OBJECTIVE

To assess the diagnostic value of tumor markers in the cerebrospinal fluid (CSF) for meningeal carcinomatosis (MC).

METHODS

Twenty-one MC patients (including 13 adenocarcinoma and 8 non-adenocarcinoma patients), 72 patients with tuberculous meningitis (TBM) and 23 with primary intracerebral tumors (PIT) were enrolled in this study. Blood and CSF tumor markers including CEA, CA125, CA15-3, CA19-9, CA72-4, CYFRA21-1, AFP and NSE were measured by Roche E170 electrochemiluminescence analyzer and sandwich assay.

RESULTS

CSF tumor markers CEA, CA125, CA199 and CYFRA21-1 and the serum tumor markers CEA, CA125, CA153, CA199 and AFP were significantly higher in MC group than in the other two groups. CSF CEA and CA15-3 were significantly higher in adenocarcinoma MC than in non-adenocarcinoma MC patients, but no significant differences were found in the serum tumor markers between the two groups (P>0.05). CSF tumor markers including CEA, CA125, CA15-3, CA72-4 and CYFRA21-1 were positively correlated to the serum tumor markers (P<0.05). CA199 was positively correlated to the disease course (P<0.05), and age was not correlated to any of the indexes (P>0.05).

CONCLUSIONS

Detection of the tumor markers in the CSF, especially CEA, CA125, CA19-9 and CYFRA21-1, may help in the early diagnosis of MC. CEA and CA15-3 can serve as indicators for differential diagnosis of adenocarcinoma and non-adenocarcinoma.

We present our assessment of CASP12 modeling efforts for targets with no obvious templates of high sequence/structure similarity in the PDB, that is for evaluation units of the free modeling (FM) and free modeling/template-based modeling (FM/TBM) categories. Models were clustered and ranked using the Global Distance Test-Total Score and 5 additional metrics developed in previous CASP rounds, producing short lists of models that were subject to visual inspection in comparison to the target structures. The whole procedure was implemented as a web app that facilitates model selection and visual inspection, and could become useful to facilitate and standardize future assessments. We describe cases of (1) targets with remarkably good predictions, (2) targets whose models captured some global shape and topology features, and (3) targets for which models fail to capture even coarse features. We note that despite this CASP being among the most challenging ones, a measurable improvement of the top predictions is apparent, that we attribute to the emergence of accurate contact prediction methods and the increased number of available sequences. We also briefly discuss current limitations in tertiary structure prediction exemplified by CASP12 targets. Overall, the Baker, Zhang, and Lee manual groups and servers were identified as the top global performing groups.

Eighty children (58 girls and 22 boys) with isosexual precocity seen in the past eight years were evaluated clinically and investigated to identify the underlying cause. Of these, 50% (29 girls and 11 boys) had centrally mediated true precocious puberty (TPP). The girls could be classified into five major groups (I) Central precocious puberty 29-subclassified into idiopathic (ITPP, 15) and organic or neurogenic (NTTP, 14), (II) Premature thelarche (PT, 20), (III) Premature menarche (PM, 2), (IV) Premature adrenarche (PA, 5), and, (V) Others: hypothyroid (n = 1), and McCune Albright Syndrome (n = 1). ITPP as a cause of precocity in girls was seen less often (52%) and NTPP more often (48%) compared to most Western series, with tubercular meningitis as the cause in 31% and hypothalamic hamartomas in 10%. Though the LH and estradiol levels were significantly higher (p < 0.05) in TPP, compared to PT, these were not helpful in differentiating because of considerable overlap. LH-predominant-response (LH/FSH ratio>> 1) to LHRH testing was seen in TPP. Amongst the 22 boys, 11 (50%) had TPP, ITPP in 27% and NTPP in 73%. Hamartomas (n = 4) and TBM (n = 3) contributed equally to NTPP; pineal tumor was seen in one. The adrenal (n = 7) and testicular (n = 2) causes together involved 41% of the boys with precocity, congenital adrenal hyperplasia (CAH) CAH, 11-beta hydroxylase being the commonest cause. Of the 6 boys witdeficiency was found in four and nonsalt losing form of 21-hydroxylase deficiency in 2. Testicular and adrenal tumors and testotoxicosis were noted in one case each. The etiologic factors were more varied in boys.

