Our goal was to use the CHANS

Our goal was to use the CHANS BLU9931 cell line approach to identify data, research needs and to set the stage for further assessment (e.g. feedbacks, time lags, surprises, sensu Liu et al., 2007) on how the socioeconomic system and the aquatic ecosystem have interacted and changed through time. Lake St. Clair (LSC), a shallow transboundary system in the Laurentian Great Lakes (Leach, 1991) (Fig. 1), connects Lakes Huron and Erie via the St. Clair River to the north and the Detroit River to the south. It is part of the Huron-Erie corridor. Lake St. Clair may seem small compared to the other Great Lakes, but it is the 11th largest lake

in surface area in the continental USA (Herdendorf, 1982 and Hunter and Simons, 2004). It also has about 1000 km of shoreline perimeter (Fig. 1). The LSC connecting channel contains three Areas of Concern as listed by the Great Lakes Water Quality Agreement, which are located in the St. Clair River, the Detroit River, and the Clinton River with a portion of the western lake shoreline (United States Environmental Protection Agency, access date Erastin 2 April 2012, http://www.epa.gov/glnpo/aoc/). The aggregate area of the local watersheds that drain to LSC (excluding the watershed of Lake Huron and other

upper Great Lakes) is 15,305 km2, with 59% of this area (8988 km2) on the Canadian side, and the remainder (6317 km2) on the USA side (Fig. 1). The USA and Canadian portions of the LSC watershed differ greatly in terms of land use according to recent satellite-derived land cover data. On the USA side in the year 2006, agricultural land use comprised 41% of the watershed and 32% percent was developed (Fry et al., 2011). In Canada as of 2000, land use in the watershed was dominated by agriculture (77%) with 5% cover each in forest and developed land (Agriculture and Agri-Food Canada, access date 8 April 2012, ftp://ftp.agr.gc.ca/pub/outgoing/aesb-eos-gg/LCV_CA_AAFC_30M_2000_V12). It is not likely that land cover change in the short

interval between 2000 and 2006 changed these percentages appreciably. The majority of the watershed is located within five counties on each side of the border (Fig. 1). Besides the St. Clair River, the other rivers that drain into the lake Casein kinase 1 include the Black, Belle and Clinton Rivers in Michigan and the Thames and Sydenham Rivers in Ontario. The largest portion of water entering the lake (98%) comes from the St. Clair River, which supports the largest freshwater delta in the Great Lakes system (Herdendorf, 1993), the St. Clair Flats which contains about 170 km2 of wetlands (Edsall et al., 1988). We used primary literature, state and federal governmental reports and websites as well as state and federal governmental data sources to compile our overview and to conduct new analyses about the characteristics of the lake and its watershed.

Documenting a stream’s sediment yield variability from the dam po

Documenting a stream’s sediment yield variability from the dam pool deposit provides for a better understanding of future down

stream impacts following dam removal. In this paper, we report a study to characterize the sediment that has accumulated in the Gorge Dam impoundment on PARP activity the Middle Cuyahoga River, Ohio. We report on the century-long sediment record of anthropogenic and natural changes occurring in the watershed. Furthermore, we use an impoundment-based estimate of the Middle Cuyahoga River sediment load to assess the output from the Spreadsheet Technique for Estimating Pollutant Loading (STEPL) watershed model. The close agreement between these two methods confirms the usefulness of the watershed modeling approach and best characterizes present-day conditions within the Middle Cuyahoga River. Because the Gorge Dam is under consideration for removal, determining the sediment load record is of practical importance. Once the dam is removed and the impoundment sediment trap no longer exists, the Middle Cuyahoga sediment load will be delivered to the Lower Cuyahoga River. Located in northeast Ohio, the headwaters of the Cuyahoga River flow south before the river turns north and finally discharges into Lake Erie (Fig. 1). Before emptying into Carfilzomib nmr Lake Erie, the Cuyahoga River is impeded by several dams (Fig. 1). Prior to

