A new diagnostic tool for rapid and accurate detection of Mycobacterium tuberculosis

Mycobacterium tuberculosis, acid fast bacilli from the family of Mycobacteriaceae, is the causative agent of most cases of tuberculosis. Tuberculosis, as a communicable disease, remains a serious public health threat, killing more than one million people globally every year. Primary diagnosis of tuberculosis bacilli (TB) relies mainly on microscopic detection of acid fast bacilli (AFB), but the method suffers from low sensitivity and the results largely depend on the technician’s skill. New diagnostic tools are necessary to be introduced for rapid and accurate detection of the bacilli in sputum samples. We, in collaboration with Anda Biologicals, have developed a new platform, named as “Patho-tb”, for rapid detection of AFB with high sensitivity and with low dependence on human skills. Evaluation of Patho-tb test performance was done in two settings: (1) primary field study conducted using 38 sputa from high TB prevalence area of Iran (Zabol city near to the Afghanistan border), and (2) main study conducted using 476 sputa from Tehran, capital of Iran. Patho-tb was applied for processed sputum samples in parallel with routine diagnostic methods (including AFB microscopy, culture and PCR). All test results were compared to final clinical diagnostic state of an individual and diagnostic sensitivity (DSe), specificity, positive predictive value, negative predictive value and accuracy of each test results were calculated using standard formulations. Analytical sensitivity and specificity of the Patho-tb test were also determined. Calculated values for five above mentioned parameters are as follows: for field study: AFB (DSe: 29.6, DSp: 81.8, PPV: 80, NPV: 23.1, AC: 44.7), Patho-tb (DSe: 63, DSp: 72.7, PPV: 85, NPV: 44.4, AC: 65.8), and for main study: AFB (DSe: 86.1, DSp: 99.4, PPV: 98.5, NPV: 93.9, AC: 95.2), Patho-tb (DSe: 97.4, DSp: 92.9, PPV: 86.5, NPV: 98.7, AC: 94.3). Reproducibility of Patho-tb test results were near to 100% (Cohen’s kappa value between 0.85 and 1). The detection limit of Patho-tb test with 100% positivity rate was 3 × 10³ cells/ml of sputum. In the field study, Patho-tb test was 33.4% more sensitive than AFB microscopy, while the improvement was only 11.3% during the main study. Patho-tb results are easy to interpret and the test can be merged with other screening tests, like AFB. Totally, Patho-tb test alone or in conjunction with AFB microscopy is a useful screening tool for TB detection especially in poor geographical lab conditions.     

THY1 as a reliable marker for enrichment of undifferentiated spermatogonia in the goat

Spermatogonial stem cells are unique cells of testes that can restore fertility upon transplantation into recipient testes. However, use of suitable markers for enrichment of these cells have important potential application. THY1, is an established conserved marker of spermatogonial stem cells in bovine, rodents, and primates, but there is no information available in goats. After three rounds of enzymatic digestion of prepubertal goat testicular tissues, undifferentiated spermatogonia positive for THY1 were isolated by magnetic-activated cell sorting and were used for immunocytochemistry, real-time polymerase chain reaction analysis for gene expression, protein expression, and transplantation into recipient mice. Immunocytochemical analyses showed that significantly higher percentage of THY1⁺ cells were positive for PLZF and VASA when compared with unselected population. This result for PLZF was further confirmed at the protein level. Real-time polymerase chain reaction analysis revealed that expression of THY1, PLZF, VASA, BCL6B, and UCHL1 as SCCs characteristic genes in THY1⁺ cells was significantly higher than in the initial population. Finally, transplantation of PKH26-labeled cells revealed that THY1⁺ cells had higher capacity for colony formation when compared with unselected cells. In conclusion, the results provide indications that THY1 surface marker can be reliably used for enrichment of undifferentiated spermatogonial in the goats.     

Balkhania balkhanica Mamontova, 1966 (benthic foraminifera) and Kopetdagaria sphaerica Maslov, 1960 (dasycladalean alga) from the Lower Cretaceous Tirgan Formation of the Kopet Dagh mountain range (NE Iran) and their paleobiogeographic significance

The poorly known larger benthic foraminifer Balkhania balkhanica Mamontova is reported from the Tirgan Formation (Upper Barremian-Lower Aptian) in the northeastern part of Iran. It was so far only known from Iran (Kopet Dagh and Central Iran), Turkmenistan, and central Afghanistan. Obviously, B. balkhanica possessed a limited distributional area overlapping largely with that of the dasycladalean alga Kopetdagaria sphaerica Maslov (Turkmenistan, Iran, Georgia, Bulgaria, Romania), Conradella bakalovae (Conrad and Peybernès) (Tibet?, Georgia, Bulgaria, Romania), and other taxa. These taxa typify the so-called Carpatho-Pontic algal bioprovince sensu Bucur (2000), which is emended and replaced with the new name Carpatho-Cimmerian bioprovince being part of the former northern (= Eurasian) margin of the Neo-Tethys. The present case study provides a further example that the investigation of the distribution pattern of larger benthic foraminifera, together with dasycladalean green algae, may provide a useful proxy in paleotectonic and paleogeographic reconstructions of the Peri-Tethyan domain.     

Assessment of the effect of biosurfactant produced by Pseudomonas aeruginosa in lethality of Bacillus thuringiensis Berl. against 3rd instars larvae of white cabbage butterfly (Pieris brassicae L.)

