The IMEx coronavirus interactome: an evolving map of Coronaviridae-host molecular interactions.

The current coronavirus disease of 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2, has spurred a wave of research of nearly unprecedented scale. Among the different strategies that are being used to understand the disease and develop effective treatments, the study of physical molecular interactions can provide fine-grained resolution of the mechanisms behind the virus biology and the human organism response. We present a curated dataset of physical molecular interactions focused on proteins from SARS-CoV-2, SARS-CoV-1 and other members of the Coronaviridae family that has been manually extracted by International Molecular Exchange (IMEx) Consortium curators. Currently, the dataset comprises over 4400 binarized interactions extracted from 151 publications. The dataset can be accessed in the standard formats recommended by the Proteomics Standards Initiative (HUPO-PSI) at the IntAct database website (https://www.ebi.ac.uk/intact) and will be continuously updated as research on COVID-19 progresses.

The IMEx Coronavirus interactome: an evolving map of Coronaviridae-Host molecular interactions.

The current Coronavirus Disease 2019 (COVID-19) pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has spurred a wave of research of nearly unprecedented scale. Among the different strategies that are being used to understand the disease and develop effective treatments, the study of physical molecular interactions enables studying fine-grained resolution of the mechanisms behind the virus biology and the human organism response. Here we present a curated dataset of physical molecular interactions, manually extracted by IMEx Consortium curators focused on proteins from SARS-CoV-2, SARS-CoV-1 and other members of the Coronaviridae family. Currently, the dataset comprises over 2,200 binarized interactions extracted from 86 publications. The dataset can be accessed in the standard formats recommended by the Proteomics Standards Initiative (HUPO-PSI) at the IntAct database website ( www.ebi.ac.uk/intact ), and will be continuously updated as research on COVID-19 progresses.

In vitro susceptibility of filamentous fungi from mycotic keratitis to azole drugs.

OBJECTIVE: The in vitro antifungal activities of azole drugs viz., itraconazole, voriconazole, ketoconazole, econazole and clotrimazole were investigated in order to evaluate their efficacy against filamentous fungi isolated from mycotic keratitis. METHODS: The specimen collection was carried out from fungal keratitis patients attending Aravind eye hospital and Post-graduate institute of ophthalmology, Coimbatore, India and was subsequently processed for the isolation of fungi. The dilutions of antifungal drugs were prepared in RPMI 1640 medium. Minimum inhibitory concentrations (MICs) were determined and MIC50 and MIC90 were calculated for each drug tested. RESULTS: A total of 60 fungal isolates were identified as Fusarium spp. (n=30), non-sporulating moulds (n=9), Aspergillus flavus (n=6), Bipolaris spp. (n=6), Exserohilum spp. (n=4), Curvularia spp. (n=3), Alternaria spp. (n=1) and Exophiala spp. (n=1). The MICs of ketoconazole, clotrimazole, voriconazole, econazole and itraconazole for all the fungal isolates ranged between 16 µg/mL and 0.03 µg/mL, 4 µg/mL and 0.015 µg/mL, 8 µg/mL and 0.015 µg/mL, 8 µg/mL and 0.015 µg/mL and 32 µg/mL and 0.06 µg/mL respectively. From the MIC50 and MIC90 values, it could be deciphered that in the present study, clotrimazole was more active against the test isolates at lower concentrations (0.12-5 µg/mL) when compared to other drugs tested. CONCLUSION: The results suggest that amongst the tested azole drugs, clotrimazole followed by voriconazole and econazole had lower MICs against moulds isolated from mycotic keratitis.

Diagnosis of arthritis through fuzzy inference system.

