Gobinda Sarkar

The use of microorganisms for the formation of metal nanoparticles and their application

Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. The development of reliable experimental protocols for the synthesis of nanomaterials over a range of chemical compositions, sizes, and high monodispersity is one of the challenging issues in current nanotechnology. In the context of the current drive to develop green technologies in material synthesis, this aspect of nanotechnology is of considerable importance. Biological systems, masters of ambient condition chemistry, synthesize inorganic materials that are hierarchically organized from the nano- to the macroscale. Recent studies on the use of microorganisms in the synthesis of nanoparticles are a relatively new and exciting area of research with considerable potential for development. This review describes a brief overview of the current research worldwide on the use of microorganisms in the biosynthesis of metal nanoparticles and their applications.

Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors

BACKGROUND The prediction of clinical behavior, response to therapy, and outcome of infiltrative glioma is challenging. On the basis of previous studies of tumor biology, we defined five glioma molecular groups with the use of three alterations: mutations in the TERT promoter, mutations in IDH, and codeletion of chromosome arms 1p and 19q (1p/19q codeletion). We tested the hypothesis that within groups based on these features, tumors would have similar clinical variables, acquired somatic alterations, and germline variants. METHODS We scored tumors as negative or positive for each of these markers in 1087 gliomas and compared acquired alterations and patient characteristics among the five primary molecular groups. Using 11,590 controls, we assessed associations between these groups and known glioma germline variants. RESULTS Among 615 grade II or III gliomas, 29% had all three alterations (i.e., were triple-positive), 5% had TERT and IDH mutations, 45% had only IDH mutations, 7% were triple-negative, and 10% had only TERT mutations; 5% had other combinations. Among 472 grade IV gliomas, less than 1% were triple-positive, 2% had TERT and IDH mutations, 7% had only IDH mutations, 17% were triple-negative, and 74% had only TERT mutations. The mean age at diagnosis was lowest (37 years) among patients who had gliomas with only IDH mutations and was highest (59 years) among patients who had gliomas with only TERT mutations. The molecular groups were independently associated with overall survival among patients with grade II or III gliomas but not among patients with grade IV gliomas. The molecular groups were associated with specific germline variants. CONCLUSIONS Gliomas were classified into five principal groups on the basis of three tumor markers. The groups had different ages at onset, overall survival, and associations with germline variants, which implies that they are characterized by distinct mechanisms of pathogenesis. (Funded by the National Institutes of Health and others.).

Artificial Cavitation Nuclei Significantly Enhance Acoustically Induced Cell Transfection

The efficiency of ultrasound-mediated gene transfection was enhanced three- to fourfold, compared to previous results, through the use of green fluorescent protein reporter gene, cultured immortalized human chondrocytes and artificial cavitation nuclei in the form of Albunex. Cells were exposed to 1.0-MHz ultrasound transmitted through the bottom of six-well culture plates containing immortalized chondrocytes, media, DNA at a concentration of 40 micrograms/mL and Albunex at 50 x 10(6) bubbles/mL. Transfection efficiency increased linearly with ultrasound exposure pressure with a transfection threshold observed at a spatial average peak positive pressure (SAPP) of 0.12 MPa and reaching about 50% of the living cells when exposed to 0.41 MPa SAPP for 20 s. Adding fresh Albunex at 50 x 10(6) bubbles/mL prior to sequential 1-s, 0.32- or 0.41-MPa exposures increased transfection with each exposure, reaching 43% transfection after four exposures. Efficient in vitro and in vivo transfection now appear possible with these enhancements.

Dideoxy fingerprinting (ddF): A rapid and efficient screen for the presence of mutations

We describe dideoxy fingerprinting (ddF), a hybrid between dideoxy sequencing and SSCP that can detect the presence of single base and other sequence changes in PCR-amplified segments. As implemented herein, ddF involves a Sanger sequencing reaction with one dideoxynucleotide followed by nondenaturing gel electrophoresis. When ddF was used to examine segments of the human factor IX gene, 84 of 84 different mutations were detected with a very low rate of false positive signals. The approximate locations of the sequence changes could be determined from the ddF pattern and samples with different sequence changes had different fingerprints. In addition, large segments could be amplified and rapidly screened by ddF in multiple smaller subsegments. The patterns observed with ddF are instructive in that they suggest an inherent limitation in the detection of certain mutations by SSCP.

Direct sequencing of the dopamine D2 receptor (DRD2) in schizophrenics reveals three polymorphisms but no structural change in the receptor

The dopamine D2 receptor gene (gene symbol DRD2) is a candidate gene for schizophrenia because the potency of certain neuroleptics correlates with their affinity for this receptor. Seven regions of likely functional significance including the coding sequences and the splice junctions were fully sequenced in the dopamine D2 receptor of 14 schizophrenics (and partially in several others) meeting DSM-III-R diagnostic criteria and in four unaffected non-Caucasians (97 kb of total sequence). No structural changes were found, suggesting that alteration in the structure of the dopamine D2 receptor is not commonly involved in the etiology of schizophrenia. However, two common and one uncommon intragenic polymorphisms were found. At least one of the polymorphisms was informative for linkage in 70% of Caucasians and 78% of Koreans.