Surfactant that is produced from cheap sources like oil sludge by biological agents such as bacteria can be used in various industrial processes. For example, it can be used in environmental processes such as bioremediation and elimination of environmental pollutants, and acts as synergistic agents and distributor pesticides on waxy leaves in agriculture. In this study, biosurfactant which is produced by Pseudomonas aeruginosa (isolated from petroleum sludge) at the intervals of 24, 48, 72 and 96?h, along with chemical surfactant Tween 80 and the biological control agent, Bacillus thuringiensis, in a pilot project for controlling one important cabbage pest (Pieris brassicae), their synergistic properties were evaluated. Statistical analysis of the results showed that B. thuringiensis in combination with biosurfactant produced at different times and B. thuringiensis in combination with chemical surfactant Tween 80 when compared with control treatments like B. thuringiensis alone and B. thuringiensis plus tween 80 as positive controls and distilled water as negative control have significant differences (p?<?0.05). This research showed that surfactant treatment produced at the intervals of 24 and 48?h in combination with B. thuringiensis has the greatest synergistic effect when compared to chemical surfactant treatment. This study concluded that biosurfactant can be used as a distributor and synergistic agent against plant pests and in addition to this, their biological roles in bioremediation can be used as a viable alternative to non-economical chemical surfactants that annually enter millions of tonnes of harmful chemical substances into the fields and underground water.     

A Convenient High Yield Synthesis of N4-Isobutyryl-2′-O-Methylcytidine and Its Monomer Units For Incorporation into Oligonucleotides

Abstract

We designed an efficient three step procedure for the synthesis of N⁴-isobutyryl-2′-O-methylcytidine. This protected nucleoside was then used to prepare a methylphosphonamidite monomer for incorporation into oligonucleotides. Transamination at the C⁴ position of cytidine using ethylenediamine, which has been reported for the N⁴-benzoyl cytidine, was not observed with N⁴-isobutyryl protected 2′-O-methylcytidine.     

Vegetation dynamics during the early to mid-Holocene transition in NW Malta, human impact versus climatic forcing

A pollen diagram was constructed for the early- to mid-Holocene transition (ca. 7350–5600 cal. b.p./5400–3650 b.c.) from the Burmarrad ria located in NW Malta. The vegetation at ca. 7350–6960 cal. b.p./5400–5010 b.c. was characterized by an almost tree-less steppe-like open landscape. Early Holocene dry climatic conditions were most probably due to intensification of the subtropical monsoon circulation that strengthened the subtropical anticyclonic descent over the central Mediterranean and blocked the penetration of humid air masses from the North Atlantic Ocean. At ca. 6950 cal. b.p./5000 b.c., the steppe-like vegetation was suddenly replaced by a Mediterranean evergreen forest or dense scrub dominated by Pistacia cf. lentiscus trees. This event, which has simultaneously been recorded in southern Sicily, was most probably caused by the southward shift of the ITCZ permitting the eastward movement of the North Atlantic cyclonic systems. Traces of human activities are evident in the pollen diagram since the beginning of the record but become more pronounced from the onset of the Temple Cultural Phase at ca. 6050 cal. b.p./4100 b.c. with a gradual decline of tree pollen. We suggest that the early- to mid-Holocene vegetation transformation was mainly controlled by a regional climatic change that occurred in a landscape only slightly impacted by human activities.     

Serotyping of Toxoplasma gondii in Cats (Felis domesticus) Reveals Predominance of Type II Infections in Germany

Background

Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany.

Methodology

To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7).

Findings

Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III.

Conclusions

Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.     

Spectroscopic and cytotoxic studies of the novel designed palladium(II) complexes: beta-lactoglobulin and K562 as the targets

Since palladium complexes have been reported to show fewer side effects relative to other heavy metal anticancer compounds, in this study a new class of four structurally related anticancer Pd(II) complexes including 2,2'-bipyridin-n-butyl dithiocarbamato Pd(II) nitrate (Com-1), 2,2'-bipyridin-n-hexyl dithiocarbamato Pd(II) nitrate (Com-2), 2,2'-bipyridin glycinato Pd(II) nitrate (Com-3) and 2,2'-bipyridin octylglycinato Pd(II) nitrate (Com-4) was designed. The effect of four synthesized ligands on the protein structure and cell proliferation were investigated. Whey carrier proteins beta-lactoglobulin-A and-B (BLG-A and-B) and chronic myelogenous leukemia cell line K562 were the targets. Fluorescence and CD instruments were used to assess effect of the ligands on the protein structure. Growth inhibitory effect of the Pd(II) complexes towards the cancer cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results of fluorescence studies revealed that the complexes had no dithiocarbamate moiety (compounds 3 and 4) could quench the intrinsic fluorescence emission of the proteins at lower concentrations than those had such moiety (compounds 1 and 2). The far-UV-CD studies revealed that the regular secondary structure of BLG-A and -B did not show any noticeable alteration upon interaction with different of Pd(II)-complexes. The results of cell proliferation assay also displayed that Com-1 and Com-2 had more growth inhibitory activity against K562, than Com-3 and Com-4. Our results suggested that addition of dithiocarbamate moiety to structure of Pd(II) complexes probably has important role to improve the antiproliferative properties of the anticancer ligands and fewer effects on the carrier protein structure.