Application of biomimetic surfaces and 3D culture technology to study the role of extracellular matrix interactions in neurite outgrowth and inhibition.

The microenvironment that cells experience during in vitro culture can often be far removed from the native environment they are exposed to in vivo. To recreate the physiological environment that developing neurites experience in vivo, we combine a well-established model of human neurite development with, functionalisation of both 2D and 3D growth substrates with specific extracellular matrix (ECM) derived motifs displayed on engineered scaffold proteins. Functionalisation of growth substrates provides biochemical signals more reminiscent of the in vivo environment and the combination of this technology with 3D cell culture techniques, further recapitulates the native cellular environment by providing a more physiologically relevant geometry for neurites to develop. This biomaterials approach was used to study interactions between the ECM and developing neurites, along with the identification of specific motifs able to enhance neuritogenesis within this model. Furthermore, this technology was employed to study the process of neurite inhibition that has a detrimental effect on neuronal connectivity following injury to the central nervous system (CNS). Growth substrates were functionalised with inhibitory peptides released from damaged myelin within the injured spinal cord (Nogo & OMgp). This model was then utilised to study the underlying molecular mechanisms that govern neurite inhibition in addition to potential mechanisms of recovery.

Development, validation, and application of an RP-HPLC method for concurrent quantification of Minoxidil and Finasteride in a topical solution for hair regrowth.

A topical solution comprising of Minoxidil (MXL) and Finasteride (FNS) for alopecia is formulated in the present work, which essentially contains a lipid-Lauroglycol FCC as a penetration enhancer. The objective of the proposed work was to develop a rapid, simple, and robust reverse phase high performance liquid chromatographic (RP-HPLC) method to determine MXL and FNS in the said formulation. Herein, the chromatographic conditions were optimized based on the theoretical principles of separation and physicochemical properties such as pKa and log P of both the Active Pharmaceutical Ingredients (APIs). The separation was accomplished on an Inertsil® ODS-3 C18 column (150mm×4.6mm; 5µm of particle size) at 25°C by using a mobile phase composed of 70:30 v/v ratio of Methanol and Milli-Q water along with 0.5% Triethylamine at pH 6.4 adjusted with Ortho Phosphoric Acid. Drug peaks showed a good resolution at 210nm. The retention times for MXL and FNS were found to be 2.40min and 6.39min, respectively. The developed method was found to be linear (R2≥0.998) in a concentration range of 5-100µg/mL for both the drugs. The method was validated according to the ICH guidelines Q2 (R1). The ability of the method to differentiate between the types formulations was demonstrated by the in vitro diffusion data performed using a highly sophisticated Strat-M® membrane. The cumulative amount of drug released (MXL and FNS) at the end of 24hours was maximum for the topical formulation containing lipids prepared using isopropyl alcohol and propylene glycol as the base.

Metastasis of squamous cell carcinoma of the head and neck to the thyroid: a single institution's experience with a review of relevant publications.

The thyroid gland is a rare site of metastasis, and in particular of those of squamous cell carcinoma (SCC) from the head and neck region. We have reviewed the aetiology, pathogenesis, clinical characteristics, radiological features, immunohistochemical profile, prognosis, and management of metastatic SCC from the head and neck region to the thyroid, and searched current publications on the Medline, Embase, and Cochrane databases using the following keywords: "SCC of thyroid", "secondary SCC of thyroid", and "metastasis to the thyroid", for papers published during the last 33 years (April 1984 to October 2017).We found a total of 19 papers that reported a total of 32 cases that were relevant. Four further cases were discovered as an incidental finding on follow-up positron emission tomographic/computed tomographic scans with magnetic resonance imaging of the head and neck at our hospital, which were confirmed with an ultrasound-guided core needle biopsy followed by immunohistochemical examination. For patients who are doing well, whose disease is controlled at the primary site, and who have no evidence of distant metastatic disease, total thyroidectomy could be considered followed by adjuvant radiation or chemoradiotherapy, depending on the presence of intermediate or high-risk features on pathological examination and previous history of radiation. This may help to control the disease and avoid local morbidity.

Spectrum of Infections in Living Donor Kidney Transplant Recipients: An Experience From a Tertiary Center in Nepal.

