Origin of Groundwater Arsenic in a Rural Pleistocene Aquifer in Bangladesh Depressurized by Distal Municipal Pumping.

Across South Asia, millions of villagers have reduced their exposure to high-arsenic (As) groundwater by switching to low-As wells. Isotopic tracers and flow modeling are used in this study to understand the groundwater flow system of a semi-confined aquifer of Pleistocene (>10 kyr) age in Bangladesh that is generally low in As but has been perturbed by massive pumping at a distance of about 25 km for the municipal water supply of Dhaka. A 10- to 15-m-thick clay aquitard caps much of the intermediate aquifer (>40- to 90-m depth) in the 3-km2 study area, with some interruptions by younger channel sand deposits indicative of river scouring. Hydraulic heads in the intermediate aquifer below the clay-capped areas are 1-2 m lower than in the high-As shallow aquifer above the clay layer. In contrast, similar heads in the shallow and intermediate aquifer are observed where the clay layer is missing. The head distribution suggests a pattern of downward flow through interruptions in the aquitard and lateral advection from the sandy areas to the confined portion of the aquifer. The interpreted flow system is consistent with 3H-3He ages, stable isotope data, and groundwater flow modeling. Lateral flow could explain an association of elevated As with high methane concentrations within layers of gray sand below certain clay-capped portions of the Pleistocene aquifer. An influx of dissolved organic carbon from the clay layer itself leading to a reduction of initially orange sands has also likely contributed to the rise of As.

Clinical Profile of Low Vision Patients Attending in Low Vision Clinic in a Tertiary Care Hospital.

The purpose of the study was to determine the disease profile of patients attending the low vision clinic of a tertiary eye care hospital at National Institute of Ophthalmology & Hospital (NIO&H), Dhaka, Bangladesh July 2016 to June 2017. Low vision and blindness are major causes of morbidity and an economic burden on the individual, family and the country. Low vision service has emerged as a major challenge faced by the developing countries .so prompt diagnosis; early treatment and early use of low vision devices can improve the quality of life. It was a prospective observational study conducted in low vision clinic at a tertiary care hospital in Bangladesh for one year. A total 419 patient, aged 6-60 years among them 267(63.7%) were male and 152(36.3%) were female. The leading causes of low vision in patient attending the low vision clinic were Retinitis pigmentosa (31.3%) macular dystrophy/stargards diseases & maculopathy (20.3%) and myopia with macular degeneration (14.8%). The percentage of visual impaired (6/18-6/60) were 38.3%, severely visual impaired (<6/60-3/60) were 24.4% & (<3/60) 37.3%. Almost all the patient was prescribed spectacles and Telescope for distant vision, Hand held magnifier and video magnifier were prescribed for near vision. Vision improved with low vision devices (6/18 or better) in 49.5%, (6/18-6/60) in 47.3%, (<6/60-3/60) in 2.8%, (<3/60) in 0.3% patient. Vision with low vision devices were significantly changes (p=0.001). The present study shows that hereditary ocular anomalies (Retinitis pigmentosa, macular dystrophy, myopic degeneration) and amblyopia were more common causes of low vision in this part of world.

An Investigation into a Gear-Based Knee Joint Designed for Lower Limb Prosthesis.

A gear-based knee joint is designed to improve the performance of mechanical-type above-knee prostheses. The gear set with the help of some bracing, and bracket arrangement, is used to enable the prosthesis to follow the residual limb movement. The motion analysis and finite-element analysis (FEA) of knee joint components are carried out to assess the feasibility of the design. The maximum stress of 29.74 MPa and maximum strain of 2.393e-004 are obtained in the gear, whereas the maximum displacement of 7.975 mm occurred in the stopper of the knee arrangement. The factor of safety of 3.5 obtained from the FE analysis indicated no possibility of design failure. The results obtained from the FE analysis are then compared with the real data obtained from the literature for a similar subject. The pattern of motion analysis results has shown a great resemblance with the gait cycle of a healthy biological limb.

