of post-wounding. One-way analysis of variance, ANOVA and Bonferroni's multiple comparison tests were done for tissue hydroxyproline levels. RESULTS: There was no significant increase in the hydroxyproline content (P < 0.005) when observed in study group and compared to controls. Whereas significant epithelizations was seen in group treated with He-Ne laser of intensity of 8 J/cm(2). CONCLUSION: The experimental observations suggest that low intensity helium-neon laser of 8 J/cm(2) intensity facilitated photo stimulation by tissue repair, but failed to show significant tissue hydroxyproline levels in excisional wound model.

A Comparative Study of Fine Needle Aspiration Cytology (FNAC) and Fine Needle Non-aspiration Cytology (FNNAC) Technique in Lesions of Thyroid Gland.

The present study was done to compare diagnostic yield by fine needle non aspiration technique (FNNAC) with Fine Needle Aspiration cytology (FNAC) of lesions in thyroid gland. FNNAC and FNAC both were performed on 69 patients presenting with thyroid lesions, except those suffering from thyrotoxicosis. Smears were then cytologically interpreted by a single pathologist as unsuitable, diagnostically adequate or diagnostically superior for opinion, without the knowledge of sampling method employed. Slides were evaluated for following aspects namely cellularity, presence of colloid, inflammatory cell, hemosiderin laden macrophages, papillary clusters nuclear overlapping, nuclear grooving, hurthle cells. FNNAC gave better results in form of better quality of cellularity and less field obscurity by blood in lesions of thyroid. Diagnostically superior specimens were obtained more frequently by FNNAC, so this technique should be used alone or in tandem with FNAC for better diagnostic yield.

Evidence for inhibitory interaction of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) with Streptococcus pneumoniae hyaluronidase.

Bacterial hyaluronan lyase enzymes are the major virulence factors that enable greater microbial ingress by cleaving hyaluronan (HA) polymers present predominantly in extracellular space of vertebrates. Based on the premise that effective inhibitors may bind to and stabilize HA thereby protecting it from degradation, here we investigated inhibitory activity of human hyaluronan-binding protein 1 (HABP1) on bacterial hyaluronidase because it is highly specific to HA and localized on the cell surface. Biochemical characterization revealed that HABP1 is a competitive inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL) with an IC50 value of 22 microm. This is thus the first report of an endogenous protein inhibitor that may be used during natural antibacterial defense. Our findings also support a novel multipronged mechanism for the high efficacy of HABP1-mediated inhibition based on structural modeling of enzyme, substrate, and inhibitor. Evidence from docking simulations and contact interface interactions showed that the inherent charge asymmetry of HABP1 plays a key role in the inhibitory activity. This novel role of HABP1 may pave the way for peptide inhibitors as alternatives to synthetic chemicals in antibacterial research.

Xeroderma pigmentosa in siblings: cystosarcoma phylloides in a case of xeroderma pigmentosa.

Xeroderma pigmentosa is a rare autosomal recessive disorder with numerous oculocutaneous neoplasms, neurological abnormalities and extremely rarely associated with neoplasms of organs other than the skin and the eyes. Here we report two cases of xeroderma pigmentosa with numerous oculocutaneous malignancies in siblings born to a consanguinously married normal parents. One of these patients had a cystosarcoma phylloides in association with xeroderma pigmentosa, hitherto not reported in literature.

Processing of Mycobacterium tuberculosis antigen 85B involves intraphagosomal formation of peptide-major histocompatibility complex II complexes and is inhibited by live bacilli that decrease phagosome maturation.

