Identification of structural fingerprints for ABCG2 inhibition by using Monte Carlo optimization, Bayesian classification, and structural and physicochemical interpretation (SPCI) analysis.

The human breast cancer resistance protein (BCRP), one of the members of the large ATP binding cassette (ABC) transporter superfamily, is crucial for resistance against chemotherapeutic agents. Currently, it has been emerged as one of the best biological targets for the designing of small molecule drugs capable of eliminating multidrug resistance in breast cancer. In order to gain insights into the relationship between the molecular structure of compounds and the ABCG2 inhibition, a multi-QSAR approach using different methods was performed on a dataset of 294 ABCG2 inhibitors with diverse scaffolds. The best models obtained by different chemometric methods have the following statistical characteristics: Monte Carlo Optimization-based QSAR (sensitivity = 0.905, specificity = 0.6255, accuracy = 0.756, and MCC = 0.545), Bayesian classification model (sensitivity = 0.735, specificity = 0.775, and concordance = 0.757); structural and physicochemical interpretation analysis-random forest method (balance accuracy = 0.750, sensitivity = 0.810, and specificity = 0.700). Additionally, structural fingerprints modulating the ABCG2 inhibitory properties were identified from the best models of each method and also validated with each other. The current modelling study is an attempt to get a deep insight into the different important structural fingerprints modulating ABCG2 inhibition.

Evaluation of microtitre plate-based Haemoglobin estimation.

INTRODUCTION: Haemoglobin estimation is one of the most important clinical investigations. Many techniques are available to measure haemoglobin; still there is a need for a haemoglobin assay technique which is cheap, robust and simple and can be used in field conditions very quickly using figure prick sample. We evaluated a cyanmethaemoglobin-based haemoglobin estimation using a microtitre plates for the purpose. METHODS: Microtitre plate-based haemoglobin estimation was developed using cyanmethaemoglobin-based assay and was compared with standard haematology analyser-based haemoglobin estimation in a large number of samples from a population of voluntary blood donors. Various tests were performed to evaluate the stability of colour, variation of the results during duplicate assay on the same days and on different days as well as linearity of the test was performed against broad range of haemoglobin values for the new microtitre plate-based technique. Standard statistical test of significance was applied to validate the assay. RESULTS: Total 200 samples from in-house and field conditions were evaluated. 10 µL blood sample in 300 µL Drabkin's solution provided optimum and comparable results after 10 minutes of incubation. The colour was stable up to 6 hours, the coefficient of variation was less than 3%, and the cost per test including everything was less than 3 cent/2P. Turnaround time for 90 samples was only 30 minutes. CONCLUSION: Cyanmethaemoglobin-based assay in microtitre plate is feasible, robust, rapid, cheap and cost-effective method for haemoglobin estimation in field conditions.

Haemophilia care in India: innovations and integrations by various chapters of Haemophilia Federation of India (HFI).

Care of persons with haemophilia (PWH) in western countries is the responsibility of the government of those countries with or without funding from health insurers. Haemophilia societies in western countries work as pressure groups to ensure better care, and they disseminate information on the disease and some of the societies even support medical research for haemophilia care. In India, Haemophilia Federation of India (HFI) was established in 1982 with few haemophilia families and sympathizers of their cause; subsequently more than 65 chapters involving more than 12 500 PWH came up under HFI. HFI and its constituent chapters are unique in the world in the sense that they are not only trying to involve state and federal government to take responsibility for delivering haemophilia care, but they are also using various innovative and integrative techniques to deliver haemophilia care to PWH themselves, till the time federal and state governments of the country make suitable arrangement for their care. In this study, several of these approaches are discussed with the understanding that 80% of worlds' haemophilia population needs similar help, and the national haemophilia organizations (NMO) of various developing countries will find some of the approaches useful and adaptable to their own circumstances.

Varied distribution of RhD epitopes in the Indian population.