Expert or knowledge-based systems are the most common type of AIM (artificial intelligence in medicine) system in routine clinical use. They contain medical knowledge, usually about a very specifically defined task, and are able to reason with data from individual patients to come up with reasoned conclusion. Although there are many variations, the knowledge within an expert system is typically represented in the form of a set of rules. Arthritis is a chronic disease and about three fourth of the patients are suffering from osteoarthritis and rheumatoid arthritis which are undiagnosed and the delay of detection may cause the severity of the disease at higher risk. Thus, earlier detection of arthritis and treatment of its type of arthritis and related locomotry abnormalities is of vital importance. Thus the work was aimed to design a system for the diagnosis of Arthitis using fuzzy logic controller (FLC) which is, a successful application of Zadeh's fuzzy set theory. It is a potential tool for dealing with uncertainty and imprecision. Thus, the knowledge of a doctor can be modelled using an FLC. The performance of an FLC depends on its knowledge base which consists of a data base and a rule base. It is observed that the performance of an FLC mainly depends on its rule base, and optimizing the membership function distributions stored in the data base is a fine tuning process.

Optimization of Antimicrobial Production by a Marine Actinomycete Streptomyces afghaniensis VPTS3-1 Isolated from Palk Strait, East Coast of India.

Totally 25 marine soil samples were collected from the region of Palk Strait of Bay of Bengal, Tamil Nadu, and were subjected to the isolation of actinomycetes. Sixty-eight morphologically distinct isolates were obtained and 37% (25) of them had antimicrobial activity. The potential producer was named as Streptomyces sp. VPTS3-1 and the phylogenetic evaluation on the basis of 16S rDNA sequence further categorized the organism as Streptomyces afghaniensis VPTS3-1. Further, the antimicrobial compound was extracted from the isolate using various solvents and the antimicrobial efficacies were tested against bacterial and fungal pathogens. In addition, in vitro optimization of parameters for the antimicrobial compound production revealed that the suitable pH as 7-8, the period of incubation as 9 days, temperature (30°C), salinity (2%), and starch and KNO3 as the suitable carbon and nitrogen sources respectively in starch-casein medium.

Antidiabetic and antihyperlipidemic potential of Abelmoschus esculentus (L.) Moench. in streptozotocin-induced diabetic rats.

OBJECTIVES: The present investigation was aimed to study the antidiabetic and antihyperlipidemic potential of Abelmoschus esculentus peel and seed powder (AEPP and AESP) in streptozotocin (STZ)-induced diabetic rats. MATERIALS AND METHODS: Acute toxicity of AEPP and AESP was studied in rats at 2000 mg/kg dose and diabetes was induced in rats by administration of STZ (60 mg/kg, i.p.). After 14 days of blood glucose stabilization, diabetic rats received AEPP, AESP, and glibenclamide up to 28 days. The blood samples were collected on day 28 to estimate the hemoglobin (Hb), glycosylated hemoglobin (HbA1c), serum glutamate-pyruvate transferase (SGPT), total protein (TP), and lipid profile levels. RESULTS: In acute toxicity study, AESP and AESP did not show any toxicity or death up to a dose of 2000 mg/kg. Therefore, to assess the antidiabetic action, one by fifth and one by tenth dose of both powders were selected. Administration of AEPP and AESP at 100 and 200 mg/kg dose in diabetic rats showed significant (P < 0.001) reduction in blood glucose level and increase in body weight than diabetic control rats. A significant (P < 0.001) increased level of Hb, TP, and decreased level of HbA1c, SGPT were observed after the treatment of both doses of AEPP and AESP. Also, elevated lipid profile levels returned to near normal in diabetic rats after the administration of AEPP and AESP, 100 and 200 mg/kg dose, compared to diabetic control rats. CONCLUSION: The present study results, first time, support the antidiabetic and antihyperlipidemic potential of A. esculentus peel and seed powder in diabetic rats.

Prevalence and antimicrobial susceptibility pattern of methicillin resistant Staphylococcus aureus: a multicentre study.