Human and livestock waste as a reduced carbon source contributing to the release of arsenic to shallow Bangladesh groundwater.

Recent studies have demonstrated that the supply of relatively young organic carbon stimulates the release of arsenic to groundwater in Bangladesh. This study explores the potential role of human and livestock waste as a significant source of this carbon in a densely populated rural area with limited sanitation. Profiles of aquifer sediment samples were analyzed for phytosterols and coprostanol to assess the relative contributions of plant-derived and human/livestock waste-derived organic carbon at two well-characterized sites in Araihazar. Coprostanol concentrations increased with depth from non-detection (<10m at Site B and <23m at Site F) to maxima of 1.3 and 0.5ng/g in aquifer sands recovered from 17m (Site B) and 26m (Site F), respectively. The commonly used sewage contamination index ([5ß-coprostanol]/([5α-cholestanol]+[5ß-coprostanol])) exceeds 0.7 between 12 and 19m at Site B and between 24 and 26m at Site F, indicating input of human/livestock waste to these depths. Urine/fecal input within the same depth range is supported by groundwater Cl/Br mass ratios >1000 compared to Cl/Br <500 at depths >50m. Installed tube wells in the area's study sites may act as a conduit for DOC and specifically human/livestock waste into the aquifer during flood events. The depth range of maximum input of human/livestock waste indicated by these independent markers coincides with the highest dissolved Fe (10-20mg/L) and As (200-400µg/L) concentrations in groundwater at both sites. The new findings suggest that the oxidation of human/livestock waste coupled to the reductive dissolution of iron-(oxy)-hydroxides and/or arsenate may enhance groundwater contamination with As.

Stimulation of Microbially Mediated Arsenic Release in Bangladesh Aquifers by Young Carbon Indicated by Radiocarbon Analysis of Sedimentary Bacterial Lipids.

The sources of reduced carbon driving the microbially mediated release of arsenic to shallow groundwater in Bangladesh remain poorly understood. Using radiocarbon analysis of phospholipid fatty acids (PLFAs) and potential carbon pools, the abundance and carbon sources of the active, sediment-associated, in situ bacterial communities inhabiting shallow aquifers (<30 m) at two sites in Araihazar, Bangladesh, were investigated. At both sites, sedimentary organic carbon (SOC) Δ(14)C signatures of -631 ± 54‰ (n = 12) were significantly depleted relative to dissolved inorganic carbon (DIC) of +24 ± 30‰ and dissolved organic carbon (DOC) of -230 ± 100‰. Sediment-associated PLFA Δ(14)C signatures (n = 10) at Site F (-167‰ to +20‰) and Site B (-163‰ to +21‰) were highly consistent and indicated utilization of carbon sources younger than the SOC, likely from the DOC pool. Sediment-associated PLFA Δ(14)C signatures were consistent with previously determined Δ(14)C signatures of microbial DNA sampled from groundwater at Site F indicating that the carbon source for these two components of the subsurface microbial community is consistent and is temporally stable over the two years between studies. These results demonstrate that the utilization of relatively young carbon sources by the subsurface microbial community occurs at sites with varying hydrology. Further they indicate that these young carbon sources drive the metabolism of the more abundant sediment-associated microbial communities that are presumably more capable of Fe reduction and associated release of As. This implies that an introduction of younger carbon to as of yet unaffected sediments (such as those comprising the deeper Pleistocene aquifer) could stimulate microbial communities and result in arsenic release.

Inequitable allocation of deep community wells for reducing arsenic exposure in Bangladesh.

