Application of Short Tandem Repeat markers in diagnosis of chromosomal aneuploidies and forensic DNA investigation in Pakistan

Purpose

Short Tandem Repeat (STR) genetic markers hold great potential in forensic investigations, molecular diagnostics and molecular genetics research. AmpFlSTR® Identifiler™ PCR amplification kit is a multiplex system for co-amplification of 15 STR markers used worldwide in forensic investigations. This study attempts to assess forensic validity of these STRs in Pakistani population and to investigate its applicability in quick and simultaneous diagnosis and tracing parental source of common chromosomal aneuploidies.

Methodology

Samples from 554 healthy Pakistani individuals from 5 different ethnicities were analyzed for forensic parameters using Identifiler STRs and 74 patients' samples with different aneuploidies were evaluated for diagnostic strengths of these markers.

Results

All STRs hold sufficient forensic applicability in Pakistani population with paternity index between 1.5 and 3.5, polymorphic information content from 0.63 to 0.87 and discrimination power ≥ 0.9 (except TPOX locus). Variation from Hardy–Weinberg equilibrium was observed at some loci reflecting selective breeding and intermarriages trend in Pakistan. Among aneuploidic samples, all trisomies were precisely detectable while aneuploidies involving sex chromosomes or missing chromosomes were not clearly detectable using Identifiler STRs. Parental origin of aneuploidy was traceable in 92.54% patients.

Conclusion

The studied STR markers are valuable tools for forensic application in Pakistan and utilizable for quick and simultaneous identification of some common trisomic conditions. Adding more sex chromosome specific STR markers can immensely increase the diagnostic and forensic potential of this system.     

Genosensor for rapid,sensitive, specific point-of-care detection of H1N1 influenza (swine flu)

A 5′ amine group-linked haemagglutinin (HA) gene-specific probe was attached over the surface of a working electrode to develop a rapid, specific, and sensitive point of care detection assay for H1N1 (swine flu) in human respiratory nasal swabs. The probe was attached with a cysteine covered screen-printed gold electrode via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). The electrochemical assay was performed using differential pulse voltammetry with the use of the redox indicator methylene blue for the detection of different concentrations of the single-stranded viral genome. The developed genosensor showed high sensitivity for H1N1 influenza virus with a detection limit of 0.002 ng/6 μL of viral nucleic acid in the sample. Samples were analysed by quantitative real-time Polymerase Chain Reaction as well as by conventional PCR. The genosensor showed high specificity, as no cross-reaction was observed with the heterologous nucleic acid of different pathogens (Salmonella typhi, Neisseria meningitides, and Streptococcus pyogenes) and human DNA, and it was specific for H1N1 with a sensitivity of ∼49 μA cm⁻² ng^-1. Genosensor is based on a very simple methodology that can be followed based on its easy-to-access approach. It is quick and could be used as a point-of-care test for the detection of influenza virus within 30 min.     

Calorimetric scrutiny of lipid binding by sticholysin II toxin mutants

The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7 × 10⁸ M^− 1). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid-water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation.     

Fluxes of dissolved and nonfossil particulate organic carbon from an Oceania small river (Lanyang Hsi) in Taiwan

Concentrations of dissolved and particulate organic carbon (DOC & POC) in river waters were measuredduring 1993–1994 in the Lanyang Hsi watershed, which representsa typical small Oceania river. The DOC concentrations varied in the range of 0.5–4 mg/l during non-typhoon period, but rose to as highas 8 mg/l during Typhoon Tim in July, 1994. Based on the log-linearrelationship between the DOC load and the discharge rate, weestimated the DOC export to be 3.4 ± 0.6 ktC/yr,and the DOC yield to be 4.1 ± 0.7 gC/m²/yr,which is considerably higher than a former estimate (ca.0.1 gC/m²/yr) for the Oceania. On the other hand, the DOC yield is less than the concurrent POC yield (21.7 ± 4.7gC/m²/yr) by a factor of five, but most of theexported POC is fossil carbon. Under the assumption that the suspended sediments contain a mean fossil POC content of0.5%, the nonfossil POC yield was calculated to be 4.6± 3.0 gC/m²/yr, comparable to theDOC yield. Since DOC and nonfossil POC are directly related to theecosystem, their combined fluxes give a biogenic organic carbonyield of 8.7 ± 3.1 gC/m²/yr.     

A robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of Schistosoma circulating anodic antigen

An earlier reported laboratory assay, performed in The Netherlands, to diagnose Schistosoma infections by detection of the parasite antigen CAA in serum was converted to a more user-friendly format with dry reagents. The improved assay requires less equipment and allows storage and worldwide shipping at ambient temperature. Evaluation of the new assay format was carried out by local staff at Ampath Laboratories, South Africa. The lateral flow (LF) based assay utilized fluorescent ultrasensitive up-converting phosphor (UCP) reporter particles, to be read by a portable reader (UPlink) that was also provided to the laboratory. Over a period of 18 months, about 2000 clinical samples were analyzed prospectively in parallel with a routinely carried out CAA–ELISA. LF test results and ELISA data correlated very well at CAA concentrations above 300 pg/mL serum. At lower concentrations the UCP–LF test indicates a better performance than the ELISA. The UCP–LF strips can be stored as a permanent record as the UCP label does not fade. At the end of the 18 months testing period, LF strips were shipped back to The Netherlands where scan results obtained in South Africa were validated with different UCP scanning equipment including a novel, custom developed, small lightweight UCP strip reader (UCP-Quant), well suited for testing in low resource settings.     

PLA2-mediated catalytic activation of its inhibitor 25-acetyl-petrosaspongiolide M: serendipitous identification of a new PLA2 suicide inhibitor

25-Acetyl-petrosaspongiolide M (PMAc) (1), a mild non-covalent PLA(2) inhibitor, unexpectedly recovers, after incubation with bvPLA(2), the ability to covalently modify the enzyme target. This study demonstrates the catalytic effect of bvPLA(2) in converting 1 in its deacetylated congener petrosaspongiolide M (PM) (2), a strong covalent PLA(2) inhibitor whose molecular mechanism of inhibition has already been clarified. Moreover, our findings outline the potential role of PMAc as anti-inflammatory pro-drug, by virtue of its ability of delivering the active PM agent at the site of inflammation, functioning as a suicide inhibitor.     

Cover crop root morphology rather than quality controls the fate of root and rhizodeposition C into distinct soil C pools

Cover crops increase carbon (C) inputs to agricultural soils, and thus have the potential to mitigate climate change through enhanced soil organic carbon (SOC) storage. However, few studies have explored the fate of belowground C inputs associated with varying root traits into the distinct SOC pools of mineral-associated organic carbon (MAOC) particulate organic carbon (POC). Therefore, a packed 0.5 m column trial was established with 0.25 m topsoil and 0.25 m subsoil with four cover crops species (winter rye, oilseed radish, chicory, and hairy vetch) known to differ in C:N ratio and root morphology. Cover crops were ¹⁴CO₂-labeled for 3 months, and then, half of the columns were sampled to quantify root and rhizodeposition C. In the remaining columns, plant shoots were harvested and the undisturbed soil and roots were left for incubation. Bulk soil from both sampling times was subjected to a simple fractionation scheme, where ¹⁴C in the <50 and >50 μm fraction was assumed to represent MAOC and POC, respectively. The fast-growing rye and radish produced the highest root C. The percentage loss of C via rhizodeposition (%ClvR) showed a distinct pattern, with 22% for the more branched roots (rye and vetch) and 6%–8% for the less branched roots (radish and chicory). This suggests that root morphology plays a key role in determining rhizodeposition C. After 1 year of incubation at room temperature, the remaining MAOC and POC were positively correlated with belowground inputs in absolute terms. However, topsoil MAOC formation efficiencies (cover crop-derived MAOC remaining as a share of belowground inputs) were higher for vetch and rye (21% and 15%, respectively) than for chicory and radish (9% and 10%, respectively), suggesting a greater importance of rhizodeposition (or indirectly, root morphology) than solely substrate C:N ratio for longer term C stabilization.     