Using human kidney cortical homogenates and long-term cultured glomerular cells as antigens, the author produced three monoclonal antibodies to glomerular components; 25C reacted with the glomerular basement membrane (GBM) and the wall of blood vessels but with neither the tubular basement membrane (TBM) nor the Bowman's capsule, 33G reacted predominantly with the mesangium, and 34F reacted with glomeruli and the tubular brush border in a granular pattern. Both 25C and 33G exhibited the species-restricted property, and 34F reacted with glomeruli and tubular brush border of all the species examined. Overnight incubation of the kidney sections with 4.0 M urea revealed the reactivity of 25C to the TBM and Bowman's capsule. Dot immunobinding assay revealed that 25C did not react with the known extracellular matrices examined in this study, but rather with collagenase-digested GBM fraction. Also, 33G recognized fibronectin. Western blotting revealed the binding of 34F to the 145-kDa polypeptide solubilized from the kidney with 0.5 M NaCl, and also showed the binding of 25C to 210-kDa polypeptide of collagenase-digested GBM. These findings revealed structural variations in the basement membrane and the existence of a common antigen between the glomeruli and tubular brush border in the human kidney.

Tyramine β-monooxygenase (TβM) belongs to a family of physiologically important dinuclear copper monooxygenases that function with a solvent-exposed active site. To accomplish each enzymatic turnover, an electron transfer (ET) must occur between two solvent-separated copper centers. In wild-type TβM, this event is too fast to be rate limiting. However, we have recently shown [Osborne, R. L.; et al. Biochemistry 2013, 52, 1179] that the Tyr216Ala variant of TβM leads to rate-limiting ET. In this study, we present a pH-rate profile study of Tyr216Ala, together with deuterium oxide solvent kinetic isotope effects (KIEs). A solvent KIE of 2 on kcat is found in a region where kcat is pH/pD independent. As a control, the variant Tyr216Trp, for which ET is not rate determining, displays a solvent KIE of unity. We conclude, therefore, that the observed solvent KIE arises from the rate-limiting ET step in the Tyr216Ala variant, and show how small solvent KIEs (ca. 2) can be fully accommodated from equilibrium effects within the Marcus equation. To gain insight into the role of the enzyme in the long-range ET step, a temperature dependence study was also pursued. The small enthalpic barrier of ET (Ea = 3.6 kcal/mol) implicates a significant entropic barrier, which is attributed to the requirement for extensive rearrangement of the inter-copper environment during PCET catalyzed by the Tyr216Ala variant. The data lead to the proposal of a distinct inter-domain pathway for PCET in the dinuclear copper monooxygenases.

OBJECTIVE

Tuberculous meningitis (TBM) is a major clinical and public health problem, both for diagnosis and management. We compare two established scoring systems, Thwaites and the Lancet consensus scoring system for the diagnosis of TB and compare the clinical outcome in a tertiary care setting.

METHODS

We analyzed 306 patients with central nervous system (CNS) infection over a 5-year period and classified them based on the unit's diagnosis, the Thwaites classification as well as the newer Lancet consensus scoring system. Patients with discordant results-reasons for discordance as well as differences in outcome were also analyzed.

RESULTS

Among the 306 patients, the final diagnosis of the treating physician was TBM in 84.6% (260/306), acute CNS infections in 9.5% (29/306), pyogenic meningitis in 4.2% (13/306) and aseptic meningitis in 1.3% (4/306). Among these 306 patients, 284 (92.8%) were classified as "TBM" by the Thwaites" score and the rest as "Pyogenic". The Lancet score on these patients classified 29 cases (9.5%) as 'Definite-TBM', 43 cases (14.1%) as "Probable-TBM", 186 cases (60.8%) as "Possible-TBM" and the rest as "Non TBM". There was moderate agreement between the unit diagnosis and Thwaites classification (Kappa statistic = 0.53), as well as the Lancet scoring systems. There is only moderate agreement between the Thwaites classification as well as the Lancet scoring systems. It was noted that 32/ 284 (11%) of patients who were classified as TBM by the Thwaites system were classified as "Non TBM" by the Lancet score and 6/258 (2%) of those who were diagnosed as possible, probable or definite TB were classified as Non TB by the Thwaites score. However, patients who had discordant results between these scores were not different from those who had concordant results when treatment was initiated based on expert clinical evaluation in the tertiary care setting.