Mycobacterium tuberculosis (MTB) inhibits phagosomal maturation to promote its survival inside macrophages. Control of MTB infection requires CD4 T cell responses and major histocompatibility complex (MHC) class II (MHC-II) processing of MTB antigens (Ags). To investigate phagosomal processing of MTB Ags, phagosomes containing heat-killed (HK) or live MTB were purified from interferon-gamma (IFN-gamma)-activated macrophages by differential centrifugation and Percoll density gradient subcellular fractionation. Flow organellometry and Western blot analysis showed that MTB phagosomes acquired lysosome-associated membrane protein-1 (LAMP-1), MHC-II, and H2-DM. T hybridoma cells were used to detect MTB Ag 85B(241-256)-I-A(b) complexes in isolated phagosomes and other subcellular fractions. These complexes appeared initially (within 20 min) in phagosomes and subsequently (>20 min) on the plasma membrane, but never within late endocytic compartments. Macrophages processed HK MTB more rapidly and efficiently than live MTB; phagosomes containing live MTB expressed fewer Ag 85B(241-256)-I-A(b) complexes than phagosomes containing HK MTB. This is the first study of bacterial Ag processing to directly show that peptide-MHC-II complexes are formed within phagosomes and not after export of bacterial Ags from phagosomes to endocytic Ag processing compartments. Live MTB can alter phagosome maturation and decrease MHC-II Ag processing, providing a mechanism for MTB to evade immune surveillance and enhance its survival within the host.

Phagosomes acquire nascent and recycling class II MHC molecules but primarily use nascent molecules in phagocytic antigen processing.

Phagosomes contain class II MHC (MHC-II) and form peptide:MHC-II complexes, but the source of phagosomal MHC-II molecules is uncertain. Phagosomes may acquire nascent MHC-II or preexisting, recycling MHC-II that may be internalized from the plasma membrane. Brefeldin A (BFA) was used to deplete nascent MHC-II in murine macrophages to determine the relative contributions of nascent and recycling MHC-II molecules to phagocytic Ag processing. In addition, biotinylation of cell-surface proteins was used to assess the transport of MHC-II from the cell surface to phagosomes. BFA inhibited macrophage processing of latex bead-conjugated Ag for presentation to T cells, suggesting that nascent MHC-II molecules are important in phagocytic Ag processing. Furthermore, detection of specific peptide:MHC-II complexes in isolated phagosomes confirmed that BFA decreased formation of peptide:MHC-II complexes within phagosomes. Both flow organellometry and Western blot analysis of purified phagosomes showed that about two-thirds of phagosomal MHC-II was nascent (depleted by 3 h prior treatment with BFA) and primarily derived from intracellular sites. About one-third of phagosomal MHC-II was preexisting and primarily derived from the plasma membrane. BFA had little effect on phagosomal H2-DM or the degradation of bead-associated Ag. Thus, inhibition of phagocytic Ag processing by BFA correlated with depletion of nascent MHC-II in phagosomes and occurred despite the persistent delivery of plasma membrane-derived recycling MHC-II molecules and other Ag-processing components to phagosomes. These observations suggest that phagosomal Ag processing depends primarily on nascent MHC-II molecules delivered from intracellular sites, e.g., endocytic compartments.

Phagocytic antigen processing and effects of microbial products on antigen processing and T-cell responses.

Processing of exogenous antigens and microbes involves contributions by multiple different endocytic and phagocytic compartments. During the processing of soluble antigens, different endocytic compartments have been demonstrated to use distinct antigen-processing mechanisms and to process distinct sets of antigenic epitopes. Processing of particulate and microbial antigens involves phagocytosis and functions contributed by phagocytic compartments. Recent data from our laboratory demonstrate that phagosomes containing antigen-conjugated latex beads are fully competent class II MHC (MHC-II) antigen-processing organelles, which generate peptide:MHC-II complexes. In addition, phagocytosed antigen enters an alternate class I MHC (MHC-I) processing pathway that results in loading of peptides derived from exogenous antigens onto MHC-I molecules, in contrast to the cytosolic antigen source utilized by the conventional MHC-I antigen-processing pathway. Antigen processing and other immune response mechanisms may be activated or inhibited by microbial components to the benefit of either the host or the pathogen. For example, antigen processing and T-cell responses (e.g. Th1 vs Th2 differentiation) are modulated by multiple distinct microbial components, including lipopolysaccharide, cholera toxin, heat labile enterotoxin of Escherichia coli, DNA containing CpG motifs (found in prokaryotic and invertebrate DNA but not mammalian DNA) and components of Mycobacterium tuberculosis.