BACKGROUND: Inhabited by more than 4000 caste and tribal groups, India has an extremely heterogenous population. For thousands of years many tribal groups have practised endogamy and are practically genetically isolated. Traditionally, polyclonal anti-D reagent has been used for RhD typing; though monoclonal antibodies are increasingly being used. As a result, blood banks find it difficult to assign the RhD status to an increasing number of people. As monoclonal anti-D typing reagents may not detect all RhD antigen epitopes, we studied the RhD antigen epitope heterogeneity in different population groups in India. METHODS: Red cells of 5315 RhD-positive individuals belonging to different castes and tribes of India were tested with 30 different epitope-specific monoclonal anti-D antibodies. RESULTS: No single monoclonal antibody could detect all RhD-positive red cells detected by polyclonal antisera. The highest proportion of D antigen was detected by LHM 76/55 and BRAD-8 (98%) monoclonal antibodies. CONCLUSION: We need to determine the correct mix of monoclonal antibodies that will detect nearly all RhD antigens detected by polyclonal anti-D sera. Similarly, before accepting monoclonal anti-D for therapeutic use, it would be necessary to determine the appropriate ones for use in the Indian population.

Low cost autologous peripheral blood stem cell transplantation performed in a municipal hospital for a patient with plasma cell leukaemia.

Autologous peripheral blood stem cell transplantation (PBSCT) is a costly procedure. In India, the cost varies from US$20000 to 25000 and most patients cannot afford it. Using several cost-cutting measures, we were able to treat a patient with plasma cell leukaemia by autologous PBSCT. A 42-year-old-male presented with plasma cell leukaemia. He was treated with VAD therapy, followed by high-dose cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) for mobilization of peripheral blood stem cells. The patient was conditioned with high dose melphalan, followed by autologous PBSCT. The procedure was performed in a municipal hospital in which there was no prior experience with stem cell transplantation. Costs were reduced by: (i) using oral medication whenever possible; (ii) having a relative of the patient prepare his food under medical guidance; (iii) starting G-CSF on day 7 rather than on day 1; (iv) short-term storage of the PBSC in an ordinary refrigerator at 4 degrees C without cryopreservation; (v) infusing a large number of CD34+ cells, which shortened the time to engraftment; (vi) delegating many of the functions of a marrow transplant nurse to a resident physician. The cost of transplantation was thereby reduced to about US$ 6000, with successful engraftment by day +13. The patient remained in remission for 7 months, after which he relapsed and was treated with chemotherapy and electron beam radiation to the skin.

Carrier detection and prenatal diagnosis in haemophilia in India: realities and challenges.

Organizing services for haemophilia in developing countries with few resources is a formidable task. There is wide variation in haemophilia care and management between developing and developed countries. The management of a genetic disorder such as haemophilia becomes difficult in developing countries where scanty resources are allocated mainly to nutrition and infectious diseases as a first priority. In a country such as India, with one billion people and with a wide diversity in cultural, educational and financial conditions, educating people about such diseases is difficult and will take a long time to have an effect on attitudes. Meanwhile, attempts are being made at several centres in India to develop carrier detection and prenatal diagnosis for this disease. Counselling a carrier for detection of her carrier status remains problematic in India because marriages are still largely arranged by the parents of prospective brides and bridegrooms. Hence, the very idea of communicating the carrier status to the bridegrooms' families may lead to cancellation of the marriages, and concealing the carrier status is associated with guilt both for the bride's parents as well as the bride. Prenatal diagnosis and carrier detection in India therefore must be discussed in this context.

The implementation of critical pathways in gynecologic oncology in a managed care setting: a cost analysis.

