γ' fibrinogen levels as a biomarker of COVID-19 respiratory disease severity.

Coronavirus disease 2019 (COVID-19) is characterized by a pro-inflammatory state associated with organ failure, thrombosis, and death. We investigated a novel inflammatory biomarker, γ' fibrinogen (GPF), in 103 hospitalized patients with COVID-19 and 19 healthy controls. We found significant associations between GPF levels and the severity of COVID-19 as judged by blood oxygen saturation (SpO2). The mean level of GPF in the patients with COVID-19 was significantly higher than in controls (69.8 (95 % CI 64.8-74.8) mg/dL compared with 36.9 (95 % CI 31.4-42.4) mg/dL, p < 0.0001), whereas C-reactive protein (CRP), lactate dehydrogenase (LDH), and total fibrinogen levels were not significantly different between groups. Mean GPF levels were significantly highest in patients with severe COVID-19 (SpO2 ≤ 93 %, GPF 75.2 (95 % CI 68.7-81.8) mg/dL), compared to mild/moderate COVID-19 (SpO2 > 93 %, GPF 62.5 (95 % CI 55.0-70.0) mg/dL, p = 0.01, AUC of 0.68, 95 % CI 0.57-0.78; Youden's index cutpoint 62.9 mg/dL, sensitivity 0.64, specificity 0.63). In contrast, CRP, interleukin-6, ferritin, LDH, D-dimers, and total fibrinogen had weaker associations with COVID-19 disease severity (all ROC curves with lower AUCs). Thus, GPF may be a useful inflammatory marker of COVID-19 respiratory disease severity.

Gamma' fibrinogen levels as a biomarker of COVID-19 respiratory disease severity.

Coronavirus disease 2019 (COVID-19) is characterized by a pro-inflammatory state associated with organ failure, thrombosis, and death. We investigated a novel inflammatory biomarker, γ' fibrinogen (GPF), in 103 hospitalized patients with COVID-19 and 19 healthy controls. We found significant associations between GPF levels and the severity of COVID-19 as judged by blood oxygen saturation (SpO 2 ). The mean level of GPF in the patients with COVID-19 was significantly higher than in controls (69.8 (95% CI 64.8-74.8) mg/dL compared with 36.9 (95% CI 31.4-42.4) mg/dL, p < 0.0001), whereas C-reactive protein (CRP), lactate dehydrogenase (LDH), and total fibrinogen levels were not significantly different between groups. Mean GPF levels were significantly highest in patients with severe COVID-19 (SpO 2 ≤ 93%, GPF 75.2 (95% CI 68.7-81.8) mg/dL), compared to mild/moderate COVID-19 (SpO 2 > 93%, GPF 62.5 (95% CI 55.0-70.0) mg/dL, p = 0.01, AUC of 0.68, 95% CI 0.57-0.78; Youden's index cutpoint 62.9 mg/dL, sensitivity 0.64, specificity 0.63). In contrast, CRP, interleukin-6, ferritin, LDH, D-dimers, and total fibrinogen had weaker associations with COVID-19 disease severity (all ROC curves with lower AUCs). Thus, GPF may be a useful inflammatory marker of COVID-19 respiratory disease severity.

Enhancement in recovery of drugs with high protein binding efficiency from human plasma using magnetic nanoparticles.

In this paper, we propose an alternate method for bioanalytical extraction of drugs from human plasma samples using bare magnetic nanoparticles. The magnetic nanoparticles (MNPs) were used for deproteination of biological samples that further assist in extraction of plasma bound drugs for bioanalytical studies. The method uses basic solvents (ethanol, methanol, etc.) rather than the expensive and toxic solvents. The MNPs provide several advantages like avoiding the use of centrifuge machine, and making extraction time effective. The average time involved for the sample preparation is around 30-40min. The developed method was examined for seven different drugs having moderate (40-70%) to high (>80%) plasma protein binding efficiency. The present study focuses on the principle of magnetic nanoparticle based extraction of drug that binds with the plasma protein. In calcitriol (protein binding efficiency >99%), it was observed that the drug extraction efficiency could be enhanced by 16% using the present method. However, we assume that still there is a scope for improving the extraction efficiency by optimizing proper solvent for the specific drug. The use of magnetic nanoparticles makes the extraction cost effective and quick with improved efficiency.

Effect of Bacillus subtilis PB6, a natural probiotic on colon mucosal inflammation and plasma cytokines levels in inflammatory bowel disease.