CONCLUSIONS

There was only moderate agreement between the Thwaites' score and the Lancet consensus scoring systems. There is need to prospectively evaluate the cost effectiveness of simple but more effective rapid diagnostic alogrithm in the diagnosis of TB, particularly in a setting without CT and MRI facilities.

Atypical clinical and CSF profiles encountered in TBM are highlighted. Acid-fast bacilli (AFB) were isolated from the CSF of 7 patients in spite of the complete absence of any cellular response and also from another 3 patients who had no clinical evidence of meningitis. M. flavescens was cultured from the CSF in 4 patients.

OBJECTIVE

The aim was to compare the effectiveness of chewing sugar-free gum after bowel resection on bowel function and length of stay.

METHODS

This was a randomized controlled trial of patients undergoing elective open or laparoscopic bowel surgery, who were allocated into two groups: a chewing gum group (CG); or a nonchewing gum group (NG). Primary outcomes were time to discharge (length of hospital stay [LOS]), time to first flatus (TFF) and time to first bowel motion (TBM). Secondary outcomes were complication rates, pain and total morphine equivalent (TMEq) medication for 7 days after the procedure.

RESULTS

Between 2010 and 2013, 162 patients were randomized; four were excluded, leaving 158 in the study (82 in the CG and 76 in the NG). There was no difference in LOS between the CG (5.8 days) and the NG (6.1 days) (P = 0.403) or in the median TFF between the CG (42.0 h) and the NG (58.0 h) (P = 0.076). The median TBM was lower in the CG (40.0 h) than in the NG (90.0 h) (P = 0.002). There was no difference in intra-operative complications between the CG (9%) and the NG (9%) (P = 0.901) or in early postoperative complications (44% for CG and 55% for NG) (P = 0.131). There was no difference in TMEq at 24 h postprocedure, but the CG had reduced TMEq from days 2 to 7 post procedure and for the 7-day total. Pain was higher among patients in the NG on day 3.

CONCLUSIONS

Chewing sugar-free gum resulted in an earlier return to bowel function and decreased analgesic requirements. There was no decrease in overall LOS or postoperative complications.

The pharmacokinetics and pharmacodynamic response to prednisolone were examined in dietary-induced obese rats and matched controls. Pharmacokinetic parameters were examined in absolute and weight normalized terms. After an i.v. dose (range, 4.0-6.3 mg/kg) of prednisolone adjusted to achieve similar initial prednisolone plasma concentrations, the time course of glucocorticoid receptors in hepatic cytosol and hepatic tyrosine aminotransferase (TAT) activity were examined. Plasma prednisolone concentrations declined biexponentially with time. Mean (S.D.) for prednisolone plasma clearance normalized for total body mass (TBM) was 2.3 (0.9) liters/hr/kg in normal rats and 2.7 (0.7) liters/hr/kg in obese rats. The volume of distribution at steady-state averaged 0.82 (0.46) liters/kg of TBM in normal rats vs. 1.08 (0.40) liters/kg of TBM in obese rats. Base-line receptor levels for obese rats were 53% higher than control levels. A model to describe simultaneously kinetics and receptor-mediated dynamics was used to analyze the data and obtain estimates for the efficiency of TAT induction. This efficiency parameter in obese rats was 22% of controls, reflecting the innate degree of diminished TAT response. This decreased response in obese animals may indicate a need for joint pharmacokinetic/dynamic considerations in dosing obese individuals with corticosteroids.

Transgenic expression of human thrombomodulin (hTBM), which has the potential to solve the problem of coagulation dysregulation in pig-to-primate xenotransplantation, may have additional benefits by neutralizing the proinflammatory cytokine high-mobility group box 1 (HMGB1). The aim of this study was to investigate HMGB1-mediated effects on porcine aortic endothelial cells (PAEC) from wild-type (WT) and hTBM transgenic pigs.

