The most significant threats to seagrass in the BHS are deforestation and coastal development causing increased turbidity and sedimentation from runoff, as well as reclamation of shallow coastal habitats that smothers seagrass beds. The BHS boasts the highest diversity of corals, reef fishes and stomatopods in the world (Veron et al., 2009, Huffard et al., 2009, Allen and Erdmann, 2009 and Allen and Erdmann, 2012). Surveys have recorded over 577 described species of scleractinian corals (75% of the world’s total), with individual reefs hosting up to 280 species per hectare GS 1101 (Veron et al., 2009 and Wallace et al., 2011). An additional 25–40 undescribed coral species have

also been collected, such that the total scleractinian diversity in the BHS is expected to exceed 600 species once taxonomic work is completed on these

collections (L. DeVantier and E. Turak, personal communication). Within the BHS the highest diversity of corals PF-02341066 clinical trial has been recorded in Raja Ampat, with 553 known species (Veron et al., 2009). Two rapid ecological assessments conducted in 2001 and 2002 in Raja Ampat also recorded 41 of the 90 Alcyonacean (soft coral) genera and 699 mollusc species (McKenna et al., 2002 and Donnelly et al., 2003), while more recent studies have documented 57 reef-associated stomatopod species in the BHS, four of which are considered endemic to the region (Huffard et al., 2009). