Netting the malaria menace: Distribution and utilization of long-lasting insecticidal net in a malaria endemic area in Bankura, West Bengal.

BACKGROUND & OBJECTIVES: Long-lasting insecticidal net (LLIN) is considered to be a highly effective intervention against malaria under National Vector Borne Disease Control Programme in India. A cross-sectional study was undertaken to assess the coverage and utilization of LLIN and the factors related thereto. METHODS: A survey of 1300 households was carried out in Ranibandh block of Bankura district in West Bengal, India, using lot quality assurance sampling (LQAS) method. Coverage/utilization of 80% was considered as minimum acceptable norm. The weighted sample size was calculated from each village of the block. The sociodemographic, economic information of the household along with the availability and use of LLIN was collected through interview and observation. RESULTS: In total, 7320 individuals including 840 children ≤ 5 yr were visited. Overall coverage of adequate LLIN was 65.4% (± 1.5%) and for children ≤ 5 yr, it was 60.5% (± 1.3%). Overall, 66.1% (± 1.4%) people of all ages and 63.7% (± 1.4%) children ≤ 5 yr slept under LLINs in the night before the survey. Out of 26 sub-centres, distribution of LLINs in 10 sub-centres was below the accepted norm, whereas utilization was sub-optimal in 19 sub-centres. In only 18.2% (± 0.5%) households, LLINs remained hanging during daytime. Poverty, caste, education, perception regarding malarial morbidity and preventive action of LLIN were associated significantly with the distribution of LLIN. Similarly, poverty (AOR = 2.14), threat perception regarding malarial morbidity (AOR = 1.51) and mortality (AOR = 2.52) were positively associated with the use of LLIN. Full utilization of bednets by under-fives of the households was higher in villages with sub-centres. INTERPRETATION & CONCLUSION: Around two-third population of the study area was effectively covered with LLIN. Higher proportion of socially marginalized people received LLIN. Threat perception regarding malaria was directly associated with both receipt and use of LLIN. Behaviour change communication on utilization along with adequate access to LLIN needs to be strengthened.

Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity.

RNA amplification with transcript sequencing (RAWTS) is a rapid and sensitive method of direct sequencing that involves complementary DNA synthesis, polymerase chain reaction (PCR) with a primer or primers containing a phage promoter, transcription from the phage promoter, and reverse transcriptase-mediated sequencing. By means of RAWTS, it was possible to sequence each of four tissue-specific human messenger RNAs (blue pigment, factor IX, phenylalanine hydroxylase, and tyrosine hydroxylase) in four cell types examined (white blood cells, liver, K562 erythroleukemia cells, and chorionic villus cells). These results indicate that there is a basal rate of transcription, splicing, and polyadenylation of tissue-specific mRNAs in adult and embryonic tissues. In addition to revealing sequence information, it is possible to generate a desired in vitro translation product by incorporating a translation initiation signal into the appropriate PCR primer. RAWTS can be used to obtain novel mRNA sequence information from other species as illustrated with a segment of the catalytic domain of factor IX. In general, the ability to obtain mRNA sequences rapidly across species boundaries should aid both the study of protein evolution and the identification of sequences crucial for protein structure and function.

Genomic amplification with transcript sequencing.

A sequencing method called genomic amplification with transcript sequencing (GAWTS) is described that is based on amplification with the polymerase chain reaction (PCR). GAWTS bypasses cloning and increases the rate of sequence acquisition by at least fivefold. The method involves the attachment of a phage promoter onto at least one of the PCR primers. The segments amplified by PCR are transcribed to further increase the signal and to provide an abundance of single-stranded template for reverse transcriptase-mediated dideoxy sequencing. An end-labeled reverse transcriptase primer complementary to the desired sequence generates the additional specificity required to generate unambiguous sequence data. GAWTS can be performed on as little as a nanogram of genomic DNA. The rate of GAWTS can be increased by coamplification and cotranscription of multiple regions as illustrated by two regions of the factor IX gene. Since GAWTS lends itself well to automation, further increases in the rate of sequence acquisition can be expected.

Regulation of soluble guanylyl cyclase activity by oestradiol and progesterone in the hypothalamus but not hippocampus of female rats.

Oestradiol and progesterone act in the hypothalamus to coordinate the timing of lordosis and ovulation in female rats in part through regulation of nitric oxide (NO) and cyclic guanosine monophosphate (cyclic GMP) signalling pathways. Soluble guanylyl cyclase is an enzyme that produces cyclic GMP when stimulated by NO and plays a crucial role in the display of lordosis behaviour. We examined the effects of oestradiol and progesterone on the stimulation of cyclic GMP synthesis by NO-dependent and independent activators of soluble guanylyl cyclase in preoptic-hypothalamic and hippocampal slices. Ovariectomised Sprague-Dawley rats were injected with oestradiol (2 microg oestradiol benzoate, s.c.) or vehicle for 2 days. Progesterone (500 microg, s.c.) or vehicle was injected 44 h after the first dose of oestradiol. Rats were killed 48 h after the first oestradiol or vehicle injection, and hypothalamus and hippocampus were obtained. NO-dependent activation of soluble guanylyl cyclase was induced by NO donors, sodium nitroprusside or diethylamine NONOate; NO-independent activation of soluble guanylyl cyclase was induced with 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole and 5'-cyclopropyl-2-[1-2fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyridine-4-ylamine. The NO-dependent activators of soluble guanylyl cyclase produced a concentration-dependent increase in cyclic GMP accumulation and induced significantly greater cyclic GMP accumulation in preoptic-hypothalamic slices from animals treated with oestradiol and progesterone than in slices from rats injected with vehicle, oestradiol or progesterone alone. Hormones did not modify soluble guanylyl cyclase activation by NO-independent stimulators or influence NO content in preoptic-hypothalamic slices. Oestradiol and progesterone did not affect activation of soluble guanylyl cyclase in hippocampal slices by any pharmacological agent, indicating a strong regional selectivity for the hormone effect. Thus, oestradiol and progesterone, administered in vivo, enhance the ability of NO to activate soluble guanylyl cyclase in brain areas modulating female reproductive function without an effect on production of NO itself.

Human papillomavirus type 16 integrations in cervical tumors frequently occur in common fragile sites.

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. HPV integration into the genome of infected cervical cells is temporally associated with the acquisition of the malignant phenotype. A relationship between the sites of HPV integration in cervical cancer and the position of the common fragile sites (CFSs) has been observed at the cytogenetic level. To explore this relationship at the molecular level, we used a PCR-based method to rapidly isolate cellular sequences flanking the sites of HPV16 integrations in primary cervical tumors. Human bacterial artificial chromosome clones were isolated based on these flanking sequences and used as probes for fluorescence in situ hybridization on metaphases derived from cells cultured in the presence of aphidicolin. Our data demonstrate that HPV16 integrations in cervical tumors frequently occur within CFSs at the molecular level. In addition, we have determined the precise molecular locations of the CFSs FRA6C and FRA17B.

Preferential stimulation of rabbit alpha globin mRNA translation by a cap-binding protein complex.

A cap-binding protein complex (Edery et al. (1983) J. Biol. Chem. 258, 11398-11403) is shown here to stimulate preferentially the translation of endogenous alpha versus beta globin mRNA in a rabbit reticulocyte lysate. Several initiation factors (eIF-2, eIF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4E and eIF-5) and elongation factor 1 were found to have no such discriminatory effect. These results are in contrast to several previous reports and demonstrate that the only factor capable of relieving translational competition between alpha and beta globin mRNAs is the cap-binding protein complex.

Large loop excision and cold coagulation for management of cervical intraepithelial neoplasia.

OBJECTIVES: To evaluate the efficacy of large loop excision of the transformation zone (LLETZ) combined with a single application of the cone probe of a Semm Cold Coagulator as a new treatment for women with cervical intraepithelial neoplasia (CIN). METHODS: Retrospective case-record review of 666 women treated with large loop excision and cold coagulation (LLECC) from 1992 to 2000. RESULTS: Of the women who had high-grade CIN at their initial consultation, 4.2% had abnormal cytologic results 6 months after treatment and 0.6% had abnormal cytologic results at 12 months. Of the women who had low-grade CIN at initial presentation, 3.8% had abnormal cytologic results 6 months after treatment and none (0%) at 12 months. Furthermore, there were no reported cases of cervical cancer in this cohort of women during the follow-up period. Short-term bleeding complications (within 24 h of the procedure) occurred in 1% of the women assessed. CONCLUSIONS: Large loop excision combined with cold coagulation is a new and effective treatment for CIN. Randomized controlled trials are required to confirm these findings and determine the long-term safety of the technique.