Porcine aortic endothelial cells were treated with HMGB1, human (h)TNFα or lipopolysaccharide (LPS). Procoagulant and proinflammatory responses were assessed by measuring expression of cell surface markers (adhesion molecules, fibrinogen-like protein 2, plasminogen activator inhibitor (PAI)-1), secretion of porcine cytokines and chemokines (HMGB1, TNFα, IL-8, monocyte chemotactic protein-1), and formation of PAI-1/tissue plasminogen activator complexes. Thrombin-mediated degradation of HMGB1 in the presence of PAEC was examined by Western blot and functional assay.

High-mobility group box 1 potently activated WT PAEC, increasing the expression of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, fibrinogen-like protein 2, and PAI-1, the secretion of TNFα, IL-8, and monocyte chemotactic protein-1 and the formation of PAI-1/tissue plasminogen activator complexes. Human TNFα- or LPS-induced activation of WT PAEC was inhibited by treatment with rabbit anti-HMGB1 antibody. Transgenic expression of hTBM significantly reduced the activation of PAEC by HMGB1 or hTNFα, and significantly enhanced thrombin-induced HMGB1 cleavage. Chemically induced shedding of the lectin-like domain of TBM resulted in significantly increased HMGB1-induced PAEC activation.

High-mobility group box 1 exerts powerful proinflammatory and procoagulant effects on WT PAEC, and appears to be an important downstream mediator for the actions of hTNFα and LPS. Human thrombomodulin transgenic PAECs are less sensitive to activation by either HMGB1 or hTNFα, an effect that appears to be dependent on the lectin-like domain of TBM.

Autoimmune tubulointerstitial nephritis (TIN) was induced in Lewis (LEW) rats by immunization with homologous Brown-Norway (BN) rat renal basement membrane (RBM), complete Freund's adjuvant and Bordetella pertussis vaccine. The BN strain has a tubular basement membrane (TBM) antigen (Ag+) detectable by immunofluorescence which is lacking in unmodified LEW rat TBM. Development of TIN in LEW rats correlated with TBM Ag+ immunogens from homologous and heterologous RBM preparations. By day 14 after immunization TIN developed characterized by elevated serum creatinine levels and by tubular destruction with focal, circumscribed lesions containing epithelioid cells, giant cells and mononuclear cell infiltrates. Approximately 60% of the mononuclear cells bore T cell antigens with most cells expressing Ia markers. Immunofluorescence and elution studies revealed no selective IgG fixation to TBM at day 14 despite high titers of circulating alloantibody reactive with the immunizing TBM. Intravenous transfer of LNC and/or splenic cells (3.5 to 7 X 10(8)) to naive LEW rats resulted in less severe but histologically identical TIN in seven days with T cell subpopulations similar to those seen in the active model. This model strongly suggests an initiating role for cell-mediated immunity in TIN in the rat and may provide a parallel to human TIN.

We determined whether the exhaled nitric oxide (eNO) level in asthmatics is age-dependent. Eighty-seven asthmatic patients aged 2-41 years were studied. Hyperreactivity to adenosine 5'-monophosphate (AMP) was used to confirm asthma (</= 200 mg/ml). In the younger group of children (2-5 years), AMP challenge was performed by the provocation concentration causing wheeze (PCW) method, while in the older groups of patients (6-41 years), regular spirometry was used. Exhaled NO was measured in the younger group by the tidal breathing method (<em>TBm</em>) and in the older subjects by the slow vital capacity method (SVCm). <em>TBm</em> and SVCm were compared in 21 other subjects, and there was a significant correlation between the two values (r = 0.96, P < 0.0001). The equation of correlation between the two methods was eNO<em>TBm</em> = 0.78eNOSVCm - 0.51. Within asthmatic patients, we found a significant increase in eNO with age (P < 0.0001), while there was no significant difference in AMP reactivity (P = 0.35). We conclude that eNO in asthmatic patients is age-dependent, with lower values in young children.

