Rapid efficient synthesis and characterization of silver, gold, and bimetallic nanoparticles from the medicinal plant Plumbago zeylanica and their application in biofilm control.

BACKGROUND: Nanoparticles (NPs) have gained significance in medical fields due to their high surface-area-to-volume ratio. In this study, we synthesized NPs from a medicinally important plant - Plumbago zeylanica. MATERIALS AND METHODS: Aqueous root extract of P. zeylanica (PZRE) was analyzed for the presence of flavonoids, sugars, and organic acids using high-performance thin-layer chromatography (HPTLC), gas chromatography-time of flight-mass spectrometry (GC-TOF-MS), and biochemical methods. The silver NPs (AgNPs), gold NPs (AuNPs), and bimetallic NPs (AgAuNPs) were synthesized from root extract and characterized using ultraviolet-visible spectra, X-ray diffraction (XRD), energy-dispersive spectrometry (EDS), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The effects of these NPs on Acinetobacter baumannii, Staphylococcus aureus, and Escherichia coli biofilms were studied using quantitative biofilm inhibition and disruption assays, as well as using fluorescence, scanning electron microscopy, and atomic force microscopy. RESULTS: PZRE showed the presence of phenolics, such as plumbagin, and flavonoids, in addition to citric acid, sucrose, glucose, fructose, and starch, using HPTLC, GC-TOF-MS, and quantitative analysis. Bioreduction of silver nitrate (AgNO3) and chloroauric acid (HAuCl4) were confirmed at absorbances of 440 nm (AgNPs), 570 nm (AuNPs), and 540 nm (AgAuNPs), respectively. The maximum rate of synthesis at 50°C was achieved with 5 mM AgNO3 within 4.5 hours for AgNPs; and with 0.7 mM HAuCl4 within 5 hours for AuNPs. The synthesis of AgAuNPs, which completed within 90 minutes with 0.7 mM AgNO3 and HAuCl4, was found to be the fastest. Fourier-transform infrared spectroscopy confirmed bioreduction, while EDS and XRD patterns confirmed purity and the crystalline nature of the NPs, respectively. TEM micrographs and DLS showed about 60 nm monodispersed Ag nanospheres, 20-30 nm Au nanospheres adhering to form Au nanotriangles, and about 90 nm hexagonal blunt-ended AgAuNPs. These NPs also showed antimicrobial and antibiofilm activity against E. coli, A. baumannii, S. aureus, and a mixed culture of A. baumannii and S. aureus. AgNPs inhibited biofilm in the range of 96%-99% and AgAuNPs from 93% to 98% in single-culture biofilms. AuNPs also showed biofilm inhibition, with the highest of 98% in S. aureus. AgNPs also showed good biofilm disruption, with the highest of 88% in A. baumannii. CONCLUSION: This is the first report on rapid and efficient synthesis of AgNPs, AuNPs and AgAuNPs from P. zeylanica and their effect on quantitative inhibition and disruption of bacterial biofilms.

Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.

Functional characterization of a flavonoid glycosyltransferase gene from Withania somnifera (Ashwagandha).

Glycosylation of flavonoids is mediated by family 1 uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs). Until date, there are few reports on functionally characterized flavonoid glycosyltransferases from Withania somnifera. In this study, we cloned the glycosyltransferase gene from W. somnifera (UGT73A16) showing 85-92 % homology with UGTs from other plants. UGT73A16 was expressed as a His(6)-tagged fusion protein in Escherichia coli. Several compounds, including flavonoids, were screened as potential substrates for UGT73A16. HPLC analysis and hypsochromic shift indicated that UGT73A16 transfers a glucose molecule to several different flavonoids. Based on kinetic parameters, UGT73A16 shows more catalytic efficiency towards naringenin. Here, we explored UGT73A16 of W. somnifera as whole cell catalyst in E. coli. We used flavonoids (genistein, apigenin, kaempferol, naringenin, biochanin A, and daidzein) as substrates for this study. More than 95 % of the glucoside products were released into the medium, facilitating their isolation. Glycosylation of substrates occurred on the 7- and 3-hydroxyl group of the aglycone. UGT73A16 also displayed regiospecific glucosyl transfer activity towards 3-hydroxy flavone compound, which is the backbone of all flavonols and also for a chemically synthesized compound, not found naturally. The present study generates essential knowledge and molecular as well as biochemical tools that allow the verification of UGT73A16 in glycosylation.

Functional characterization and differential expression studies of squalene synthase from Withania somnifera.