A clinico epidemiological study on acute viral infection of brain among children admitted in North Bengal Medical College, West Bengal.

A clinico epidemiological prospective study was carried out on acute viral infection of brain among children admitted in a rural based medical college from September '99 to Oct '01. Out of 80 cases, 8 cases (10%) of aseptic meningitis, 35 cases (43.75%) of encephalitis and 37 cases (6.25%) of meningo-encephalitis were found. Overall case fatality was 47.5% and found higher (77%) among normally nourished children in comparison to malnourished children (47.5%). Virological investigation did not isolate any known Flavivirus, Herpes Simplex virus (HSV) and Measles virus; nor any serological evidence against these viruses.

Iodine deficiency disorders among school children of Dakshin Dinajpur District, West Bengal.

Iodine deficiency disorders (IDD) are major public health problems in India, including West Bengal. Existing programme to control IDD needs to be continuously monitored through recommended methods and indicators. Thus we undertook the study to assess the prevalence of goiter, status of urinary iodine excretion (UIE) level and to estimate iodine content of salts at the household level in Dakshin Dinajpur district, West Bengal. We conducted a community-based, cross-sectional study in 2004; among 2250 school children, aged 8-10 years. The '30 cluster' sampling methodology and indicators for assessment of IDD, as recommended by the joint WHO/UNICEF/ICCIDD consultation, were used for the study. Goitre was assessed by standard palpation technique, UIE was analyzed by wet digestion method and salt samples were tested by spot iodine testing kit. Of the 2250 children, 419 (18.6%) had goitre (95% CI = 17.0 - 20.2%). Total goitre rate (TGR) was not significantly different in respect of gender, age and religion. Visible goitre rate was 2.5%. Median urinary iodine excretion level was 16 mcg/dL (normal: > or = 10 mcg/dl.) and 16.5% children had value less than 5 mcg/dL. Only 67.4% of the salt samples tested had adequate iodine content of > or = 15 ppm, with significant difference between Hindus and Muslims (chi2 = 12.68, d.f. = 1, p < 0.01). TGR of 18.6% indicate the district is still endemic for IDD, but median urinary iodine within normal range reflects no current iodine deficiency. The district is in the transition phase from iodine-deficient to iodine-sufficient. Measures are to be sustained for successful transition towards elimination.

Isolation and characterization of osteoblast precursor cells from human bone marrow.

Osteoblasts are derived from precursor cells present in low frequency in the stromal element of bone marrow. Because of the lack of a practical procedure to isolate osteoblast precursors from early cultures of plastic adherent cells from bone marrow, previous studies of marrow stromal cells have been made in confluent cultures of bone marrow when the osteoblast (OB) precursors are already differentiated. Also these studies utilized cultures containing mixed populations of cells including hematopoietic cells. Thus we have employed a negative immunoselection procedure to remove contaminating hematopoietic cells and to isolate nearly homogeneous populations of early human stromal cells derived from the plastic-adherent mononuclear marrow cells cultured in the presence of serum. By reverse transcriptase polymerase chain reaction (RT-PCR) analysis for mRNA, and by immunocytochemical study for protein, we studied the sequential expression in culture of multiple markers of the osteoblast phenotype--alkaline phosphatase, osteopontin, parathyroid hormone receptor, types I and III procollagen, and osteocalcin--as well as lipoprotein lipase (LPL), a marker of the adipocyte phenotype. At an early stage of culture (7-9 days), human OB precursors formed colonies of variable sizes that expressed low levels of mRNA and protein concentrations of OB markers, and their concentration increased on growth to a confluent monolayer (approximately 14 days). LPL mRNA was expressed at high levels in the colony stage, and its level decreased upon confluency, suggesting a loss of potential for commitment to the adipocyte lineage. Interestingly, treatment with dexamethasone at 10(-8) M increased the expression for some of the osteoblast markers and for the LPL gene and was required for the deposition of mineralized matrix and for the formation of adipocytes containing cytoplasmic lipid droplets in confluent cultures. Cloned single early colonies were able to coexpress the osteoblast and adipocyte markers (as assessed by RT-PCR). Thus these immunoselected marrow stromal cells have the characteristics of authentic human osteoblast precursor cells which also are capable of differentiating into adipocytes.