The pathophysiology of inflammatory bowel disease (IBD) involves the production of diverse lipid mediators, namely eicosanoid, lysophospholipids, and platelet-activating factor, in which phospholipase A2 (PLA2) is the key enzyme. Thus, it has been postulated that control of lipid mediators production by inhibition of PLA2 would be useful for the treatment of IBD. This hypothesis has been tested in the present study by examining the therapeutic effect of a novel natural probitic Bacillus subtilis PB6 (ATCC- PTA 6737). B. subtilis PB6 is found to secrete surfactins (cyclic lipopeptides) which have anti-bacterial potential. These surfactins inhibit PLA2, a rate-limiting enzyme involved in the arachidonic acid associated inflammatory pathway and could downregulate the inflammatory response by regulating the eicosanoid and cytokine pathways. With this concept, an experimental animal trial has been conducted in a rat model of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. The oral administration of PB6 suppresses the colitis as measured by mortality rate, changes in the weight gain, colon morphology and the levels of plasma cytokines. The animals treated orally with PB6 at 1.5 x 10(8) CFU/kg thrice daily from day 4 to 10 significantly improve gross pathology of the colon and regain the colon weight to normal (p < 0.05), compared to TNBS-induced positive control. The plasma levels of pro-inflammatory cytokines (TNF-alpha, 1L-1beta, IL-6 and IFN-gamma) are also significantly lowered (p < 0.05) and anti-inflammatory cytokine (IL-I0 and TGF-beta) significantly (p < 0.05) increased after the oral administration of PB6 on day 11. The present study supports the concept that PB6 inhibits PLA2 by the secreting surfactins. In a clinical investigation, it is found to be well tolerated by all the healthy volunteers.

Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support.

In recent years, techniques employing magnetizable solid-phase supports (MSPS) have found application in numerous biological fields. This magnetic separation procedure offers several advantages in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap, and often highly scalable. The current paper details a genomic DNA isolation method optimized in our laboratory using magnetic nanoparticles as a solid-phase support. The quality and yields of the isolated DNA from all the samples using magnetic nanoparticles were higher or equivalent to the traditional DNA extraction procedures. Additionally, the magnetic method takes less than 15 min to extract polymerase chain reaction (PCR) ready genomic DNA as against several hours taken by traditional phenol-chloroform extraction protocols. Moreover, the isolated DNA was found to be compatible in PCR amplification and restriction endonuclease digestion. The developed procedure is quick, inexpensive, robust, and it does not require the use of organic solvents or sophisticated instruments, which makes it more amenable to automation and miniaturization.

Activity and stability of alkaline phosphatase (ALP) immobilized onto magnetic nanoparticles (Fe3O4).

Direct binding of alkaline phosphatase (ALP) on magnetic nanoparticles (Fe(3)O(4)) in the presence of carbodiimide (CDI) using two different methods is described. The activity and stability of immobilized ALP with both shaking and sonication method were compared. The results indicated the ALP binding efficiency to be in the range of 80-100% with both the immobilization techniques. The activities retained were in the range of 20-38% with shaking method and 30-43% with sonication method, respectively. The activities of the immobilized ALP preparations were found to be stable compared to the free (unbound) ALP for at least 16-week storage period. Moreover, ALP immobilized on magnetic nanoparticles was successfully used for dephosphorylation of plasmid DNA before it was used for ligation reaction. The use of immobilized ALP for plasmid dephosphorylation allows easy manipulation, reduces the procedural time, and also avoids exposure of reaction mixture to high temperature.

Polyunsaturated fatty acid levels in red cell membranes of unmedicated schizophrenic patients.

There are several reports of reduced levels of polyunsaturated fatty acids (PUFA), particularly arachidonic acid (AA) and docosahexaenoic acid (DHA), in membrane phospholipid from various tissues including red blood cells (RBC) taken from schizophrenic patients. However, reports have not been entirely consistent and most studies have been confounded by the potential effects of environmental factors including antipsychotic medication and diet. We measured PUFA levels in RBC from two separate groups of unmedicated patients and control subjects from India and Malaysia, populations which have substantial differences in diet. We found no significant difference in levels of AA between patients and control subjects in either population. Levels of adrenic acid were significantly reduced, and levels of DHA significantly increased in both clinical populations. However, diet-related differences in DHA between the populations from India and Malaysia were much greater than differences between schizophrenic patients and controls. It is concluded that reduced RBC membrane levels of AA and DHA are not pathognomic of schizophrenia but that variations in cell membrane fatty acid levels are an epiphenomenon which may reflect underlying abnormalities of phospholipid and fatty acid metabolism and their interaction with environmental factors including medication and diet.

Two double-blind placebo-controlled pilot studies of eicosapentaenoic acid in the treatment of schizophrenia.