湄洲湾夏季生物有机碳的研究

湄洲弯夏季浮游植物有机碳的调查结果表明,活体有机碳「（ＰＯＣ（Ｂ）」含量为４２．５０－１８５．０μｇ／ｄｍｄｍ＾３,平均值为７７．５ｇ／ｄｍ＾３ＰＯＣ（Ｂ）和叶绿素（Ｃｈｌａ）的转移系数为６２．４;由三磷酸腺苷（ＡＴＰ）计算所得ＰＯＣ（Ｂ）要比由光合作用速率（ＰＲ）所计算的结果大,同时,同化系数（ＰＩ）的变化为０．７８－２．２２ｈ＾－１,它的垂直变化与ＰＯＣ（Ｂ）分布有关。氮是湄洲湾初级生产力的限     

Water chemistry variability in the lower intertidal zone [-2pt] of an estuary in the Seto Inland Sea,Japan: seasonal patterns of nutrients and particulate compounds

From April 1994 to April 1996, we carried out monthly surveys on the physical and chemical variability of ebbing water at two intertidal stations near the emerged tidal flat and surface water at a nearby subtidal station, in a tidal estuary of the Seto Inland Sea, Japan. The fresh water runoff was a major source of new nitrogen (nitrate+nitrite = –2.1 × salinity + 74.3, r ² = 0.76, p < 0.001, at the inner intertidal station). In contrast, the absence or weak correlation of ammonium, phosphate and silicate with salinity in ebbing water highlighted the importance of in situ biologically-mediated processes, including the excretory activity of intertidal dominant bivalves (Ruditapes philippinarum and Musculista senhousia) and primary producer nutrient uptake. Among suspended particulate matter (SPM), the negative correlation of Chl a with salinity (p< 0.001) suggested that an upper intertidal zone, where high microphytobenthic assemblages occur, may represent a considerable source of microalgal biomass. Whereas, a stronger correlation of POC with pheo-pigments (r ² 0.92) than with Chl a (r ² 0.44), a low fraction of living phyto-Carbon (ca. 5%) and a high pheo-pigment/Chl a ratio (ca. 3) indicated a high portion of refractory algal material and a close water–sediment coupling on the flat, in which abundant macrozoobenthos is likely to play an important role. In spite of the strong variability of this ecosystems, this study also demonstrated that highly significant seasonal patterns can be recognized in our study area. However, patterns varied depending on the different nutrient species, SPM and sites. In ebbing water, ammonium concentrations were significantly higher in the cold period (i.e. November–April: water temperature 10.0 ± 4.4 °C), when primary producer biomass decreases, while Chl a and pheo-pigment content were 4.3 and 4.8 higher in the warm period (May–October: water temperature 23.5 ± 4.2 °C), respectively. At the subtidal site, nutrients were 1.9 (silicate)–2.9 (phosphate) and SPM was 1.8 (POC)–2.1 (pheo-pigments) times higher in the warm period. On an annual basis, mean pheo-pigment and POC content was 12–25 times and 4–8 times higher on the intertidal zone than on the subtidal zone, respectively, while nutrient concentrations were within the same order of magnitude. Such a qualitative difference (i.e. nutrients vs. SPM) between sites suggests that particle deposition and/or removal is likely to occur along the estuary, resulting in a limited SPM content at the surface layer of the subtidal site, while nutrients are more directly transported into the surface layer of the subtidal zone by lower salinity water mass intrusion from the intertidal zone, most importantly during the warm period.     

Synthesis and antiproliferative activity of two diastereomeric lignan amides serving as dimeric caffeic acid-l-DOPA hybrids

Two new diastereomeric lignan amides (4 and 5) serving as dimeric caffeic acid-l-DOPA hybrids were synthesized. The synthesis involved the FeCl₃-mediated phenol oxidative coupling of methyl caffeate to afford trans-diester 1a as a mixture of enantiomers, protection of the catechol units, regioselective saponification, coupling with a suitably protected l-DOPA derivative, separation of the two diastereomers thus obtained by flash column chromatography and finally global chemoselective deprotection of the catechol units. The effect of hybrids 4 and 5 and related compounds on the proliferation of two breast cancer cell lines with different metastatic potential and estrogen receptor status (MDA-MB-231 and MCF-7) and of one epithelial lung cancer cell line, namely A-549, was evaluated for concentrations ranging from 1 to 256 μM and periods of treatment of 24, 48 and 72 h. Both hybrids showed interesting and almost equipotent antiproliferative activities (IC₅₀ 64–70 μM) for the MDA-MB-231 cell line after 24–48 h of treatment, but they were more selective and much more potent (IC₅₀ 4–16 μM) for the MCF-7 cells after 48 h of treatment. The highest activity for both hybrids and both breast cancer lines was observed after 72 h of treatment (IC₅₀ 1–2 μM), probably as the result of slow hydrolysis of their methyl ester functions.