Because the glomerular basement membrane (GBM) is subject to damage in a multitude of renal diseases, a model of basement membrane permeability properties would be useful for learning more about this important barrier. Isolated, perfused tubular basement membrane (TBM) allows measurement of permeability, but it is not known whether TBM is similar enough to GBM for data to be extrapolated from this model to the glomerulus. As a first approach to assessing differences between GBM and TBM, we looked at composition. Renal glomeruli and tubules were isolated from Swiss-Webster mice by sucrose-gradient centrifugation. GBM and TBM were isolated by sonication in 1% deoxycholate and then subjected to a sequential extraction procedure. Analysis of the solubilized basement membranes by electrophoresis revealed a complex mixture of proteins. Immunoblot analysis demonstrated that, among the proteins, laminin and fibronectin were found exclusively in the guanidine and guanidine/dithiotreitol extracts. The total amount of laminin extracted in GBM, 1.8 +/- 0.001 micrograms/mg dry weight (n = 2 groups animals, by inhibitory ELISA), was significantly less than in TBM, 3.4 +/- 0.1 micrograms/mg dry weight (n = 2); however, the total amount of fibronectin extracted did not differ between GBM and TBM, 8.2 +/- 0.8 and 7.7 +/- 1.0 micrograms/mg dry weight (n = 2) respectively. Examination of deoxycholate supernatants was carried out to see if components of GBM or TBM were solubilized during isolation of basement membranes. Immunoblot analysis revealed loss of some laminin and fibronectin occurred during the detergent isolation of GBM and TBM. We conclude that GBM and TBM are qualitatively similar in that they have the same protein components, but differ significantly in content of laminin and probably other macromolecular components.

Immunopathology contributes to high mortality in tuberculous meningitis (TBM) but little is known about the blood and cerebrospinal fluid (CSF) immune response. We prospectively characterised the immune response of 160 TBM suspects in an Indonesian cohort, including 67 HIV-negative probable or definite TBM cases. TBM patients presented with severe disease and 38% died in 6 months. Blood from TBM patients analysed by flow cytometry showed lower αβT and γδT cells, NK cells and MAIT cells compared to 26 pulmonary tuberculosis patients (2.4-4-fold, all p < 0.05) and 27 healthy controls (2.7-7.6-fold, p < 0.001), but higher neutrophils and classical monocytes (2.3-3.0-fold, p < 0.001). CSF leukocyte activation was higher than in blood (1.8-9-fold). CSF of TBM patients showed a predominance of αβT and NK cells, associated with better survival. Cytokine production after ex-vivo stimulation of whole blood showed a much broader range in TBM compared to both control groups (p < 0.001). Among TBM patients, high ex-vivo production of TNF-α, IL-6 and IL-10 correlated with fever, lymphocyte count and monocyte HLA-DR expression (all p < 0.05). TBM patients show a strong myeloid blood response, with a broad variation in immune function. This may influence the response to adjuvant treatment and should be considered in future trials of host-directed therapy.

Earthworms are soil engineers that alter the soil bio-physical properties to favor plant growth whereas pesticides represent a significant threat to their abundance and soil health. Thus, we investigated the toxic effects of tribenuron-methyl (TBM) and tebuconazole (TEB) on the soil earthworm, Eisenia fetida. The TBM demonstrated low toxicity to E. fetida in the contact filter paper and artificial soil tests, with median lethal concentrations (LC50) of 135.6 μg cm-2 at 48 h and 511 mg kg-1 on day 14, respectively. Similarly, TEB also showed low toxicity to E. fetida in the artificial soil test with LC50 of 287 mg kg-1 on day 14. However, TEB was highly toxic to earthworm in the contact filter paper test with LC50 of 5.7 μg cm-2 at 48 h. The mixture of two pesticides had an antagonistic effect on the earthworm. Under 0.1 LC50 of TBM and TEB, either single or combined application of pesticides induced oxidative stress and inhibited cellulase activity in early days of the earthworm exposure. However, both pesticides did not damage the earthworm DNA. Our results suggest that pesticides can negatively affect soil earthworms and provide valuable information regarding the responses of soil biological engineers to the lethal agrochemicals.