the construction of the Gorge Dam, the river in this reach

flowed in a gorge over shale, siltstone, and sandstone of the Cuyahoga Group and between steep cliffs of Sharon Formation (Coogan et al., 1974, Evans, 2003 and Wells, 2003). Early settlers to Ohio were drawn to the gorge by the waterpower provided by the Cuyahoga River (Hannibal and Foos, 2003). By 1854, five mill dams were present in the narrower portion of the gorge MEK inhibitor upstream of the present study area (Whitman et al., 2010, p. 20). The recreational value of the river gorge was recognized early, and several amusement parks operated between the 1870s and 1930s, attracting thousands of people daily in the warmer months (Hannibal and Foos, 2003 and Whitman et al., 2010, pp. 59–72; Vradenburg, 2012). By 1933 the amusement parks had all closed due to declining attendance, and the site became the county Gorge Metro Park (Whitman et al., 2010, pp. 59–60; Vradenburg, 2012). Beginning in 1911 and finishing in 1912, the Northern Ohio Power and Light Company constructed the Gorge Dam (Whitman et al., 2010, p. 80). The dam pool provided cooling-water storage for a coal-fired power plant and water for a hydroelectric power generating station. The dam is located at river kilometer 72.6 in present-day Gorge Metro Park, Summit County, Ohio (Fig. 1). The dam was built on Big Falls, the largest waterfall in the gorge. The 17.4-m-tall, reinforced concrete Gorge Dam is the tallest dam on the Cuyahoga River.

Stratigraphic sequences on Tikopia reveal extensive burning (mark

Stratigraphic sequences on Tikopia reveal extensive burning (marked by charcoal in sediments), erosion of the volcanic slopes, and deposition of terrigenous sediments on the coastal plain as the island’s forest was cleared for gardening during the Kiki Phase (950–100 B.C.). During the island’s Sinapupu Phase (∼100 B.C. to A.D. 1200) the use of fire in agriculture gradually declined as the population developed the sophisticated system of arboriculture RO4929097 purchase or “orchard gardening” for which Tikopia is known ethnographically. This arboricultural system mimics the multi-story layering of the tropical rainforest, allowing for extremely high population

densities (∼250 persons/km2). Virtually every hectare of the Tikopia land surface consists of intensively managed orchard gardens, a classic case of the total transformation of an island landscape into an anthropogenic ecosystem.

Mangaia, like other islands within central Eastern Polynesia, was not colonized by Polynesians until ca. A.D. 900–1000. With a land area of 52 km2, the island consists of a 20-million year old central volcanic core surrounded by a ring of upraised coral limestone or makatea. The old, laterized volcanic terrain is nutrient depleted and was highly vulnerable to intensive human land use activities. Archeological investigation of several stratified rockshelters (especially the large MAN-44 site) and sediment coring and palynological analysis of valley-bottom Selleck BMS-754807 swamps and lakes revealed a detailed history of land cAMP use and human impacts on Mangaia ( Steadman and Kirch, 1990, Ellison, 1994, Kirch et al., 1995 and Kirch, 1996). The sediment cores and pollen records reveal rapid deforestation following Polynesian colonization, with an initial spike in microscopic charcoal particles indicative of anthropogenic burning, probably in an effort to cultivate the volcanic slopes

using shifting cultivation. Once the thin organic A horizon had been stripped off of hillslopes through erosion, the lateritic soils were incapable of supporting forest regrowth; the island’s interior became a pyrophytic fernland dominated by Dicranopteris linearis fern and scrub Pandanus tectorius. Agricultural efforts were then directed at the narrow valley bottoms, which were developed into intensive pondfield irrigation systems for taro (Colocasia esculenta) cultivation. The faunal record from the Mangaia rockshelters, especially site MAN-44, exhibits an especially well-documented sequence of significant impacts on the native biota, as well as the introduction of invasive and domestic species (Steadman and Kirch, 1990 and Steadman, 2006). Of 17 species of native land birds present in the early phases of the sequence, 13 became extinct or extirpated.