BACKGROUND: Kidney transplant recipients are always at risk of infections because they are on lifelong immunosuppressive medications. The spectrum of infections in this special population is not the same as in the general population. Post-transplant infections are extensively studied in the developed world. Publications about post-transplant infections from Nepal are scarce. This study was carried out to study the spectrum of infections, the trends in treatment, and the incidence of tuberculosis in kidney transplant recipients. METHODS: This is a retrospective analysis of the patient data in Tribhuvan University Teaching Hospital, Kathmandu, Nepal. Follow-up data from the first 100 kidney transplant recipients was recorded in a Microsoft Excel worksheet and descriptive analysis was done. RESULTS: In the first 100 transplants done until 21 September 2011, 92 patients' data were recorded and 8 patients' data were missing. The mean follow-up period was 61.03 months. The population was 76.09% male (n = 70) and 23.91% female (n = 22). A total of 641 episodes of infections were recorded. Urinary tract infections were the most common type of infection. Escherichia coli was the most common organism isolated (36% of all cultures). There were 17 (2.65%) episodes of viral and 42 (6.6%) episodes of fungal infections. Tuberculosis was diagnosed in 6 (6.5%) patients. CONCLUSION: Urinary tract infection is the most common type of infection in post-kidney transplant patients. Quinolones were the most common agents used to treat urinary tract infections. The incidence of tuberculosis in kidney transplant recipients is 6.5% in 5 years' follow-up.

Severe immune thrombocytopenic purpura treated with plasma exchange.

Immune thrombocytopenic purpura (ITP) is a hematological disorder characterized by immunologically mediated destruction of platelets and absence of other causes of thrombocytopenia. Treatment is required when the low platelet count entails risk of serious bleeding. Steroid is the first line of management. Acute refractory ITP with very low platelet count is variably treated with high dose steroid, intravenous immunoglobulin (IVIg), anti D or emergency splenectomy. Here, we present a case of steroid resistant ITP with severe thrombocytopenia treated with plasma exchange and low dose IVIg who responded dramatically to the therapy with maintained platelet count till one month from the institution of therapy.

Urological complication after kidney transplantation.

BACKGROUND: Renal transplantation is a regular service at Tribhuvan University Teaching Hospital and complications have been known to occur after it. This study was conducted to assess complications after transplantation. OBJECTIVES: To determine the incidence of urological complications after living related renal transplantation at Tribhuvan University Teaching Hospital. METHODS: A clinical study was performed (from August 2008 to July 2010) which included 50 living-related renal transplantations at Tribhuvan University Teaching Hospital. All the donors and recipients were evaluated preoperatively with necessary investigations and followed up postoperatively with standard hospital transplant protocol. The incidence of urological complications were documented and analyzed. RESULTS: Fifty living-related, renal transplantations were carried out during the study period. Seven doors had minor post operative complications; three had post operative fever, two had chest infections and each one had superficial surgical site infections and severe pain at incision site. Ureteroneocystostomy was performed with double J stent in all recipients. Urological complications were noted in 12 (24%) recipients. Clinical significant hematuria occurred in four cases. One patient had ureteric necrosis and urinary leak which required re-exploration post operatively. Two patients developed delayed ureteric stricture which were managed by antegrade Double J stenting and ureteric reimplantation. Peri-graft abscess occurred in two cases, which were drained percutaneously. surgical site infections was seen in one case. CONCLUSIONS: Urological complications are inevitable in renal transplantation and our complications rate appears similar to that reported in literature.

Neural differentiation regulated by biomimetic surfaces presenting motifs of extracellular matrix proteins.

The interaction between cells and the extracellular matrix (ECM) is essential during development. To elucidate the function of ECM proteins on cell differentiation, we developed biomimetic surfaces that display specific ECM peptide motifs in a controlled manner. Presentation of ECM domains for collagen, fibronectin, and laminin influenced the formation of neurites by differentiating PC12 cells. The effect of these peptide sequences was also tested on the development of adult neural stem/progenitor cells. In this system, collagen I and fibronectin induced the formation of beta-III-tubulin positive cells, whereas collagen IV reduced such differentiation. Biomimetic surfaces composed of multiple peptide types enabled the combinatorial effects of various ECM motifs to be studied. Surfaces displaying combined motifs were often predictable as a result of the synergistic effects of ECM peptides studied in isolation. For example, the additive effects of fibronectin and laminin resulted in greater expression of beta-III-tubulin positive cells, whereas the negative effect of the collagen IV domain was canceled out by coexpression of collagen I. However, simultaneous expression of certain ECM domains was less predictable. These data highlight the complexity of the cellular response to combined ECM signals and the need to study the function of ECM domains individually and in combination.

Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins.

Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory.

Self-assembling layers created by membrane proteins on gold.