Collagen studies in newborn rats with incomplete ureteric obstruction were performed to describe and quantify changes in collagen deposition resulting from urinary tract obstruction at an early developmental age. Incomplete ureteric obstruction was created in three-day-old rats by placing the left ureter in a tunnel formed by the psoas muscle, and sham-operated controls underwent a laparotomy. The rats were sacrificed at 10, 17, 24 or 31 days. Collagen types I, III, IV, and V were localized by indirect immunofluorescence microscopy, the total collagen content of the kidney was quantitated using hydroxyproline analysis, and collagen types I and III were quantitated using cyanogen bromide (CNBr) peptide analysis. Increased immunofluorescent staining for all of the collagens was found in the diffusely widened medullary interstitium of the obstructed kidney, and more focally in the cortical interstitium. Collagen types I, III and V, but not collagen type IV, were also found in bands in the interstitium at the junction of the cortex with the medulla. Increased staining for collagen type IV was found in thickened and tortuous tubular basement membranes (TBM) of the obstructed kidneys. The total collagen content of the obstructed kidney was significantly increased compared to the amounts in both the contralateral kidneys and in the kidneys from sham-operated controls at 24 and 31 days of age (P < 0.01 in each case, Wilcoxon matched pairs rank sum test and Mann Whitney U-test, respectively). The amount of collagen in the kidneys correlated with the degree of hydronephrosis (Spearman correlation test, r = 0.78, P < 0.02). CNBr peptide analysis demonstrated that over 50% of the collagen in the normal neonatal rat kidney was collagen type I and approximately 25% was collagen type III. In the obstructed kidneys most of the collagen was also collagen type I and collagen type III, although the proportion of total collagen comprised by these collagen types was decreased compared with the controls. The amount of collagen type III in the contralateral kidneys was reduced compared to that in the controls. Thus, the neonatal renal response to obstruction resulted in increased amounts of a range of collagens in the interstitium and TBM, and the extent of this response was partially related to the degree of hydronephrosis.

OBJECTIVE

Tuberculous meningitis (TBM) is a life-threatening disease and is difficult to diagnose. We aim to promote the role of magnetic resonance imaging (MRI) in TBM diagnosis and survey.

METHODS

This was a retrospective study undertaken between 1996 and 2003 in which we reviewed all cases of TBM that had undergone cerebral computed tomography (CT) and MRI performed with and without contrast.

RESULTS

We reviewed 29 patients; all had had subacute lymphocytic meningitis. Diagnosis was definite in only 11 cases and presumptive in 18 cases. MRI was performed showing one or more abnormalities in 26 cases. The use of MRI allowed the detection of CNS lesions in both brain and spine.

CONCLUSIONS

Cerebrospinal MRI performed when TBM is suspected aids in its diagnosis and is also a useful means of monitoring the course of the disease under treatment.

Molecular mechanisms underlying Alzheimer's disease (AD) are difficult to investigate, partly because diagnosis lags behind the insidious pathological processes. Therefore, identifying AD neuroimaging markers and their genetic modifiers may help study early mechanisms of neurodegeneration. We aimed to identify brain regions of the highest vulnerability to AD using a data-driven search in the AD Neuroimaging Initiative (ADNI, n = 1,100 subjects), and further explored genetic variants affecting this critical brain trait using both ADNI and the younger UK Biobank cohort (n = 8,428 subjects). Tensor-Based Morphometry (TBM) and Independent Component Analysis (ICA) identified the limbic system and its interconnecting white-matter as the most AD-vulnerable brain feature. Whole-genome analysis revealed a common variant in SHARPIN that was associated with this imaging feature (rs34173062, p = 2.1 × 10^-10 ). This genetic association was validated in the UK Biobank, where it was correlated with entorhinal cortical thickness bilaterally (p = .002 left and p = 8.6 × 10^-4 right), and with parental history of AD (p = 2.3 × 10^-6 ). Our findings suggest that neuroanatomical variation in the limbic system and AD risk are associated with a novel variant in SHARPIN. The role of this postsynaptic density gene product in β1-integrin adhesion is in line with the amyloid precursor protein (APP) intracellular signaling pathway and the recent genome-wide evidence.

Keywords: Alzheimer's disease; brain atrophy; independent component analysis; synaptic adhesion; tensor-based morphometry; whole-genome sequencing.