When this is done the correlation between inflow residuals and te

When this is done the correlation between inflow residuals and temperature (r = −0.02) effectively

disappears. From this analysis we conclude that the direct relationship CT99021 between inflows and temperature is misleading because (a) rainfall and temperature tend to be inversely related and (b) there exist long-term trends in the data sets. Once these have been accounted for, there is no evidence that SWWA temperature has any significant effect on total inflows to Perth dams. Estimates of SWWA annual rainfall from each model were made by averaging the results from grid squares representing the wider SWWA region and generating continuous time series over the period 1901–2100. For a variety of reasons (e.g. different model resolutions, physical parameterizations, and overall skill) model results for regional rainfall tend to differ (both in means and variability) from observations. Fig. 6 shows an example of a time series of raw values from one particular CMIP5 model (MPI-ESM-LR) which is characterized by a consistent underestimate

of both the mean and interannual variance. While it is tempting to discriminate amongst the model results depending on their see more skill at reproducing these fundamental characteristics of rainfall there is little evidence that this has much of an effect on projections (e.g. Smith and Chandler, 2009). Instead, we assume in the first instance that all model results are of equal value but transform them to remove any biases relative to observations. If Y   denotes a model value for rainfall, O denotes an observed value, overbars denote averages over the 20th century (1901–2000) and σ   denotes the associated interannual standard deviation, then the transformation equation(1) Y*=(Y−Y¯)σoσy+O¯provides

a bias correction and makes the projected values from the different models comparable ( Smith et al., 2013). Note that it is not necessary to use observations for the transformation since setting O¯=0 and σo = 1 yields time series with zero mean and unit variance. A potential problem with this type of linear transformation is that it can sometimes lead to small, physically unrealistic, Etoposide concentration negative values for rainfall. However, these situations are rare and replacing any such occurrences with zeroes has negligible impact on the findings presented in this study. While other techniques exist for transforming model time series to obtain a closer match with observed time series (e.g. quantile–quantile matching), this is usually done at the daily time scale (c.f. Bennett et al., 2012 and Kokic et al., 2013) where there can be relatively large discrepancies between model and observed values.

116 There are several strategies to use exosomes as a (therapeuti

116 There are several strategies to use exosomes as a (therapeutic) vaccine. Tumor-derived exosomes carrying tumor antigens and plasmacytoma cell-derived exosomes may be used to induce tumor-specific immunity and thus to prevent tumor development.117 Despite the extensive studies on EVs, until now there are no protocols available for standardized collection, isolation and storage of EVs. Such standardized protocols are important to be able to compare results between laboratories. Despite the fact that blood is probably our most complex body fluid, EVs present in

or isolated from blood or fractions thereof have been most extensively studied so far. Although there are several recommendations Buparlisib nmr regarding the collection of blood with regard to EVs,118 for other body fluids no protocols are available. In most studies EVs have been isolated from body fluids by differential centrifugation.[3] and [47] Differential centrifugation involves multiple sequential centrifugation steps where in each step the centrifugal force is increased to separate smaller and less dense components from the previous step. Another type of separation by means of centrifugation

Akt inhibitor is density-gradient ultracentrifugation, which separates vesicles based on density.[20] and [119] Although different types of vesicles have been distinguished based on density,[3], [20] and [41] differences in density are likely too small to allow full separation of EV species. Differential centrifugation and density-gradient centrifugation protocols

are unlikely to Cyclin-dependent kinase 3 isolate only a single type of vesicle. Immunoaffinity-based assays, usually coated with a specific CD-antibody, are also used.[84] and [120] Theoretically, this method isolates only one subpopulation of vesicles. Unfortunately, in daily practice successful isolation and purification of a single population with an acceptable recovery by this technique are usually very difficult. Ideally, EVs are measured directly in freshly collected samples, but in a clinical setting this is hardly feasible at present. When samples are frozen and thawed before analysis, concentrations and exposure of PS can markedly increase in samples containing PMVs.[35] and [118] As EVs may expose one or more surface antigens of their parent cell, the cellular origin of EVs can be assessed by using antibodies directed against such cell-type specific surface antigens. Flow cytometry (FCM) is still commonly used to estimate the number of EVs. Due to the fact that the refractive index of vesicles is low, only the larger vesicles will be detected as single vesicles and the smaller vesicles will be detected only as a swarm.121 Thus, FCM will underestimate the number and concentration of vesicles. Although many researchers use annexin V to identify or isolate MVs, PS exposure by MVs is still ambiguous because exposure of PS can be due to isolation and handling procedures such as centrifugation and storage.