Phagosomes are fully competent antigen-processing organelles that mediate the formation of peptide:class II MHC complexes.

During the processing of particulate Ags, it is unclear whether peptide:class II MHC (MHC-II) complexes are formed within phagosomes or within endocytic compartments that receive Ag fragments from phagosomes. Murine macrophages were pulsed with latex beads conjugated with OVA. Flow or Western blot analysis of isolated phagosomes showed extensive acquisition of MHC-II, H-2M, and invariant chain within 30 min, with concurrent degradation of OVA. T hybridoma responses to isolated subcellular fractions demonstrated OVA (323-339):I-Ad complexes in phagosomes and plasma membrane but not within dense late endocytic compartments. Furthermore, when two physically separable sets of phagosomes were present within the same cells, OVA(323-339):I-Ad complexes were demonstrated in latex-OVA phagosomes but not in phagosomes containing latex beads conjugated with another protein. This implies that these complexes were formed specifically within phagosomes and were not formed elsewhere and subsequently transported to phagosomes. In addition, peptide:MHC-II complexes were shown to traffic from phagosomes to the cell surface. In conclusion, phagosomes are fully competent to process Ags and generate peptide:MHC-II complexes that are transported to the cell surface and presented to T cells.

Phagocytic processing of antigens for presentation by class II major histocompatibility complex molecules.

Microbes and other particulate antigens (Ags) are internalized by phagocytosis and then reside in plasma membrane-derived phagosomes. The contribution of phagosomes to the degradation of Ags has long been appreciated. It has been unclear, however, whether peptides derived from these degraded antigens bind class II major histocompatibility complex (MHC-II) molecules within phagosomes or within endocytic compartments that receive Ag fragments from phagosomes. Recent experiments have demonstrated that phagosomes containing Ag-conjugated latex beads express a full complement of Ag-processing molecules, e.g. MHC-II molecules, invariant chain, H2-DM and proteases sufficient to degrade bead- associated Ag. These phagosomes mediate the formation of peptide-MHC-II complexes, which are transported to the cell surface and presented to T cells. Phagosomes acquire both newly synthesized and plasma membrane-derived MHC-II molecules, but the formation of peptide-MHC-II complexes in phagosomes primarily involves newly synthesized MHC-II molecules. The content and traffic of phagosomal proteins vary considerably with the type of Ag ingested. Pathogenic microbes can alter phagosome composition and function to reduce Ag processing. For example, Mycobacterium tuberculosis blocks the maturation of phagosomes and reduces the ability of infected cells to present exogenous soluble protein Ags.

Flow analysis of MHC molecules and other membrane proteins in isolated phagosomes.

A method was developed to apply flow cytometry analysis to the characterization of individual phagosomes. Macrophages were incubated with latex beads and homogenized to release the phagosomes. Intact cells and nuclei were removed by low speed centrifugation, and a crude phagosome preparation was fixed with paraformaldehyde. Distinct optical properties of latex bead phagosomes allowed their analytic isolation from other organelles and cell fragments by flow analysis using a narrow gate based on scatter parameters. Furthermore, separate gates were established for phagosomes containing one, two and even three beads, which were sorted and examined by electron microscopy (EM). EM showed that the phagosomal membrane was closely apposed to the latex bead in most phagosomes, but some more spacious phagosomes were also observed. Phagosomes were immunolabeled and subjected to flow analysis for MHC-I and MHC-II molecules and lysosomal membrane markers (LAMPs). The proportion of LAMP-positive phagosomes increased with incubation time, reflecting maturation of phagolysosomes. Significant staining for MHC-I and MHC-II was demonstrated and remained relatively constant with time. Flow analysis of phagosomes allows the characterization and comparison of individual phagosomes, and the identification of subpopulations of phagosomes with differing membrane compositions. It also provides the advantage of analytically isolating phagosomes from other components of the cell without the need for extensive prior physical purification. Thus, it can be used to rapidly assess changes in phagosomal membrane composition as a function of phagosome maturation.