PURPOSE: Methicillin resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen. We report the prevalence and antibiotic susceptibility pattern of MRSA in major southern districts of Tamilnadu. METHODS: A total of 7172 clinical specimens and 1725 carrier screening samples were collected from different centers and subjected to MRSA screening using conventional microbiological methods. Subsequently the antibiotic sensitivity test was performed for the confirmed MRSA isolates. RESULTS: Out of 906 strains of S. aureus isolated from clinical and carrier samples, 250 (31.1%) and 39 (37.9%) were found to be methicillin resistant respectively. Almost all clinical MRSA strains (99.6%) were resistant to penicillin, 93.6% to ampicillin, and 63.2% towards gentamicin, co-trimoxazole, cephalexin, erythromycin, and cephotaxime. All MRSA strains (100%) of carrier screening samples had resistance to penicillin and about 71.8% and 35.9% were resistant to ampicillin and co-trimoxazole respectively. Multidrug resistance was observed among 63.6% of clinical and 23% of carrier MRSA isolates. However, all strains of clinical and carrier subjects were sensitive to vancomycin. CONCLUSION: The determination of prevalence and antibiotic sensitivity pattern of MRSA will help the treating clinicians for first line treatment in referral hospitals.

Acanthamoeba keratitis - a six year epidemiological review from a tertiary care eye hospital in south India.

PURPOSE: This study analyses the prevalence, demography, predisposing factors and seasonal variation of Acanthamoeba keratitis. METHODS: A retrospective review of all cases presenting with keratitis at the cornea clinic, Aravind Eye Hospital, Coimbatore, from August 1997 to July 2003, was done for screening patients with a provisional diagnosis of Acanthamoeba keratitis. Their records were further analyzed for microbiological details. Cases with culture proven Acanthamoeba keratitis were included for epidemiological analysis. RESULTS: From a total of 4519 patients who attended cornea clinic 32 (33 eyes) patients were confirmed to be positive for Acanthamoeba keratitis. Twenty cases (62.5%) were males. Majority (18; 54.2%) of the Acanthamoeba keratitis eyes reported corneal trauma by solid objects. No peak period was observed in a year, as the number of cases was almost uniform in all months. CONCLUSION: This study indicates the increasing prevalence of Acanthamoeba keratitis among non-contact lens users in this region during the 6-year period.

Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells.

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

A novel anionic modification of N-glycans on mammalian endothelial cells is recognized by activated neutrophils and modulates acute inflammatory responses.

We previously reported an unusual carboxylated modification on N:-glycans isolated from whole bovine lung. We have now raised IgG mAbs against the modification by immunization with biotinylated aminopyridine-derivatized glycans enriched for the anionic species and screening for Abs whose reactivities were abrogated by carboxylate neutralization of bovine lung glycopeptides. One such Ab (mAb GB3.1) was inhibited by carboxylated bovine lung glycopeptides and other multicarboxylated molecules, but not by glycopeptides in which the carboxylate groups were modified. The Ab recognized an epitope constitutively expressed on bovine, human, and other mammalian endothelial cells. Stimulated, but not resting, neutrophils bound to immobilized bovine lung glycopeptides in a carboxylate-dependent manner. The binding of activated neutrophils to immobilized bovine lung glycopeptides was inhibited both by mAb GB3.1 and by soluble glycopeptides in a carboxylate-dependent manner. The Ab also inhibited extravasation of neutrophils and monocytes in a murine model of peritoneal inflammation. This inhibition of cell trafficking correlated with the increased sequestration but reduced transmigration of leukocytes that were found to be adherent to the endothelium of the mesenteric microvasculature. Taken together, these results indicate that these novel carboxylated N:-glycans are constitutively expressed on vascular endothelium and participate in acute inflammatory responses by interaction with activated neutrophils.

Crystallization of agglutinin from the seeds of Abrus precatorius.

Agglutinin protein purified from the seeds of Abrus precatorius has a high antitumour activity and was crystallized at room temperature with polyethylene glycol 8000 as the precipitant. The agglutinin crystal diffracted to 3.45 A and belongs to one of two possible tetragonal space groups, P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 141.91, c = 105.63 A. The asymmetric unit contains a heterotetrameric protein molecule of molecular weight 134 kDa and has a solvent content of approximately 38%.

Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG).