Community wells that extend deeper than most private wells are crucial for reducing exposure to groundwater arsenic (As) in rural Bangladesh. This study evaluates the impact on access to safe drinking water of 915 such intermediate (90-150 m) and deep (>150 m) wells across a 180 km2 area where a total of 48,790 tubewells were tested with field kits in 2012-13. Half the shallow private wells meet the Bangladesh standard of 50 µg/L for As in drinking water, whereas 92% of the intermediate and deep wells meet the more restrictive World Health Organization guideline for As in drinking water of 10 µg/L. As a proxy for water access, distance calculations show that 29% of shallow wells with >50 µg/L As are located within walking distance (100 m) of at least one of the 915 intermediate or deep wells. Similar calculations for a hypothetical more even distribution of deep wells show that 74% of shallow wells with >50 µg/L As could have been located within 100 m of the same number deep wells. These observations and well-usage data suggest that community wells in Araihazar, and probably elsewhere in Bangladesh, were not optimally allocated by the government because of elite capture.

Evidence for Elevated Levels of Arsenic in Public Wells of Bangladesh Due To Improper Installation.

One of the mainstays of mitigation to reduce the exposure of the rural population of Bangladesh to arsenic (As) from private, mostly <90-m deep wells over the past 15 years has been the installation of over 300,000 deeper community wells. A comprehensive testing campaign previously conducted across a 180 km2 of area of Bangladesh identified 9 out of total of 927 wells >90 m deep that contained >50 µg/L arsenic. We show here that for five of these nine wells, conductivity profiles obtained after spiking the well bore with salt indicate a shallow leak that could explain the high As in the well water. In two of the five leaky wells, the presence of additional screens at the depth of the leak was documented with a downhole camera. The downhole camera did not detect anomalies in the construction of the remaining three leaky wells or in the four wells that did not leak. The four wells that did not leak were all >150-m deep and located in two villages separated by less than 500 m. Excluding these two villages and a handful of leaky wells, the results indicate an aquifer that is consistently low in As over a sizeable area at depths >90 m. Isolated cases of public wells that are elevated in As that have been reported elsewhere in Bangladesh may therefore reflect improper installation rather than actual contamination of the deep aquifer.

VULNERABILITY OF LOW-ARSENIC AQUIFERS TO MUNICIPAL PUMPING IN BANGLADESH.

Sandy aquifers deposited >12,000 years ago, some as shallow as 30 m, have provided a reliable supply of low-arsenic (As) drinking water in rural Bangladesh. This study concerns the potential risk of contaminating these aquifers in areas surrounding the city of Dhaka where hydraulic heads in aquifers >150 m deep have dropped by 70 m in a few decades due to municipal pumping. Water levels measured continuously from 2012 to 2014 in 12 deep (>150m), 3 intermediate (90-150 m) and 6 shallow (<90 m) community wells, 1 shallow private well, and 1 river piezometer show that the resulting drawdown cone extends 15-35 km east of Dhaka. Water levels in 4 low-As community wells within the 62-147 m depth range closest to Dhaka were inaccessible by suction for up to a third of the year. Lateral hydraulic gradients in the deep aquifer system ranged from 1.7×10-4 to 3.7×10-4 indicating flow towards Dhaka throughout 2012-2014. Vertical recharge on the edge of the drawdown cone was estimated at 0.21±0.06 m/yr. The data suggest that continued municipal pumping in Dhaka could eventually contaminate some relatively shallow community wells.

Recharge of low-arsenic aquifers tapped by community wells in Araihazar, Bangladesh, inferred from environmental isotopes.