In the present study, we fabricated a label-free avian influenza (AIV H5N1) detection biosensor composed of a multi-functional DNA 3 way-Junction (3 W J) on a hollow Au spike-like nanoparticle (hAuSN) using a localized surface plasmon resonance (LSPR) method. To construct the multi-functional DNA (MF-DNA) as a bioprobe, the 3 W J was introduced. The proposed AIV detection bioprobe should contain three functionalities: target recognition, signal amplification, and connection to substrate. To achieve this goal, each piece of the DNA 3 W J was tailored to a hemagglutinin (HA) binding aptamer, FAM dye and thiol group, respectively. The assembly of each DNA 3 W J functional fragment was then confirmed by TBM-Native PAGE. Moreover, the hAuSN was immobilized on the indium-tin-oxide (ITO) substrate for LSPR measurement. The DNA 3 W J was immobilized onto the hAuSN electrode through the thiol-group of DNA 3 W J. The fabricated DNA 3 W J/hAuSN heterolayer on the ITO substrate was investigated by field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). LSPR experiments were conducted to confirm HA protein binding to the DNA 3 W J/ hAuSN -modified electrode. The proposed biosensor can detected the HA protein in PBS buffer (LOD: 1 pM) as well as in the diluted chicken serum (LOD: 1 pM). The present study details a label-free, simple fabrication method consisted of DNA 3 W J/ hAuSN heterolayer that uses easy-to-tailor elements to detect not only AIV but also various viruses detection platform easily.

OBJECTIVE

Structure-activity studies were carried out with the model bioreductive alkylating agent benzoquinone mustard (BM) and its structural analogs. The specific objectives were: (1) to investigate the effects of functional group substitutions to the benzoquinone ring on DNA crosslink and strand break formation subsequent to reduction of the analogs by DT-diaphorase (DTD) in vitro, (2) to correlate DNA crosslink and strand break formation by the analogs with anaerobic reduction of the BM analogs by DTD and their redox cycling in vitro, and (3) to correlate DNA crosslink and strand break formation by the BM analogs with their cytotoxic effects in cancer cells.

METHODS

DNA interstrand crosslink and single-strand break formation were assessed using agarose gel assays. To determine DNA interstrand crosslinks or single-strand breaks, linearized or supercoiled plasmid DNA, respectively, were incubated with purified human DTD and increasing concentrations of each BM analog. Subsequently, DNA was electrophoresed on an agarose gel and DNA crosslink and strand break formation were quantified using densitometry. The rates of reduction of the BM analogs by purified human DTD were measured in vitro under hypoxic conditions, and the redox cycling potential was determined under aerobic conditions using HPLC analysis. The cytotoxic activities of these agents in human tumor cell lines were measured by the MTT assay, with and without the DTD inhibitor, dicoumarol.

RESULTS

BM analogs with electron-donating groups (MeBM, MBM, m-MeBM), electron-withdrawing groups (CBM, FBM), sterically bulky groups (PBM, m-PBM, m-TBM) and positional isomers (MeBM, m-MeBM, PBM, m-PBM) were synthesized. After reduction by DTD, the BM analogs produced a concentration-dependent increase in DNA crosslink and DNA strand break formation. The E(10) (extent of DNA crosslink formation produced by 10 micro M BM analog) for DNA crosslink formation displayed the rank order MeBM approximately MBM>m-MeBM approximately PBM approximately BM>CBM>FBM>m-PBM approximately m-TBM. For DNA strand break formation, the E(10) values (extent of DNA strand break formation produced by 10 micro M BM analog) displayed the rank order MeBM>MBM>m-MeBM>PBM>BM approximately CBM>FBM>m-PBM approximately m-TBM. Importantly, the cytotoxic activity of the BM analogs in SK-Mel-28 human melanoma cells correlated positively with the E(10) values for DTD-mediated DNA crosslink formation ( r(s)=0.87, P<0.05) and DNA strand break formation ( r(s)=0.95, P<0.05). Similar correlations were observed in NCI-H661 human lung carcinoma cells. Furthermore, the D(10) values (concentration of BM analog that decreased the surviving cell fraction to 0.1) for cytotoxic activity of the BM analogs correlated with the maximum levels of DNA crosslinks formed with each BM analog, with r(s) values of -0.85 ( P<0.05) for the NCI-H661 cell line, and -0.81 ( P<0.05) for the SK-MEL-28 cell line. The half-time of reduction (t(1/2)) of the BM analogs by DTD did not correlate with DNA crosslink formation, DNA strand break formation, or cytotoxic potency of the analogs.