For example, one might consider 3 forms of gait training in which

For example, one might consider 3 forms of gait training in which a patient is given feedback on every step during a walking task, is given feedback at the end of each short walk, Panobinostat or is shown a videotape of the day’s walking

for discussion. An initial taxonomy might group all of these in a category defined by repeated performance of an activity with feedback. If, however, subsequent research shows that step-by-step feedback has a qualitatively different impact than end-of-walk feedback, this category might require further subdivision. Alternatively, if the mode of feedback appears to have similar effects across a range of therapies focused on skilled performance of a routine task, this might become a nonessential ingredient for a range of treatments, rather than a way of subdividing each of those treatments. (It should be clear that because of the hierarchical structure of a taxonomy, all levels above the moderate level of granularity will,

by definition, be developed.) The practical requirements of an RTT have received little specification to date because the rehabilitation field is, as yet, too far from having a useable RTT to concern itself with ease of use. However, as the RTT is constructed, this issue clearly will loom larger. This feature PS-341 mw of the RTT should have a secondary priority in the early phases of the RTT construction because ease of use of a conceptually inappropriate classification scheme is worth little, and shortcuts that enhance utility can be developed over time. However, the experience

obtained in the PBE projects should be harvested. Analysis of the methods used in those studies will be of benefit in developing an RTT, especially where it concerns the fit between how clinicians select treatments and the design and presentation of the components that are part of the taxonomy.87 The idea of constructing a taxonomy of rehabilitation interventions has been around for quite some time, but other than small efforts focused on a limited area, not much progress has been made, in spite of articulate pleas by some well-respected clinician scholars. The pragmatic nature of rehabilitation, and insufficient attention to the Montelukast Sodium theoretical underpinning of the why and how of treatments, are partly to blame. It would seem that with recent developments in many areas, the time is ripe to achieve broad-based consensus on the framework for an RTT, which should be followed by a cross-disciplinary effort to actually build the RTT. Various issues that need to be taken into account were discussed in this article, and other articles in this supplement offer extensive suggestions for the framework that rehabilitation clinicians, educators, researchers, and administrators might adopt to lay the foundation for what, without doubt, will be a multiyear effort.

When ‘ + 1′ score is assigned to the control tissue sections, ACB

When ‘ + 1′ score is assigned to the control tissue sections, ACB stained piroxicam treated animal tissue sections scored ‘ + 3′. Such observation revealed a pathological change in mucin secretion type on piroxicam treatment. The histopathological finding clearly indicates that piroxicam treatment increases acid mucin secretion in otherwise neutral mucin secreting normal gastric tissue. Reduction in

ACB staining intensity in tissue sections from only Cu LE treated group indicates that mucin secretion pattern did not alter significantly selleck chemicals llc on pre-treatment with the extract. The free neutral mucin content depletes appreciably and the nature of secreted mucin turns acidic on ulcerated stomach. Figure 3D shows that piroxicam also mediates its ulcerative damage through reduction in mucin level by Crizotinib mouse 22.3% (*P≤ 0.001 Vs control). Cu LE pre-treated piroxicam fed animals had no such reduction in mucin content clearly indicating protection from ulcerative damage rendered

by the pre-feeding of the extract. Biomarkers of oxidative stress include lipid peroxidation level, protein carbonyl content, reduced glutathione (GSH), non enzymatic total sulfhydryl group content (TSH), oxidized glutathione (GSSG) content and GSH-GSSG ratio. Table 1 represents the changes in biomarkers of oxidative stress on piroxicam treatment and protection rendered on pre-treatment of rats with Cu LE at a dose of 200 mg/kg body weight. Lipid peroxidation level and protein carbonyl content increased in piroxicam treated rats by 2.16 folds and 5.57 folds respectively, compared to values obtained (P≤0.001Vs

control) in control rats. Levels of TSH and GSH decreased significantly in piroxicam fed rats by 59.17% and 59.63% respectively from control rats (*P ≤ 0.001 Vs control in each case). GSSG content increased by 51.16% and the ratio (GSSG: GSH) increased by 4.3 folds from control in piroxicam treated rats (*P≤0.001 Vs control in each case). Values in the table ID-8 no.1 clearly indicate that no biomarkers altered on feeding rats with only aqueous extract of curry leaves at 200 mg/kg BW dose. Table 1 further indicate that altered biomarkers were restored to control values when rats were pre-treated with aqueous curry leaf extract at 200 mg/kg BW dose before feeding piroxicam at 30 mg/kg BW dose. Table 2 depicts the alterations in activities of different gastric antioxidant enzymes on piroxicam administration. Piroxicam feeding inhibits activities of key gastric antioxidant enzyme called gastric peroxidise and glutathione–S-transferase. Increased activities of gastric glutathione reductase, glutathione peroxidise, superoxide dismutases (Cu-Zn SOD and Mn SOD) and catalase are also observed on piroxicam feeding. Pre-treatment of piroxicam fed rats with Cu LE protected the activity of these antioxidant enzymes from being altered.