Osteoblasts Express Types I and II Activin Receptors During Early Intramembranous and Endochondral Bone Formation

Increasing evidence suggests a potential role for activin in bone formation. However, the cognate receptors through which activins function with respect to skeletal tissues have not yet been identified. Identification and regulation of expression of these receptors are necessary prerequisites to understanding the role of activins in bone metabolism. We detected mRNAs for three activin receptors, type I (ActRI), type II (ActRII), and type IIB (ActRIIB), in multiple skeletal tissues in rat, including tibia and costochondral growth plate, and also in cultured osteoblasts. To gain information about the relationship between receptor expression and different skeletal cell functions, we evaluated expression of the three receptors in a semiquantitative manner during the early stages of fracture healing, a model for rapid bone formation. Relatively high levels of ActRI and ActRII expression were detected in the callus at 7, 10, and 14 days after fracture, times that correlate with the interval of rapid intramembranous bone formation and the initiation of endochondral bone formation. Expression of the ActRIIB in the fracture callus was strikingly lower than either ActRI or ActRII. Immunostaining of the fracture callus and the newborn rat femur with an anti‐ActRII antibody localized the receptor to osteoblasts at regions of membranous and endochondral bone formation. No staining of osteoblasts in fracture callus or bone was seen with an anti‐ActRIIB antibody. These results provide strong evidence of the identification of the principal receptors through which activins could function in the skeletal system and further shed light on activins mechanism of action in bone formation.

Polymerase Chain Reaction Amplification of Specific Alleles: A General Method of Detection of Mutations, Polymorphisms, and Haplotypes

Publisher Summary The polymerase chain reaction (PCR) can be adapted for the rapid detection of known single-base changes in DNA by using specially designed oligonucleotides in a method called “PCR amplification of specific alleles (PASA).” PASA has been further modified to include two allele-specific primers in the same PCR reaction. By varying the lengths of the allele-specific oligonucleotides, the amplified products can be distinguished from each other by gel electrophoresis. This method, called PCR amplification of multiple specific alleles (PAMSA), allows the rapid detection of more than one allele in a single PCR reaction. PASA is a generally applicable technique for the detection of point mutations or polymorphisms. The method may also be used to detect the presence of small deletions or insertions. PASA has the advantages of being rapid, reproducible, nonisotopic, and amenable to automation.

Cathepsins and osteosarcoma: Expression analysis identifies cathepsin K as an indicator of metastasis

Osteosarcoma is the most frequent malignant bone tumor with a poor survival rate for patients with metastasis. Previous studies have shown that beside other proteases, distinct sets of cathepsins are involved in the process of metastasis of different tumors. In this study we investigated the expression of cathepsin proteases in human osteosarcoma metastasis. First, the mRNA expression of 14 human cathepsins was studied in SAOS‐2 osteosarcoma cells and the highly metastatic LM5 and LM7 sublines by reverse transcriptase (RT)‐polymerase chain reaction (PCR). The expression of cathepsin D, K, and L mRNA was found upregulated and that of cathepsin F, H, and V downregulated in the highly metastatic LM5 and LM7 cells. A subgroup of the cathepsin proteases was further studied at the protein level by Western blot analysis of cell extracts. The expression of cathepsin B and H was decreased and that of cathepsin D, K, and L was increased in the highly metastatic cell lines as compared to the SAOS‐2 cell line. Diagnostic relevance of cathepsin K expression in osteosarcoma was revealed upon correlation of survival and metastasis with immunohistochemical cathepsin K staining of biopsies collected from 92 patients prior to chemotherapy. Patients with metastatic high‐grade osteosarcoma and low cathepsin K expression at diagnosis had a better prognosis than those with high expression. Thus, it appears that cathepsin K expression is of predictive prognostic value for patients with high‐grade tumors and metastasis at diagnosis.

PIG-B: A Homemade Monophasic Cocktail for the Extraction of RNA

An inexpensive monophasic reagent has been developed for the extraction of total RNA from cells or tissues. The main ingredients of the reagent arePhenol,Isoamyl alcohol,Guanidinium isothiocyanate, andBeta-mercaptoethanol (PIG-B). The quality and yield of RNA obtained by this reagent is at par with that obtained by TRIzol, an expensive but widely used monophasic reagent available commercially. The complete composition and method of preparation of PIG-B is provided to aid preparation of the reagent in the laboratory.

Removal of DNA contamination in polymerase chain reaction reagents by ultraviolet irradiation

Publisher Summary This chapter discusses the removal of DNA contamination in polymerase chain reaction (PCR) reagents by ultraviolet (UV) irradiation. The experiments described demonstrate the efficacy of UV irradiation as a decontamination agent. The method involves mixing the reagents, irradiating with UV for the desired time, adding template DNA, and amplifying with PCR. Although the experiments focus on PCR, UV irradiation can inactivate undesired nucleic acids in other protocols, such as in vitro transcription and certain alternative amplification systems. Two major practical conclusions emerge from these studies. First, it is advantageous not to amplify small segments of DNA whenever feasible because these are likely to be poorly inactivated by UV if these segments become contaminants. Second, UV inactivation is much less effective in eliminating dried DNA, suggesting that the decontamination of laboratory equipment may require the aid of a photosensitizer or even a completely different approach.