Membrane systems are based on several types of organization. First, amphiphilic lipids are able to create monolayer and bilayer structures which may be flat, vesicular or micellar. Into these structures membrane proteins can be inserted which use the membrane to provide signals for lateral and orientational organization. Furthermore, the proteins are the product of highly specific self-assembly otherwise known as folding, which mostly places individual atoms at precise places in three dimensions. These structures all have dimensions in the nanoscale, except for the size of membrane planes which may extend for millimetres in large liposomes or centimetres on planar surfaces such as monolayers at the air/water interface. Membrane systems can be assembled on to surfaces to create supported bilayers and these have uses in biosensors and in electrical measurements using modified ion channels. The supported systems also allow for measurements using spectroscopy, surface plasmon resonance and atomic force microscopy. By combining the roles of lipids and proteins, highly ordered and specific structures can be self-assembled in aqueous solution at the nanoscale.

Purification and characterization of the flagellar basal body of Rhodobacter sphaeroides.

Flagellar hook-basal body (HBB) complexes were purified from Rhodobacter sphaeroides. The HBB was more acid labile but more heat stable than that of Salmonella species, and protein identification revealed that HBB components were expressed only from one of the two sets of flagellar gene clusters on the R. sphaeroides genome, under the heterotrophic growth conditions tested here.

3-Nitrotyrosine immunoreactivity in olfactory receptor neurons of patients with Alzheimer's disease: implications for impaired odor sensitivity.

Olfactory sensory function is impaired in patients with the diagnosis of probable Alzheimer's disease (AD) compared to elderly controls, and the olfactory epithelium (OE) of AD patients exhibits several pathological changes characteristic of the AD brain. To confirm that the populations from whom our postmortem tissues are obtained exhibit similar decrements in sensory function, threshold testing was performed; probable AD patients had significantly higher olfactory thresholds than controls. To determine if oxidative stress contributes to decreased olfactory function in AD, we localized 3-nitrotyrosine (3-NT) immunoreactivity in OE obtained postmortem from patients with neuropathologically confirmed AD and age-matched controls with brains free of significant neurodegenerative pathology. In AD patients, immunoreactivity was localized in olfactory receptor neurons (ORNs), including dendritic knobs where ion channels that participate in sensory transduction are located, suggesting a direct mechanism for olfactory impairment. In controls, immunoreactivity occurred in blood vessel endothelium, suggesting age-related vascular dysfunction. Immunohistochemistry for CD68, a macrophage scavenger receptor, demonstrated activated macrophages, a source of free radicals contributing to 3-NT formation, in the OE of AD patients but not controls. These results demonstrate increased oxidative stress and modification of ORN proteins that may contribute directly to olfactory impairment in AD patients.

Chromosomal insertions and deletions in Streptococcus mutans.

Many isolates of Streptococcus mutans lack the ability to ferment melibiose and other sugars. We previously reported that this was commonly due to a chromosomal deletion and, in the present study, sequence information from the S. mutans genome project was used to design PCR primers to explore the nature and extent of the deletion. In all melibiose-negative strains examined, there was an incomplete insertion element, ISSmu3, in place of the 18-kb stretch of chromosome encoding the msm and GAL operons. Strains that were also unable to utilise beta-glucosides were found to have a separate 4 kb deletion in the BGL regulon that is proposed to be due to homologous recombination between two short stretches of identical sequence. The evidence is consistent with all the melibiose-negative strains examined being derived from a common ancestor.

CheR- and CheB-dependent chemosensory adaptation system of Rhodobacter sphaeroides.

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in three major operons and other, unlinked, loci. These include cheA(1) and cheR(1) (che Op(1)) and cheA(2), cheR(2), and cheB(1) (che Op(2)). In-frame deletions of these cheR and cheB homologues were constructed and the chemosensory behaviour of the resultant mutants examined on swarm plates and in tethered cell assays. Under the conditions tested, CheR(2) and CheB(1) were essential for normal chemotaxis, whereas CheR(1) was not. cheR(2) and cheB(1), but not cheR(1), were also able to complement the equivalent E. coli mutants. However, none of the proteins were required for the correct polar localization of the chemoreceptor McpG in R. sphaeroides. In E. coli, CheR binds to the NWETF motif on the high-abundance receptors, allowing methylation of both high- and low-abundance receptors. This motif is not contained on any R. sphaeroides chemoreceptors thus far identified, although 2 of the 13 putative chemoreceptors, McpA and TlpT, do have similar sequences. This suggests that CheR(2) either interacts with the NWETF motif of E. coli methyl-accepting chemotaxis proteins (MCPs), even though its native motif may be slightly different, or with another conserved region of the MCPs. Methanol release measurements show that R. sphaeroides has an adaptation system that is different from that of Bacillus subtilis and E. coli, with methanol release measurable on the addition of attractant but not on its removal. Intriguingly, CheA(2), but not CheA(1), is able to phosphorylate CheB(1), suggesting that signaling through CheA(1) cannot initiate feedback receptor adaptation via CheB(1)-P.