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 activin's mechanism of action in bone formation.

A genomic point mutation in the extracellular domain of the thyrotropin receptor in patients with Graves' ophthalmopathy.

Orbital and pretibial fibroblasts are targets of autoimmune attack in Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD). The fibroblast autoantigen involved in these peripheral manifestations of Graves' disease and the reason for the association of GO and PTD with hyperthyroidism are unknown. RNA encoding the full-length extracellular domain of the TSH receptor has been demonstrated in orbital and dermal fibroblasts from patients with GO and normal subjects, suggesting a possible antigenic link between fibroblasts and thyrocytes. RNA was isolated from cultured orbital, pretibial, and abdominal fibroblasts obtained from patients with severe GO (n = 22) and normal subjects (n = 5). RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction, using primers spanning overlapping regions of the entire extracellular domain of the TSH receptor. Nucleotide sequence analysis showed an A for C substitution in the first position of codon 52 in 2 of the patients, both of whom had GO, PTD, and acropachy. Genomic DNA isolated from the 2 affected patients, and not from an additional 12 normal subjects, revealed the codon 52 mutation by direct sequencing and AciI restriction enzyme digestions. In conclusion, we have demonstrated the presence of a genomic point mutation, leading to a threonine for proline amino acid shift in the predicted peptide, in the extracellular domain of the TSH receptor in two patients with severe GO, PTD, acropachy, and high thyroid-stimulating immunoglobulin levels. RNA encoding this mutant product was demonstrated in the fibroblasts of these patients. We suggest that the TSH receptor may be an important fibroblast autoantigen in GO and PTD, and that this mutant form of the receptor may have unique immunogenic properties.

Differential mRNA fingerprinting by preferential amplification of coding sequences.

The need for rapid identification of differentially expressed genes will persist even after the complete human genomic sequence becomes available. The most popular method for identifying differentially expressed genes acquires expressed sequence tags (ESTs) from the extreme 3' non-coding end of mRNAs. Such ESTs have limitations for downstream applications. We have developed a method, termed preferential amplification of coding sequences (PACS), that was applied to identify differentially expressed coding sequence tags (dCSTs) between osteoblasts and osteosarcoma cells. PACS was achieved by PCR with a set of primers to anchor at sequences complementary to AUG sequences in mRNAs and another set of primers to anchor at a PCR-amplifiable distance from AUG sequences. An initial screen identified 103 candidate dCSTs after screening approximately 15% of the expressed genes between the two cell types. Of these sequences, 27 represent CSTs of known genes and two are from 3'-ESTs of known mRNAs. Thus, PACS identified CSTs approximately 13.5 times more often than it identified 3' ESTs, attesting to the objective of the method. Since many of the dCSTs represent known genes, their identity and potential relevance to osteosarcoma could be immediately hypothesized. Differential expression of many of the dCSTs was further demonstrated by northern blotting or RT-PCR. Since PACS is not dependent on the existence of a poly A tail on an mRNA, it should have application to identify dCSTs for both prokaryotic and eukaryotic organisms. Additionally, PACS should aid in the identification of cell-specific or tissue-specific genes and bidirectional acquisition of cDNA sequence enabling rapid retrieval of full-length cDNA sequence of novel genes.

Identification of twenty-two candidate markers for human osteogenic sarcoma.