Evidence that the metabolism of phospholipids and polyunsaturated fatty acids (PUFA) is abnormal in schizophrenia provided the rationale for intervention studies using PUFA supplementation. An initial open label study indicating efficacy for n-3 PUFA in schizophrenia led to two small double-blind pilot studies. The first study was designed to distinguish between the possible effects of two different n-3 PUFA: eicosapentaenoic acid (EPA) and docohexaenoic acid (DHA). Forty-five schizophrenic patients on stable antipsychotic medication who were still symptomatic were treated with either EPA, DHA or placebo for 3 months. Improvement on EPA measured by the Positive and Negative Syndrome Scale (PANSS) was statistically superior to both DHA and placebo using changes in percentage scores on the total PANSS. EPA was significantly superior to DHA for positive symptoms using ANOVA for repeated measures. In the second placebo-controlled study, EPA was used as a sole treatment, though the use of antipsychotic drugs was still permitted if this was clinically imperative. By the end of the study, all 12 patients on placebo, but only eight out of 14 patients on EPA, were taking antipsychotic drugs. Despite this, patients taking EPA had significantly lower scores on the PANSS rating scale by the end of the study. It is concluded that EPA may represent a new treatment approach to schizophrenia, and this requires investigation by large-scale placebo-controlled trials.

Plasma homovanillic acid in untreated schizophrenia--relationship with symptomatology and sex.

Plasma homovanillic acid (pHVA) concentrations are considered to reflect, in part, central dopamine metabolism and thus may be of value in assessing the role of dopamine neurotransmission in schizophrenia. Furthermore, some recent studies have suggested a relationship of pHVA with symptomatology. We have undertaken a study of pHVA in a large cohort of unmedicated DSM-IV schizophrenic patients in order to assess the relationship of pHVA to various clinical parameters. pHVA in 58 drug-free patients (10.11+/-0.52 ng/ml) was significantly elevated in comparison with 62 matched control subjects (8.77+/-0.39 ng/ml). pHVA was found to be higher in patients with a more negative syndrome. No significant correlation of pHVA with overall SAPS or SANS scores was apparent in the patients although, within the SANS subscales, a significant relationship to anhedonia-asociality was apparent. Interestingly, the male drug-free patients showed a correlation of pHVA with negative symptoms defined by SANS and several SANS subscales, while females showed no significant relationship with any SANS subscales. The results may suggest that an increased dopaminergic turnover is apparent in (male) schizophrenic patients with predominantly negative symptoms, providing some support for reports that this change in neuronal activity may be related to the neuropathological abnormalities seen in the disease, which may themselves differ between males and females. Such neuronal deficits of developmental or degenerative origin may thus result in an elevation/disinhibition of central dopamine metabolism in schizophrenia.

Association of the Ban I dimorphic site at the human cytosolic phospholipase A2 gene with schizophrenia.

There is evidence of increased phospholipid breakdown in cell membranes of patients suffering from schizophrenia. This may be related to increased levels of the enzyme cytosolic phospholipase A2 (cPLA2) which have been reported in schizophrenic subjects. We have identified a Ban I dimorphic site on the first intron of the cPLA2 gene. Schizophrenic subjects were found to have a significant excess of the A2/A2 homozygote relative to healthy control subjects. Genetically determined alterations in phospholipase activity may thus underlie the reported abnormalities of phospholipid metabolism in schizophrenia.

TaqI polymorphic sites at the human dopamine beta-hydroxylase gene possibly associated with biochemical alterations of the catecholamine pathway in schizophrenia.

Two parts of the dopamine beta-hydroxylase (DBH) gene, one a 7.5-kb single copy fragment (F1) spanning the 5'-flanking region to exon 3 and the second a 9.0-kb single copy fragment (F2) spanning exon 3 to exon 7, were amplified by a long PCR procedure in 161 unrelated patients with schizophrenia and 67 unrelated control subjects. The PCR products were completely digested with the restriction enzyme TaqI. These subjects were classified into genetic subgroups according to the TaqI restriction fragment length polymorphisms (RFLPs) for the human DBH gene, and the association of the TaqI RFLPs with biochemical alterations of the catecholamine pathway in schizophrenia was then examined. The frequencies of the two TaqI polymorphic sites did not show significant differences between the patients and control subjects, but the TaqI RFLPs were found to be associated with biochemical alterations of the catecholamine pathway in schizophrenia.

Nitric oxide, free radicals and polyamines may have a role in the membrane pathology of schizophrenia.

Studies on skin fibroblasts in culture derived from schizophrenic and control subjects showed that polyamines are increased, nitrate levels are reduced and thiobarbituric acid reacting substances did not alter in cultured cells from schizophrenic patients compared to control subjects. Results seem to indicate some alteration in membrane functions in schizophrenia, which is susceptible to neuroleptic treatment. Significantly increased levels of polyamines in drug-treated schizophrenic cells indicates a possible role of polyamines in the activation of proposed hypofunctional NMDA subtype of glutamate receptor systems in schizophrenia.