Squalene synthase (SQS: EC 2.5.1.21) is a potential branch point regulatory enzyme and represents the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In the present study, cloning and characterization of Withania somnifera squalene synthase (WsSQS) cDNA was investigated subsequently followed by its heterologous expression and preliminary enzyme activity. Two different types of WsSQS cDNA clones (WsSQS1and WsSQS2) were identified that contained an open reading frames of 1,236 and 1,242 bp encoding polypeptides of 412 and 414 amino acids respectively. Both WsSQS isoforms share 99 % similarity and identity with each other. WsSQS deduced amino acids sequences, when compared with SQS of other plant species, showed maximum similarity and identity with Capsicum annuum followed by Solanum tuberosum and Nicotiana tabacum. To obtain soluble recombinant enzymes, 24 hydrophobic amino acids were deleted from the carboxy terminus and expressed as 6X His-Tag fusion protein in Escherichia coli. Approximately 43 kDa recombinant protein was purified using Ni-NTA affinity chromatography and checked on SDS-PAGE. Preliminary activity of the purified enzymes was determined and the products were analyzed by gas chromatograph-mass spectrometer (GC-MS). Quantitative real-time PCR (qRT-PCR) analysis showed that WsSQS expresses more in young leaves than mature leaves, stem and root.

Use of epidural analgesia for pain management after major spinal surgery.

OBJECTIVE: This is a retrospective study of the role of postoperative epidural analgesia in major spinal surgical procedures. With the number and complexity of the procedures performed on the spine ever-increasing, this method of analgesia is becoming more important. METHODS: Results of 74 consecutive cases of major spinal surgeries between January 2000 and January 2001 at the Spine Division, Amritha Institute of Medical Sciences and Research Centre, Kochi, India, were studied. 32 cases were posterior procedures and the other 42 were anterior procedures of the thoracic and lumbar regions. The use of various combinations of local anaesthetic and opioid to control postoperative pain after spinal surgery were analysed. RESULTS: 36 (49%) of 74 patients did not require any parenteral supplements. Of the remaining 38 patients who required supplementary parenteral analgesia in the first 48 hours, 25 (34%) received a single dose and 13 (18%) required more than one dose. The number of patients requiring parenteral analgesia immediately after operation were 11; between 2 and 6 hours were 12; and between 6 and 24 hours were 11. Of the 74 patients, 67 had a sound sleep after epidural administration. There were 2 cases of respiratory depression and 2 of transient hypotension. CONCLUSION: Most epidural analgesic regimens significantly reduced postoperative pain, and the requirement for supplementary parenteral analgesics was minimal. Adverse effects were rare, yet we recommend that patients treated with this protocol be managed in high-dependency units.

Splenosis in a port site after laparoscopic splenectomy.

Splenosis, the autotransplantation of splenic tissue, is most commonly seen after traumatic splenic rupture and splenectomy. It also can occur during embryonic development. Intraperitoneal, intrathoracic, and retroperitoneal sites have been reported. Although the presence of the splenic tissue often is asymptomatic and an incidental finding, it may present with pain or be confused with various pathologies including neoplasia. Because most pediatric splenectomies are performed for hemolytic disorders, parenchymal disruption must be contained to avoid recurrent disease. We present a case in which the devascularized spleen was contained in a bag and fragmented in situ. Splenosis developed in the retrieval port site after laparoscopic splenectomy and cholecystectomy. Port-site splenosis needs to be considered in the differential diagnosis of port-site pain and a palpable nodule postsplenectomy.

Management of extravasation injuries.

BACKGROUND: Various agents have been implicated in causing tissue necrosis after intravenous infusions have extravasated. These include solutions of calcium, potassium, bicarbonate, hypertonic dextrose, cytotoxic drugs and antibiotics. Views on management of these injuries differ, and range from a non-operative conservative approach to early debridement and grafting. METHODS: A retrospective review was undertaken of the hospital files of patients with extravasation injuries seen in three Australian hospitals. Nine patients were identified, and their management and long-term follow up are reported. RESULTS: Age ranged from 17 days to 60 years. Two patients received their injuries from solutions containing isotonic dextrose/saline. The other seven patients received injuries from a variety of solutions including calcium gluconate (n = 1), parenteral nutrition (n = 1), sodium bicarbonate (n = 1), immunoglobulin (n = 1), gentamicin and penicillin (n = 1), flucloxacillin (n = 1), and the chemotherapeutic agents epirubicin and cyclophosphamide (n = 1). The sites involved included the dorsum of the right foot (n = 3), the dorsum of the left foot (n = 3), the right groin (n = 1), the right hand (n = 1) and the left hand (n = 1). Four patients were managed by delayed debridement and split skin grafting, while five were treated non-operatively. Prolonged scar management was necessary in seven of the nine patients. Final results were satisfactory in all patients who received skin grafting and in all patients who were managed conservatively. CONCLUSIONS: Management of extravasation injuries should be conservative if possible. Delayed debridement and split skin grafting is required if the area of skin loss is extensive. Scar management remains a problem. Prevention of these injuries with the education of both medical and nursing staff remains the ultimate aim.