CONCLUSIONS

Functional groups on the benzoquinone ring affect the ability of BM to produce DNA crosslinks and strand breaks following reduction by DTD. Electron-donating groups increased DNA damage, whereas electron-withdrawing groups and sterically bulky groups at the C6 position had no effect or decreased the ability of the compounds to produce DNA damage compared to BM. Moreover, both DNA crosslink and strand break formation appear to have an important impact on the cytotoxicity of the BM analogs. These results may have significance for optimal use of BM-based antitumor agents and for rationalization of the development of novel therapeutic compounds that require bioactivation by DTD.

OBJECTIVE

Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12).

METHODS

CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA.

RESULTS

Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity.

CONCLUSIONS

CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation.

Intracranial tuberculomas are known to develop during treatment of tuberculous meningitis (TBM). However, they usually develop within weeks or a couple of months after the start of antituberculous therapy (ATT). We present a case of an 18-month-old boy who developed tuberculomas after 9 months of ATT, which subsequently responded to the reintroduction of steroids. Thus, one must keep a watch for neurological deterioration in a child of TBM and if it is due to tuberculomas, one may have to continue steroids and ATT for a long time.

BACKGROUND

Confirmation of tuberculous meningitis (TBM) and initiation of treatment are often delayed due to limitations in isolating Mycobacterium tuberculosis from cerebrospinal fluid (CSF).

OBJECTIVE

To evaluate the role of the BACTEC radiometric method in a clinical setting for the early diagnosis of TBM.

METHODS

Patients meeting criteria for clinically probable TBM over a 3 year period were included. Clinical features, results of CSF investigations (protein, glucose, cell count, Ziehl-Neelsen staining, culture in Löwenstein-Jensen (LJ) medium and BACTEC) and brain CT imaging were reviewed. Drug sensitivity was tested using BACTEC. Patients were started on standard treatment and functional outcome, and response at discharge and follow-up were assessed. Patients were divided according to whether or not M. tuberculosis was isolated by BACTEC and the clinical, radiological, and laboratory features compared.

RESULTS

Sixty patients were evaluated. The mean age was 30 years +/- 11.7 years. Headache and fever were the most common symptoms and the mean duration was 26 days. CT findings were hydrocephalus (n=21), basal exudates (n=16), and tuberculoma (n=14). In 40 patients, M. tuberculosis was isolated by BACTEC and average 15 days was required for detection, whereas it was 30 days in LJ medium. Results of drug-sensitivity testing (n=32) were obtained average 7 days after isolation. Patients from whom M. tuberculosis had been isolated by BACTEC more often had tuberculomas in CT imaging (P=0.018).

CONCLUSIONS

Use of the BACTEC method allows early confirmation in patients with clinically probable TBM. It can guide clinicians in the rational use of anti-tuberculosis treatment by confirming diagnosis and identifying drug- sensitivity.

Tuberculous meningitis (TBM) is the most severe manifestation of tuberculosis and its diagnosis remains a challenge even today due to the lack of an adequate test. HspX antigen of Mycobacterium tuberculosis was previously established as a reliable diagnostic biomarker for TBM in an ELISA test format using anti-HspX polyclonal antibodies. Towards overcoming the limitations of batch-to-batch variation and challenges of scalability in antibody generation, we utilized Systematic Evolution of Ligands by EXponential enrichment (SELEX) to develop high affinity DNA aptamers against HspX as an alternative diagnostic reagent. Post-SELEX optimization of the best-performing aptamer candidate, H63, established its derivative H63 SL-2 M6 to be superior to its parent. Aptamer H63 SL-2 M6 displayed a specific and high affinity interaction with HspX (Kd ∼9.0 × 10-8 M). In an Aptamer Linked Immobilized Sorbent Assay (ALISA), H63 SL-2 M6 significantly differentiated between cerebrospinal fluid specimens from TBM and non-TBM subjects (n = 87, ***p < 0.0001) with ∼100% sensitivity and ∼91% specificity. Notably, ALISA exhibited comparable performance with previously reported antibody-based ELISA and qPCR. Altogether, our findings establish the utility of HspX aptamer for the reliable diagnosis of TBM and pave the way for developing an aptamer-based point-of-care test for TBM.