EST-SSR markers derived from transcribed regions of DNA were show

EST-SSR markers derived from transcribed regions of DNA were shown to produce high rates of transferability in cotton species [17] and other related plant groups Selleck ATR inhibitor [18] and [19]. In this study, a total of 707 SSR markers developed from G. arboreum (A genome), G. raimondii (D genome), and G. hirsutum (AD genome) were chosen to amplify DNA from G. hirsutum, G. anomalum, and the synthetic. All 707 primer pairs yielded microsatellite products in G. hirsutum and the synthetic, but 14 failed to produce a band in G. anomalum. However the transferability rate from the three species to G. anomalum was very high (98.0%). D-genome-derived SSR markers showed slightly lower rates of transferability than A- and AD-genome species-derived

markers. Although all selected SSR markers expressed high levels of transferability and polymorphism, almost half of the markers (47.24%) were dominant in G. hirsutum. Since G. hirsutum will be used as the recurrent parent in future backcrossing programs, those dominant markers in G. hirsutum cannot be used to monitor the introgression of TGF-beta inhibitor G. anomalum-specific segments in backcross populations. However, the A-genome-derived markers produced more codominant loci (56.38%) than the D-genome-derived

markers (42.59%), indicating that the A-genome-derived markers were more powerful for distinguishing genomic differences between G. hirsutum and G. anomalum. In addition, the A-genome-derived SSR markers have a higher level of transferability between G. hirsutum and G. anomalum than the D-genome-derived SSR markers. These phenomena suggest that SSR markers developed from close relatives of the wild species are more applicable to the analysis of the transfer of chromosome segments from the wild species to cultivated cotton than other types of markers. Although hybrids are expected to have additive banding profiles of the two parents, previous work has demonstrated that allopolyploid speciation in plants may be associated with non-Mendelian genomic changes in the early generations following polyploid synthesis in crops such as wheat [20] and [21] and rapeseed [22]. However, there is no evidence for structural genomic

changes or de novo DNA methylation modifications in newly synthesized allopolyploid cotton [23]. In this study, 9 SSR primer pairs failed to amplify G. anomalum-specific oxyclozanide bands in hexaploid plants. Possible explanations for this include chromosome loss, heterozygosity at some loci in a parental plant, chromosome rearrangement, and sequence discrepancy between the different species. Loss of chromosomes was not the causative factor because the synthetic plants had the expected chromosome number (2n = 78). The presence of heterozygous loci in a parental plant was also unlikely since there was no evidence of variation among the tested parental plants. We also ruled out the possibility of chromosome rearrangement since one SSR marker (NAU2954) that failed to amplify G.

Rotavirus infection in infants and young

children can rap

Rotavirus infection in infants and young

children can rapidly lead to severe diarrhoea and dehydration, electrolyte imbalance and metabolic acidosis. In developing countries, severe gastroenteritis caused by rotavirus is a leading cause of childhood illness and death. Eighty two per cent of the annual rotavirus deaths in children occur in low income countries, most likely due to limited healthcare infrastructure and inadequate domestic sanitation conditions. Rotavirus causes a substantial disease burden; natural infection with one or several serotypes of rotavirus does not afford 100% protection against subsequent infection, although it can mitigate the severity of subsequent attacks. The burden CDK inhibitor of disease is largely associated with children below 5 years of age, with a higher rate of severe cases in children below 2 years of age (mostly in infants). In older children, previous encounters with rotaviruses make individuals less

susceptible to infection AZD9291 in vivo and more likely to develop a milder form of disease. Therefore, a realistic goal for a vaccine candidate would be to provide at least the degree of protection that follows natural infection at an earlier age, ie to prevent the severe and life-threatening complications of rotavirus diarrhoeas in infancy. In August 1998, rhesus rotavirus tetravalent vaccine (RRV-TV) (Rotashield™) was licensed by the FDA and recommended for inclusion in the regular childhood immunisation schedule. The efficacy and safety of this vaccine, which incorporated three reassortant (human/simian) rotaviruses, was tested in seven large efficacy trials in which approximately 7000 infants received the vaccine. DOCK10 The data showed efficacy against rotavirus disease and the only significant safety outcome of note was fever. In the first year following licensure of the vaccine in the USA however,