More than 100,000 community wells have been installed in the 150-300 m depth range throughout Bangladesh over the past decade to provide low-arsenic drinking water (<10 µg/L As), but little is known about how aquifers tapped by these wells are recharged. Within a 25 km2 area of Bangladesh east of Dhaka, groundwater from 65 low-As wells in the 35-240 m depth range was sampled for tritium (3H), oxygen and hydrogen isotopes of water (18O/16O and 2H/1H), carbon isotope ratios in dissolved inorganic carbon (DIC, 14C/12C and 13C/12C), noble gases, and a suite of dissolved constituents, including major cations, anions, and trace elements. At shallow depths (<90 m), 24 out of 42 wells contain detectable 3H of up to 6 TU, indicating the presence of groundwater recharged within 60 years. Radiocarbon (14C) ages in DIC range from modern to 10 kyr. In the 90-240 m depth range, however, only 5 wells shallower than 150 m contain detectable 3H (<0.3 TU) and 14C ages of DIC cluster around 10 kyr. The radiogenic helium (4He) content in groundwater increases linearly across the entire range of 14C ages at a rate of 2.5×10-12 ccSTP 4He g-1 yr-1. Within the samples from depths >90 m, systematic relationships between 18O/16O, 2H/1H, 13C/12C and 14C/12C, and variations in noble gas temperatures, suggest that changes in monsoon intensity and vegetation cover occurred at the onset of the Holocene, when the sampled water was recharged. Thus, the deeper low-As aquifers remain relatively isolated from the shallow, high-As aquifer.

Comparative study of early and conventional catheter removal following buccal mucosal graft urethroplasty.

The duration of catheterization period following urethroplasty is a great controversy. For buccal mucosal graft urethroplasty recommended catheterization period ranges from 2-4 weeks. In recent years, early catheter removal has been advocated for several reasons. Early catheter removal improves both patient comfort, mobility and reduces catheter related complications and shortens post operative hospital stay. This prospective observational study was done from January 2007 and December 2008, 50 patients underwent dorsal onlay buccal mucosal graft urethroplasty for stricture urethra. Among them 25 cases were selected for early catheter removal on 7th postoperative day and 25 cases for conventional catheter removal at 3 weeks. Three patients (12%) of early catheter removal group and 2 patients (8%) of conventional catheter removal group exhibited periurethral leakage. One patient (4%) and three patients (12%) developed wound infection in early and conventional catheter removal group respectively. No significant difference was observed in urinary flow (p=0.089). In these two groups the mean postoperative hospital stay was significant (p<0.001). Early catheter removal on 7th postoperative day is safe and has better impact on patient comfort with minimum catheter related complications and shortens hospital stay.

Development of a control system for artificially rehabilitated limbs: a review.

Development of an advanced control system for prostheses (artificial limbs) is necessary to provide functionality, effectiveness, and preferably the feeling of a sound living limb. The development of the control system has introduced varieties of control strategies depending on the application. This paper reviews some control systems used for prosthetics, orthotics, and exoskeletons. The advantages and limitations of different control systems for particular applications have been discussed and presented in a comparative manner to help in deciding the appropriate method for pertinent application.

The outcome of a cohort of tuberculin-positive predominantly South Asian new entrants aged 16-34 to the UK: Blackburn 1989-2001.

SETTING: Blackburn, Hyndburn and Ribble Valley Local Government areas of England and Wales, the former a high tuberculosis (TB) prevalence district. BACKGROUND: The incidence of tuberculosis in new entrants aged 16-34 with positive tuberculin skin tests but normal chest X-rays after initial entry is not definitely known, and was previously estimated from cross-sectional national surveys and derived data for the 2006 and 2011 NICE economic appraisals of new entrant TB screening. METHODS: New entrants aged 16-34 years predominantly from South Asia (India, Pakistan and Bangladesh), with tuberculin tests inappropriately positive for their BCG history were identified for the years 1989-2001 inclusive from a new entrant database. These entrants were compared with the current GP registration database to see if local residence could be confirmed and the local TB notification database to October 2008. Survival analysis was carried out using Kaplan-Meier survival curves and a Cox Regression model. RESULTS: Four hundred and seventy-nine such new entrants with normal initial chest X-rays were identified. Of these 402 (84%) registered with a General Practitioner in East Lancashire for a period of time and could be followed up by this study. The crude incidence density of active TB amongst these individuals with latent disease was 1297 per 100 000 person-years (95% CI; 991-1698 per 100 000 person-years). After 10 and 15 years of follow-up 13.5 and 16.3% of individuals, respectively, had progressed on to active disease. CONCLUSION: This patient-derived, rather than estimated, data shows a minimum risk of TB disease of 16.3% at 15 years. The 2006 NICE economic appraisal, suggested that treatment for latent TB infection (LTBI) was cost-effective when the incidence of clinical TB over 15 years surpassed 18% in these populations. The 2011 NICE economic appraisal reduced this to 12% active TB over 15 years, and showed that at 16% active TB over 15 years a single interferon gamma release assay was the most cost-effective strategy. Further cohort studies are urgently needed to confirm or revise the assumptions behind the 2011 NICE economic appraisal.