Since osteogenic sarcoma (OGS) predominantly affects children, its etiology and progression may be determined more by genetic than environmental factors. A few genes have been associated with OGS, however, their value in the diagnosis and/or prognosis of the disease remains poor. Evidently, more markers need to be identified for improving management of patients with OGS. To identify potential genetic markers for OGS, we have extended preferential amplification of coding sequences (PACS) to screen multiple samples simultaneously. The extended method is termed multi-PACS. Multi-PACS was applied between a normal osteoblast and four OGS-derived cell lines to identify differentially expressed coding sequence tags (dCST) that identified 145 dCSTs. Subsequently, differential mRNA expression was validated for a chosen subset of 22 dCSTs. These chosen dCSTs include among others cyclins D and E, two cyclin dependent kinases, two other kinases, transcription factors E2F4, E2F5, and p130, a DNA repair gene, a gene for the signalosome subunit, and potential guanine nucleotide binding factors. We infer that these genes could be so easily identified because PACS preferentially identifies coding instead of non-coding sequences. We also infer that these genes identify signaling pathways pertinent to OGS. mRNA expression profile of these 22 genes/dCSTs generated distinct expression signature of the OGS-derived cell lines suggesting that further work on clinical samples with these dCSTs will yield valuable information for OGS. We conclude that these 22 genes/dCSTs are candidate markers for OGS.

The "megaprimer" method of site-directed mutagenesis.

We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.

Parameters affecting susceptibility of PCR contamination to UV inactivation.

Contamination of reagents used for PCR is a serious problem. We have recently reported the remarkable effectiveness of UV light in successful decontamination of PCR reagents when the reagents were contaminated with a 6-kb plasmid, followed by amplification of 750-bp segment from the insert. However, further investigation reveals that segment size, sequence and hydration can have a dramatic effect on the efficiency of UV inactivation. Despite some limitations, UV remains a highly effective means of decontamination.

Haplotyping by double PCR amplification of specific alleles.

Haplotypes are useful in population genetics and medicine. Determining haplotypes in the absence of DNA samples from appropriate family members can be difficult and laborious. We have developed a rapid and reproducible method for haplotyping an individual in the absence of relatives. The method utilizes pairs of allele-specific PCR primers to differentially amplify each haplotype. Four amplifications can distinguish the haplotypes produced by two biallelic polymorphisms.

Gene identification by arrested primer extension.

This paper reports a novel method for the identification of nucleic acid target sequences when these targets have high sequence identity. Homologous genes are currently identified by sequencing. We hypothesize that by primer extension in the presence of selected nucleotides, genes with similar sequence can be identified by the length of the extension products on gel electrophoresis. This simple procedure eliminates the much-demanding process of sequencing. We term this process Arrested Primer Extension (APE). As a demonstration of the feasibility of this method, we have used APE to speciate a known set of cultured mycobacteria. There should be many other applications of this method.

Two-step "hot" PCR amplification of GC-rich avian c-myc sequences.

A new two-step cycle PCR method has been developed for amplification of GC-rich DNA sequences. Using this method, termed "hot PCR," 111 and 179 bp regions (GC contents of 74% and 76%, respectively) of the avian c-myc proto-oncogene were specifically amplified from cloned and genomic DNA. This method uses high-melting primers (Tm between 70 degrees and 74 degrees C), a two-step cycle that employs a 94 degrees C denaturation step and an annealing-elongation step between 70 degrees and 80 degrees C with or without formamide.

Absence of chromosome fragility at 19q13.3 in patients with myotonic dystrophy.

Myotonic dystrophy (DM) is an autosomal dominant disorder characterized by a trinucleotide (AGC) amplification at 19q13.3. The degree of trinucleotide amplification may increase in successive generations and generally correlates with severity of the disorder. Because amplification of a trinucleotide repeat is also associated with the observation of a fra(X)(q27.3) in the fragile X syndrome, we investigated whether chromosome fragility at 19q13.3 might be inducible in patients with DM. Using 3 different culture stress systems (medium 199, RPMI 1640 with excess TdR, and RPMI 1640 with FudR) and high resolution chromosome analyses, we studied 6 individuals with DM and 5 unaffected relatives representing two unrelated families. Molecular studies were done on two of the most affected patients and showed AGC repeat sequences of 970 and 1,260 at 19q13.3. The normal range of AGC repeat is 5 to 30. We found no indication of fragility at 19q13.3 in any of these individuals.