15 cases of intussusception (a reversible invagination of a section of the small intestine into itself) occurred among infants who had received RRV-TV (13 following the first dose, two within 1 week of any dose). Background estimates of the incidence of intussusception before RRV-TV licensure ranged from 39 to 74 per 100,000 person-years among children ≤12 months of age. A study was performed to identify other cases occurring in vaccinees, using hospital discharge records and other databases, and then intussusception rates were compared between vaccinees and non-vaccinees, correcting for variables such as age and time elapsed since vaccination ( Centers for Disease Control and Prevention, 1999). The rate of intussusception mainly after the first dose among vaccinated children was significantly higher compared with children who were not vaccinated (125 versus 45 per 100,000 person-years). Subsequently, the recommendation for RRV-TV immunisation in infancy was reversed and the vaccine was voluntarily withdrawn from the market by the manufacturer, thereby prompting the need for other candidate vaccines.

We report here improvement in functional Fab expression into the

We report here improvement in functional Fab expression into the E. coli periplasm as a result of its co-expression with FkpA lacking a signal sequence (cytFkpA). The secretion of active Fabs into the periplasm was higher when co-expressed with cytFkpA either on a separate vector under control of an l-arabinose-inducible promoter, or as part of a tricistronic message that includes the chaperone, Fd and light chains on a single plasmid. We also examined the effect of cytFkpA expression on selection of scFv or Fab candidates from large phage libraries and have demonstrated increased expression levels

and diversity of displayed antibodies targeting the selected antigens, resulting in selection of a larger number of functional, sequence-unique antibody fragments screening assay with slower dissociation

constants. XL1-Blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]) and TG1 cells (supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5 (rK-mK–) [F′ traD36 proAB lacIqZΔM15]) were purchased from Agilent (Santa Clara, CA). In order to generate the plasmids responsible for cytoplasmic expression of chaperones, the native signal sequences were excised from the genes encoding the chaperones FkpA (Swiss-Prot accession no. P65764) and Skp (Swiss-Prot Small molecule library accession no. P0AEU7). Chaperones were also allowed to express in the bacterial periplasm with their native signal sequences. To generate the plasmid constructs of the cytoplasmic or periplasmic versions of the chaperones Skp and FkpA, and the bicistronic Skp-FkpA, the chaperone gene fragments were amplified by PCR and then cloned into the plasmid vector pAR3 (ATCC accession no. 87026). The vector pAR3 (Perez-Perez and Gutierrez, 1995) contains the pBAD promoter and the cat gene which confers chloramphenicol antibiotic resistance.

This plasmid harbors the p15A origin of replication which is compatible with the origin ColE1 included in all the vectors co-expressing Fabs or scFvs in our experiments. Two different forward primers and one reverse primer were designed in order to amplify FkpA from XL-1Blue cells by PCR amplification with or without the N-acetylglucosamine-1-phosphate transferase native leader peptide. Similarly, two forward primers and one reverse primer were designed to amplify Skp from XL1Blue cells by PCR with or without its native signal sequence. To generate the chaperone plasmid constructs pAR3-FkpA and pAR3-Skp for periplasmic expression and pAR3-cytFkpA and pAR3-cytSkp for cytoplasmic expression, the products of the previous PCR reactions were used as templates for PCR re-amplification using forward primers to incorporate a BglII restriction site followed by the enhancer sequence GAATTCATTAAAGAGGAGAAATTAACT upstream from the chaperone encoding gene fragment. Reverse primers were used to incorporate the V5 tag sequence (GGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACG) into pAR3-Skp and pAR3-cytSkp and the FLAG tag sequence (GACTACAAGGACGATGACGACAAG) into the pAR3-FkpA and pAR3-cytFkpA, followed by the restriction site HindIII.