Arsenic migration to deep groundwater in Bangladesh influenced by adsorption and water demand.

Drinking shallow groundwater with naturally elevated concentrations of arsenic is causing widespread disease in many parts of South and Southeast Asia. In the Bengal Basin, growing reliance on deep (>150 m) groundwater has lowered exposure. In the most affected districts of Bangladesh, shallow groundwater concentrations average 100 to 370 µg L(-1), while deep groundwater is typically < 10 µg L(-1). Groundwater flow simulations have suggested that, even when deep pumping is restricted to domestic use, deep groundwater in some areas of the Bengal Basin is at risk of contamination. However, these simulations have neglected the impedance of As migration by adsorption to aquifer sediments. Here we quantify for the first time As sorption on deeper sediments in situ by replicating the intrusion of shallow groundwater through injection of 1,000 L of deep groundwater modified with 200 µg L(-1) of As into a deeper aquifer. Arsenic concentrations in the injected water were reduced by 70% due to adsorption within a single day. Basin-scale modelling indicates that while As adsorption extends the sustainable use of deep groundwater, some areas remain vulnerable; these areas can be prioritized for management and monitoring.

Matrix metalloproteinase-13 influences ERK signalling in articular rabbit chondrocytes.

OBJECTIVE: Matrix metalloproteinase-13 (MMP-13) is an extracellular MMP that cleaves type II collagen, the major protein component of cartilage, with high specificity and has been implicated in the pathology of osteoarthritis. The present study aimed to characterize the binding and internalization kinetics of MMP-13 in normal rabbit chondrocytes and whether MMP-13 affected cell signalling. METHODS: Rabbit chondrocytes were used in [125I]-MMP-13 binding assays to investigate the MMP-13 binding kinetics and Western analysis allowed for the assessment of intracellular signalling cascades. RESULTS: Rabbit chondrocytes were found to express the cartilage-specific genes aggrecan and type II collagen throughout their in vitro culture period. Appreciable specific cell-association of [125I]-MMP-13 was detected after 10 min of exposure to the ligand and equilibrium was obtained after 2 h. Binding of [125I]-MMP-13 to chondrocytes was specific and approached saturation at 75 nM. Internalization of MMP-13 was evident after 20 min, reached a maximum at 30 min and had returned to baseline by 90 min. Addition of receptor-associated protein (RAP) inhibited the internalization of MMP-13 indicating a likely role for low-density lipoprotein receptor-related protein-1 (LRP1) in this process. Interestingly the presence of MMP-13 induced phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2) protein showing that there is initiation of a signalling process in response to MMP-13 being bound and internalized by rabbit chondrocytes. However, this activation does not involve the MMP-13 internalization receptor LRP1. CONCLUSION: These studies demonstrate and characterize the MMP-13 binding and internalization system in rabbit chondrocytes and indicate that MMP-13 may regulate the phenotype of the chondrocytes through this receptor system.

Mortality in severely malnourished children with diarrhoea and use of a standardised management protocol.