Congenital Disorders of Glycosylation (CDG) are human deficiencies in glycoprotein biosynthesis. Previous studies showed that 1 mM mannose corrects defective protein N-glycosylation in cultured fibroblasts from some CDG patients. We hypothesized that these CDG cells have limited GDP-mannose (GDP-Man) and that exogenous mannose increases the GDP-Man levels. Using a well established method to measure GDP-Man, we found that normal fibroblasts had an average of 23.5 pmol GDP-Man/10(6) cells, whereas phosphomannomutase (PMM)-deficient fibroblasts had only 2.3-2.7 pmol/10(6) cells. Adding 1 mM mannose to the culture medium increased the GDP-Man level in PMM-deficient cells to approximately 15.5 pmol/10(6) cells, but had no significant effect on GDP-Man levels in normal fibroblasts. Similarly, mannose supplementation increased GDP-Man from 4.6 pmol/10(6) cells to 24.6 pmol/10(6) cells in phosphomannose isomerase (PMI)-deficient fibroblasts. Based on the specific activity of the GDP-[(3)H]Man pool present in [2-(3)H]mannose labeled cells, mannose supplementation also partially corrected the impaired synthesis of mannosylphosphoryldolichol (Man-P-Dol) and Glc(0)(-)(3)Man(9)GlcNAc(2)-P-P-Dol. These results confirm directly that deficiencies in PMM and PMI result in lowered cellular GDP-Man levels that are corrected by the addition of mannose. In contrast to these results, GDP-Man levels in fibroblasts from a CDG-Ie patient, who is deficient in Man-P-Dol synthase, were normal and unaffected by mannose supplementation even though mannose addition was found to correct abnormal lipid intermediate synthesis in another study (Kim et al. [2000] J. Clin. Invest., 105, 191-198). The mechanism by which mannose supplementation corrects abnormal protein N-glycosylation in Man-P-Dol synthase deficient cells is unknown, but this observation suggests that the regulation of Man-P-Dol synthesis and utilization may be more complex than is currently understood.

Structure determination of porcine haemoglobin.

To investigate a potential candidate material for making artificial red blood cells to supplement blood transfusion, the X-ray structure of porcine haemoglobin at 1.8 A resolution was determined as part of research towards synthesizing human blood. Porcine haemoglobin was crystallized by the vapor-diffusion method, producing crystals of dimensions 0.3-0.5 mm after successive seeding. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 68.10, b = 72.27, c = 114.85 A. The initial phase was determined by the molecular-replacement method, using human oxyhaemoglobin as a model. The final R factor was 21.1% for 36 820 reflections after validation of 574 water molecules. The r.m.s. deviations of bond lengths, angles, torsion angles and improper angles from their ideal values are 0.017 A, 3.0, 20.6 and 1.8 degrees, respectively. The average B factor is 33.63 A(2) for the haemoglobin molecule and 50.53 A(2) for the water molecules. The structure could be superimposed on a 2.8 A resolution structure with an r.m.s. difference of 0.59 A in main-chain atomic positions and 1. 27 A in side-chain atomic positions. Porcine and human haemoglobins are compared. A tentative model for artificial blood is proposed based on the complementarity relationship of the surface charges between haemoglobin and the surrounding cell membrane.

Direct utilization of mannose for mammalian glycoprotein biosynthesis.

Direct utilization of mannose for glycoprotein biosynthesis has not been studied because cellular mannose is assumed to be derived entirely from glucose. However, animal sera contain sufficient mannose to force uptake through glucose-tolerant, mannose-specific transporters. Under physiological conditions this transport system provides 75% of the mannose for protein glycosylation in human hepatoma cells despite a 50- to 100-fold higher concentration of glucose. This suggests that direct use of mannose is more important than conversion from glucose. Consistent with this finding the liver is low in phosphomannose isomerase activity (fructose-6-P<->mannose-6-P), the key enzyme for supplying glucose-derived mannose to the N-glycosylation pathway. [2-3H] Mannose is rapidly absorbed from the intestine of anesthetized rats and cleared from the blood with a t1/2of 30 min. After a 30 min lag, label is incorporated into plasma glycoproteins, and into glycoproteins of all organs during the first hour. Most (87%) of the initial incorporation occurs in the liver, but this decreases as radiolabeled plasma glycoproteins increase. Radiolabel in glycoproteins also increases 2- to 6-fold in other organs between 1-8 h, especially in lung, skeletal muscle, and heart. These organs may take up hepatic-derived radiolabeled plasma glycoproteins. Significantly, the brain, which is not exposed to plasma glycoproteins, shows essentially no increase in radiolabel. These results suggest that mammals use mannose transporters to deliver mannose from blood to the liver and other organs for glycoprotein biosynthesis. Additionally, contrary to expectations, most of the mannose for glycoprotein biosynthesis in cultured hepatoma cells is derived from mannose, not glucose. Extracellular mannose may also make a significant contribution to glycoprotein biosynthesis in the intact organism.