Is the polymorphic microsatellite repeat of the dopamine beta-hydroxylase gene associated with biochemical variability of the catecholamine pathway in schizophrenia?

Six allelic fragments were typed by a polymerase chain reaction process with a pair of primers specific for a sequence containing the polymorphic (GT)n repeat, a microsatellite repeat, in the human dopamine beta-hydroxylase (DBH) gene. Their frequencies in unrelated patients with schizophrenia were 0.003 (A1), 0.114 (A2), 0.343 (A3), 0.526 (A4), 0.006 (A5), and 0.009 (A6), and in unrelated control subjects, 0.012 (A1), 0.086 (A2), 0.309 (A3), 0.574 (A4), 0.006 (A5), and 0.012 (A6). Kruskal-Wallis analysis revealed significant differences among the three groups, the drug-free and drug-treated patients, and the control subjects, in serum DBH activity of the subjects whose genotype was A2/A3 (H = 6.0, p < .05) or A3/A3 (H = 9.8, p < .01), in serum homovanillic acid concentration of those whose genotype was A3/A4 (H = 7.7, p < .025), and in serum tyrosine concentration of those whose genotype was A4/A4 (H = 8.3, p < .02). Mann-Whitney U test showed that in the subjects carrying the A3/A4 genotype, serum noradrenaline concentration of drug-treated patients was significantly higher than that of control subjects (N = 58, p < .02). These results suggest that genotypic polymorphism of the human DBH is likely to be associated with biochemical variability of the catecholamine pathway in schizophrenia.

Possible control of dopamine beta-hydroxylase via a codominant mechanism associated with the polymorphic (GT)n repeat at its gene locus in healthy individuals.

Six allelic fragments were typed by a PCR-based process with a pair of primers specific for a sequence containing the polymorphic (GT)n repeat at the human dopamine beta-hydroxylase (DBH) locus in 125 unrelated healthy individuals. Their frequencies among these individuals were 0.012 (A1), 0.08 (A2), 0.344 (A3), 0.548 (A4), 0.004 (A5) and 0.012 (A6); the two major alleles, A3 and A4, made up nearly 90% of the alleles. These individuals were divided into four groups according to the genotype they possessed, i.e. A3/A3, A4/A4, A3/A4 and others (mixed group). Kruskal-Wallis analysis revealed a significant difference in serum DBH activity among these four genetic groups (H = 32.7, P < 0.0001). The homozygotic genotypes, A3/A3 and A4/A4, were associated with low and high DBH activity, respectively, and the heterozygotic genotype, A3/A4, seemed to play a role in keeping the DBH activity at a moderate level. The present work suggests that the human DBH is likely to be controlled via a codominant mechanism associated with the dinucleotide repeat polymorphism at its gene locus.

Possible association of catecholamine turnover with the polymorphic (TCAT)n repeat in the first intron of the human tyrosine hydroxylase gene.

Five allelic fragments were typed by a PCR-based process with a pair of primers specific for the polymorphic sequence due to (TCAT)n tetranucleotide repeat, a microsatellite repeat, in the first intron of the human tyrosine hydroxylase gene, i.e. A1, A2, A3, A4 and A5. Comparisons of some neurochemical parameters of the catecholamine pathway were then made between the unrelated individuals genotypically classified into six subgroups, five homozygotic and one heterozygotic. Among the six subgroups, the individuals with the A2/A2 genotype had the highest levels of serum noradrenaline and those with the A4/A4 genotype had the lowest, and the individuals with the A1/A1 genotype had the highest levels of serum homovanillic acid. These findings suggest that polymorphism of the (TCAT)n repeat in the first intron of the human tyrosine hydroxylase gene may be associated with catecholamine turnover. Possibly, the two alleles, A2 and A4, may be related to the high and low excitabilities of the noradrenergic nerves, respectively, and the allele, A1, may be associated with the up-regulation of dopamine turnover.

Individuals with schizophrenia have an increased incidence of the H2R649G allele for the histamine H2 receptor gene.

We have previously described an allelic variant of the human H2R, nominated the H2R649G allele. This allele contains an adenine-->guanidine substitution at base 649, which introduces an additional TaqI restriction endonuclease site into the gene. With this in mind, we have investigated allelic polymorphism of this receptor and its association with schizophrenia. H2R DNA from 47 schizophrenic patients and 46 control subjects was amplified by the polymerase chain reaction (PCR). These PCR products were analyzed by observing TaqI cleavage patterns and single-stranded conformational polymorphisms. It was found that the H2R649G allele was 1.8 times more frequent in the schizophrenic population (chi 2 test P < 0.01). In addition, schizophrenic individuals were 2.8 times more likely to be homozygous for the H2R649G allele than the control population, (chi 2 test P < 0.05). These data place the attributable fraction for possession of the H2R649G allele at 28.4%.