BACKGROUND: Severely malnourished children have high mortality rates. Death commonly occurs during the first 48 h after hospital admission, and has been attributed to faulty case-management. We developed a standardised protocol for acute-phase treatment of children with severe malnutrition and diarrhoea, with the aim of reducing mortality. METHODS: We compared severely malnourished children with diarrhoea aged 0-5 years managed by non-protocol conventional treatment, and those treated by our standardised protocol that included slow rehydration with an emphasis on oral rehydration. The standardised-protocol group included children admitted to the ICDDR,B Hospital, Dhaka between Jan 1, 1997, and June 30, 1997, while those admitted between Jan 1, 1996, and June 30, 1996, before the protocol was implemented, were the non-protocol group. FINDINGS: Characteristics on admission of children on standardised protocol (n=334) and non-protocol children (n=293) were similar except that more children on standardised protocol had oedema, acidosis, and Vibrio cholerae isolated from stools. 199 (59.9%) of children on standardised protocol were successfully rehydrated with oral rehydration solution, compared with 85 (29%) in the non-protocol group (p<0.0001). Use of expensive antibiotics was less frequent in children on standardised protocol than in the other group (p<0.0001). Children on standardised protocol had fewer episodes of hypoglycaemia than non-protocol children (15 vs 30, p=0.005). 49 (17%) of children on non-protocol treatment died, compared with 30 (9%) children on standardised protocol (odds ratio for mortality, 0.49, 95% CI 0.3-0.8, p=0.003). INTERPRETATION: Compared with non-protocol management, our standardised protocol resulted in fewer episodes of hypoglycaemia, less need for intravenous fluids, and a 47% reduction in mortality. This standardised protocol should be considered in all children with diarrhoea and severe malnutrition.

PIP: Severely malnourished children have high mortality rates, with death commonly occurring during the first 48 hours after hospital admission. Such mortality has been attributed to faulty case management. The authors therefore developed a standardized protocol for the acute-phase treatment of children with severe malnutrition and diarrhea, with the aim of reducing mortality. This paper compares severely malnourished children with diarrhea aged 0-5 years managed by nonprotocol conventional treatment to those treated by the authors' standardized protocol, which included slow rehydration with an emphasis upon oral rehydration. The characteristics on admission of the 334 children on the standardized protocol and the 293 nonprotocol children were similar, except that more children on the standardized protocol had edema, acidosis, and Vibrio cholerae isolated from stools. 59.9% of the children on the standardized protocol were successfully rehydrated with oral rehydration solution, compared with 29% of those in the nonprotocol group. The use of expensive antibiotics was less frequent in children on the standardized protocol than in the other group, and children on the standardized protocol had fewer episodes of hypoglycemia than nonprotocol children. 17% of nonprotocol and 9% of standardized protocol children died. These findings suggest that the standardized protocol should be considered for all children with diarrhea and severe malnutrition.

Emesis induced by inhibitors of type IV cyclic nucleotide phosphodiesterase (PDE IV) in the ferret.

Emesis induced by inhibitors of type IV cyclic nucleotide phosphodiesterase (PDE IV) has been investigated in the ferret. The PDE IV inhibitors studied were: RS14203, R-rolipram and CT-2450 (i.e. (R)-N-[4-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-(4-pyridyl)ethyl]phenyl ]N'-ethylurea), in addition to the less active enantiomers S-rolipram and CT-3405. Following oral administrations, different emetic profiles were observed with time. Emesis induced by RS14203 exhibited a dose-response relationship but no such relationship was seen for R-rolipram or CT-2450. The incidence of emesis was positively influenced by the dose of PDE IV inhibitors administered, allowing a rank order of potency: RS14203 > R-rolipram > S-rolipram > CT-2450 > CT-3405. PDE IV inhibitor-induced emesis was abolished by the tachykinin NK1 receptor antagonist, CP-99,994. No peripheral release of substance P by PDE IV inhibitors seems to be involved in triggering the emetic reflex since L-743,310, which only has peripheral NK1 receptor antagonist activity, was without effect. The implication of 5-HT3 receptors in PDE IV inhibitor-induced emesis was variable. Our results suggest that the PDE IV inhibitors studied are mixed peripheral-central emetogens. PDE IV inhibition itself could be plausible mechanism of action of these agents. However, whether emesis is mediated via a specific isoform of PDE IV remains to be established.