DNA transfection and transfected cell viability using amphipathic asymmetric dendrimers.

Amphipathic asymmetric dendrimers have been investigated for use in delivery of genes into cells, with the objective of optimising transfection efficiency and maintaining cell viability. We have synthesised amphipathic asymmetric dendrimers by solid phase methods. The ability of two of these to transfect BHK cells in culture with beta-galactosidase gene was determined by X-gal staining. Cell viability was measured by the MTT assay for BHK cells, and by spectroscopy for lysis of erythrocytes. Interactions between dendrimer and DNA were investigated by agarose gel electrophoresis. BHK cells were optimally transfected at 5:1 +/- charge ratio yielding 20% cells receiving at least one copy of the plasmid. Cell viability decreased when the dendrimer to DNA ratio exceeded 5:1. Raising the pH significantly affected the electrophoretic mobility of complexes of dendrimer and DNA. We conclude that amphipathic asymmetric dendrimers enable efficient plasmid DNA uptake into BHK cells. Cell viability is maintained at high concentrations of dendrimer when complexed with DNA at a 5:1 +/- charge ratio. Efficiency of transfection and cell viability suggest the system may be suitable for gene delivery in vivo.

Fine tuning bacterial chemotaxis: analysis of Rhodobacter sphaeroides behaviour under aerobic and anaerobic conditions by mutation of the major chemotaxis operons and cheY genes.

Rhodobacter sphaeroides chemotaxis is significantly more complex than that of enteric bacteria. Rhodobacter sphaeroides has multiple copies of chemotaxis genes (two cheA, one cheB, two cheR, three cheW, five cheY but no cheZ), controlling a single 'stop-start' flagellum. The growth environment controls the level of expression of different groups of genes. Tethered cell analysis of mutants suggests that CheY(4) and CheY(5) are the motor-binding response regulators. The histidine protein kinase CheA(2) mediates an attractant ('normal') response via CheY(4), while CheA(1) and CheY(5) appear to mediate a repellent ('inverted') response. CheY(3) facilitates signal termination, possibly acting as a phosphate sink, although CheY(1) and CheY(2) can substitute. The normal and inverted responses may be initiated by separate sets of chemoreceptors with their relative strength dependent on growth conditions. Rhodobacter sphaeroides may use antagonistic responses through two chemosensory pathways, expressed at different levels in different environments, to maintain their position in a currently optimum environment. Complex chemotaxis systems are increasingly being identified and the strategy adopted by R.sphaeroides may be common in the bacterial kingdom.

The flagellar filament of Rhodobacter sphaeroides: pH-induced polymorphic transitions and analysis of the fliC gene.

Flagellar motility in Rhodobacter sphaeroides is notably different from that in other bacteria. R. sphaeroides moves in a series of runs and stops produced by the intermittent rotation of the flagellar motor. R. sphaeroides has a single, plain filament whose conformation changes according to flagellar motor activity. Conformations adopted during swimming include coiled, helical, and apparently straight forms. This range of morphological transitions is larger than that in other bacteria, where filaments alternate between left- and right-handed helical forms. The polymorphic ability of isolated R. sphaeroides filaments was tested in vitro by varying pH and ionic strength. The isolated filaments could form open-coiled, straight, normal, or curly conformations. The range of transitions made by the R. sphaeroides filament differs from that reported for Salmonella enterica serovar Typhimurium. The sequence of the R. sphaeroides fliC gene, which encodes the flagellin protein, was determined. The gene appears to be controlled by a sigma(28)-dependent promoter. It encodes a predicted peptide of 493 amino acids. Serovar Typhimurium mutants with altered polymorphic ability usually have amino acid changes at the terminal portions of flagellin or a deletion in the central region. There are no obvious major differences in the central regions to explain the difference in polymorphic ability. In serovar Typhimurium filaments, the termini of flagellin monomers have a coiled-coil conformation. The termini of R. sphaeroides flagellin are predicted to have a lower probability of coiled coils than are those of serovar Typhimurium flagellin. This may be one reason for the differences in polymorphic ability between the two filaments.

Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway.

The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 x CheA, 3 x CheW and 3 x CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild-type E. coli (RP437) by increasing its smooth-swimming bias. While some R. sphaeroides proteins restore tumbling to smooth-swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.

Bronchogenic cyst and its progress in a premature infant.

The progressive course of a congenital bronchogenic cyst in a very low birth weight infant with respiratory distress is presented. A bronchogenic cyst, while uncommon, should be in the differential diagnosis of pneumomediastinum or medial pneumothorax even in premature infants who are on ventilators.