OBJECTIVES: The aim of the study is to determine whether critical pathways can be implemented at an academic institution to limit cost, without compromising patient satisfaction and quality of care. PATIENTS AND METHODS: Patients undergoing a hysterectomy with either cervical or endometrial cancer were placed on specific critical pathways consecutively for an 18-month study period. Preoperative teaching was intensified to educate the patient regarding expectations during the postoperative period. All patients were started on early feeding and patients were also placed on separate care pathways addressing pain and deep vein thrombosis prophylaxis. Total direct costs and patient satisfaction were obtained throughout the study period. During the year prior to care pathway implementation, patient data and direct costs were obtained for the preintervention group utilized for comparison. Postintervention groups were summarized every 6 months during the study period. RESULTS: From January 1997 through June 1998, 63 patients with cervical carcinoma undergoing a radical hysterectomy (DRG 353) and 21 patients with endometrial cancer who underwent a hysterectomy and lymph node sampling (DRG 355) were utilized as the preintervention group. During the 18-month study period (July 1998-December 1999), 42 patients (DRG 353) and 25 patients (DRG 355) were accrued. The average length of stay was reduced from 5.2 (DRG 353) and 4.7 days (DRG 355) prior to implementation of pathways to 3.4 days in both groups. In addition, total direct costs were reduced by 29 (DRG 353) and 32% (DRG 355) after implementation of care pathways. Patient satisfaction data recorded during the study did not demonstrate any change throughout the study period nor were there any higher rates of readmission after implementation of the care pathways. CONCLUSIONS: Critical pathways in gynecologic oncology can be implemented in a managed care environment in order to maintain high quality of care, maintain outcomes, and help reduce costs.

Use of the dual force system to correct chronic knee deformities due to severe haemophilia.

In this study, the use of the dual force system to correct recent or relatively longstanding knee deformities in ten patients is described. (Nine of the patients had severe haemophilia and one had severe von Willebrand's disease.) The mean duration of deformity in these patients was 10 months. The mean range of movement at the affected knee joints increased from 50 degrees at pre-intervention to 110 degrees following 6 weeks of application of the dual force system. In nine of ten patients (90%) the residual flexion deformity ranged from 0 degrees to 10 degrees. The dual force system offers an easily affordable and effective means of correcting a flexion deformity of the knee joint in severely affected haemophilia and allied disorders. More extensive use of this technique in different centres is required to determine its place in the day-to-day management of such patients.

See-and-treat in the management of high-grade squamous intraepithelial lesions of the cervix: a resource utilization analysis.

OBJECTIVE: To use activity-based costing techniques to compare see-and-treat with conventional evaluation and treatment of women presenting with a screening Papanicolaou smear demonstrating high-grade squamous intraepithelial lesion (SIL). METHODS: A total of 4000 theoretical patients were assumed to be evaluated and treated following one of four management algorithms: conventional algorithm I, with colposcopy and directed biopsies, followed by cryotherapy or cold-knife conization; conventional algorithm II, substituting the loop electrosurgical excision procedure for cold-knife conization; conventional algorithm III, substituting the loop electrosurgical excision procedure for cold-knife conization and cryotherapy; or see-and-treat algorithm IV, using the loop electrosurgical excision procedure. Costs associated with patient management in each algorithm were calculated including those for the procedure, patient time, physician time, and disposable expenses, as well as costs to manage complications, treatment failures, and follow-up for 1 year. RESULTS: Algorithm I was the most expensive, costing $899,405 for 1000 patients with high-grade SIL. Substituting the loop electrosurgical excision procedure for cold-knife conization (algorithm II) decreased the cost by 32%, whereas substituting it for cryotherapy also (algorithm III) reduced the cost by only 25%. The most cost-effective management was the see-and-treat single visit of algorithm IV. This strategy cost $531,281, offering a 41% cost reduction compared with algorithm I. CONCLUSION: A see-and-treat approach to the management of women with high-grade SIL, although incorporating more procedures, offers significant cost savings over conventional management algorithms.

Design and models for estimating antagonist potency (pA2, Kd and IC50) following the detection of antagonism observed in the presence of intrinsic activity.