Translational effects of peptide antagonists of Tat protein of human immunodeficiency virus type 1.

The Tat (trans-activator of transcription) regulatory protein of human immunodeficiency virus (HIV-1) acts by interacting with the TAR RNA domain of nascent viral transcripts and with cellular proteins to increase viral transcription. In Jurkat-derived HCLE-D36 cells, which are stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene expressed from the TAR-encoding long terminal repeat (LTR) of HIV-1, CAT protein expression is dependent on Tat. The Tat9-K-biotin peptide antagonist of Tat binds specifically to TAR RNA and competes with Tat for binding. In the cellular expression system, Tat9-K-biotin reduces Tat-dependent CAT expression. However, while the Tat antagonist greatly reduces CAT protein production and polysome association of CAT mRNA, it has little effect on CAT mRNA levels, suggesting that the antagonist works at the post-transcriptional level.

A polyethylene glycol copolymer for carrying and releasing multiple copies of cysteine-containing peptides.

Two different methods were developed to prepare an adduct of a poly(ethylene glycol)-lysine copolymer with either cysteamine or 1-amino-2-methyl-2-propanethiol. Cysteine-containing peptides could then be disulfide-linked to the thiol groups on the polymer in a facile manner. In the described procedures, a coupling ratio of about 8 peptides/molecule of poly(ethylene glycol)-lysine copolymer (Mw = 27 000) was typically attained. The products were stable at neutral pH, but the peptides could be released from the polymer in a physiologically relevant reducing environment. The release rate was highly dependent on the linker used for forming the disulfide bond. To illustrate the potential biomedical usefulness of this polymer carrier, a Tat peptide-PEG conjugate was shown to inhibit expression of a reporter gene fused to the TAR element of human immunodeficiency virus in a model cell assay.

Inhibition of HIV-1 replication by a Tat RNA-binding domain peptide analog.

The peptidic compound, N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys(biotin)-NH2 (Tat10-biotin), contains the 9-amino acid sequence from the basic domain of the Tat protein responsible for specific interaction with TAR RNA. The cysteine residue provides an attachment site for biotin, which acts as a cellular uptake enhancer. Tat10-biotin binds a fragment of TAR RNA (deltaTAR) avidly and specifically, as measured in an electrophoretic gel shift assay. Tat10-biotin inhibited tat gene-induced expression of a stably transfected chloramphenicol acetyl transferase (CAT) reporter gene linked to the HIV-1 long terminal repeat (LTR) in a model cell assay, but did not inhibit phorbol ester-induced expression of CAT, thereby demonstrating a Tat-dependent mechanism of inhibition. Inhibition of HIV-1 replication after acute infection of MT2 cells was demonstrated by absence of HIV-induced syncytium formation and cytotoxicity, as well as by suppression of reverse transcriptase production. These results suggest that a peptide or peptide mimetic capable of competing with the TAR RNA-binding domain of Tat protein might be useful as a therapeutic agent for AIDS.

Use of a polyethylene glycol-peptide conjugate in a competition gel shift assay for screening potential antagonists of HIV-1 Tat protein binding to TAR RNA.

Interference of binding of Tat protein to TAR RNA in HIV-1-infected cells may be a useful therapeutic strategy for AIDS. An electrophoretic assay to screen potential low-molecular-weight (< 2 kDa) Tat antagonists has been established. A radiolabeled TAR RNA fragment (delta TAR) is retarded in mobility when bound by a Tat peptide-polyethylene glycol conjugate (Tat-PEG), which is used in place of the Tat protein. The assay determines the ability of a potential antagonist to compete with Tat-PEG for binding to delta TAR, as measured by interference with the gel shift of delta TAR. To discriminate between specific and nonspecific interactions, the assay is done in the absence or the presence of a 250-fold molar excess of tRNA.