Human fibroblasts prefer mannose over glucose as a source of mannose for N-glycosylation. Evidence for the functional importance of transported mannose.

Mannose in N-linked oligosaccharides is assumed to be derived primarily from glucose through phosphomannose isomerase (PMI). The discovery of mammalian mannose-specific transporters that function at physiological concentrations suggested that mannose might directly contribute to oligosaccharide synthesis. To determine the relative contribution of glucose and mannose, human fibroblasts were labeled with either [2-3H]mannose or [1,5,6-3H]glucose at the same specific activity, and the N-linked chains were released by PNGase F digestion. Most of the trichloroacetic acid-precipitable [3H]mannose label was released by this digestion, but only about 10% of the trichloroacetic acid-precipitable material was released from cells labeled with [1,5,6-3H]glucose. Both sugars labeled a similar array of oligosaccharides, and acid hydrolysis of these chains showed that [2-3H]mannose contributed 65-75% of the [3H]mannose in cells labeled for 1 h, despite the 100-fold higher concentration of exogenous glucose. Mannose consumption and [2-3H]mannose utilization were within the range of rates expected for mannose transport via the mannose-specific transporter. About 7-14% of the [2-3H]mannose is used for glycosylation, while the rest (86-93%) is catabolized to 3H2O via PMI. Increasing the exogenous mannose concentration beyond mannose transporter saturation results in the conversion of >99% of [2-3H]mannose into 3H2O. Long term labeling of cells with [2-3H]mannose showed that the specific activity of mannose in glycoproteins reached 77% of the specific activity of [2-3H]mannose added to the medium. These results show that when fibroblasts are provided with physiological concentrations of mannose, they use the mannose-specific transporter to supply the majority of mannose needed for glycoprotein synthesis. PMI may normally be used to catabolize excess mannose rather than to primarily supply Man-6-P for glycoprotein synthesis.

Abnormal metabolism of mannose in families with carbohydrate-deficient glycoprotein syndrome type 1.

Patients with carbohydrate-deficient glycoprotein syndrome (CDGS) Type 1 underglycosylate many glycoproteins by failing to add entire N-linked carbohydrate chains to them. The primary defect in these patients has been reported as a > 90% deficiency in phosphomannomutase activity (PMM), the enzyme that converts mannose-6-phosphate to mannose-1-phosphate. This lesion reduces both the amount and the size of the lipid-linked oligosaccharide precursor. We have now analyzed the activity of PMM and the level of glycosylation in cultured fibroblasts as well as the level of blood mannose in seven CDGS Type 1 patients and their parents. All of these patients were approximately 95% deficient in PMM activity and their parents had an average of 51% of control PMM activity. Furthermore, parental fibroblasts showed reduced glycosylation and a higher proportion of truncated N-linked chains compared to those made by control fibroblasts. Addition of 0.25 mM mannose to the culture medium corrected both the underglycosylation and size of the oligosaccharide chains in CDGS Type 1 patients and their parents. Finally, serum from CDGS patients had considerably reduced mannose levels (5-40 microM) compared to normal controls (40-80 microM) and some parents were below normal (16-103 microM). These results suggest that the reduced blood mannose level is a consequence of the PMM deficiency. This is the first inherited disorder in human metabolism that shows a decrease in available mannose. Increasing blood mannose levels might correct some protein underglycosylation in these patients.