The antagonist activity of the metabotropic glutamate receptor ligand (2S,1'S,2'S)-2-methyl-2(carboxycyclopropyl)glycine (MCCG) was examined using the [35S]GTPgammaS binding and forskolin (FSK)-stimulated adenosine 3':5'-cyclic monophosphate (cAMP) assays with recombinant Chinese hamster ovary (CHO) cells expressing the G protein-coupled human subtype 2 metabotropic glutamate (hmGlu2) receptor. Whereas MCCG proved to be a partial agonist in the GTPgammaS binding assay, it not only antagonized the agonist effect of (IS,3R)-ACPD in the cAMP assay but further produced an anomalous increase of the cAMP level relative to baseline. The anomalous MCCG response was also observed following treatment of the cells with MCCG in the absence of added agonist. Determination of the glutamate concentration in the incubate at the start and end of the cAMP reaction revealed the existence of micromolar concentrations of cellularly released glutamate throughout the course of the assay, reaching levels which exceeded its reported affinity for the mGlu2 receptor. Considering MCCG's partial agonist effect in the GTPgammaS binding assay and its pseudo-inverse agonist effect in the cAMP assay, available methods of estimating its antagonist potency were inappropriate since the classical Schild method and the alternative model suggested by Waud both assume the antagonist to lack a concentration-response relationship. We derived an alternate design and models that permit estimation of the pA2 (pAx), Kd and IC50 for antagonists which produce a concentration related effect when applied by themselves. With their use, the data acquired in both assays support the designation of MCCG as a competitive antagonist of the hmGlu2 receptor and provide similar pA2 estimates between assays. In addition, the newly derived models and design permit the determination of antagonist potency for partial and inverse agonists so characterized in studies employing the Schild design.

A [35S]GTPgammaS binding assessment of metabotropic glutamate receptor standards in Chinese hamster ovary cell lines expressing the human metabotropic receptor subtypes 2 and 4.

The activities of metabotropic glutamate receptor (mGluR) standards were evaluated in the [35S]GTPgammaS binding assay and in the forskolin (FSK)-enhanced cyclic AMP assay using Chinese hamster ovary (CHO) cells or homogenates which expressed the human mGluR (hmGluR) subtypes 2 and 4. Though distinct rank orders of activities were determined for the agonists between the cell lines expressing individual hmGluRs, similar rank orders of agonist activities were determined for the standards between assays. O-phospho-L-serine (L-SOP) and (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) antagonized agonist EC90 responses in the cell lines expressing the hmGluR 2 and 4 subtypes, respectively. In addition to its antagonist effect, L-SOP increased the baseline level of cAMP when tested in the absence of agonist. In spite of this anomalous effect, L-SOP was found to be a competitive antagonist in the cAMP assay as well as in the [35S]GTPgammaS binding assay with a pA2 value of 5.2 in both assays. MAP4 was a competitive antagonist of L(+)-2-amino-4-phosphonobutyric acid (L-AP4)-induced responses in the CHO cell line expressing hmGluR4 with pA2 values of 4.4 and 4.5 determined in the [35S]GTPgammaS binding and cAMP assays, respectively.

Phosphorothioate-phosphodiester oligonucleotide co-polymers: assessment for antisense application.

Efforts have been made to reduce the disadvantages associated with the natural oligonucleotides (all-PO) for antisense application by introducing phosphorothioate (PS) linkages into the molecule. A series of such oligodeoxynucleotide copolymers (17-mers) complementary to the coding region of the rabbit beta-globin mRNA, and containing different proportions and arrangements of PO and PS bonds, were synthesized and tested for their protein-binding properties, nuclease stability in vitro, hybridizing ability with the complementary DNA (cDNA), ability to form RNase H-sensitive substrates and antisense activity in cell-free systems. The melting temperatures (Tm) of the co-polymers were reduced by up to 6 degrees C relative to the all-PO oligo, compared to 11 degrees C for the all-PS compound, indicating intermediate hybridizing abilities of the co-polymers. The protein-binding studies with human serum albumin exhibited a linear correlation with the percentage of PS linkage present in the molecule. Nuclease susceptibilities of the co-polymers were also improved, but the number and position of the PS linkages played a significant role in such improvement. Translation inhibition by these oligonucleotides was only found in wheat germ agglutinin (WGA) extract, but not in rabbit reticulocyte lysate (RRL) cell-free system, suggesting the involvement of RNase H in their antisense activities. Provided they have > or = 50% PS linkages, the co-polymers produced almost the same increased inhibition in the WGA system as that of the all-PS oligo. The translation arrest in WGA extract is in good agreement with the in vitro cleavage found for rabbit globin mRNA in the oligo:mRNA duplex by RNase H alone. It is concluded that a copolymer of PO and PS might be preferable to either all-PO or all-PS for antisense applications.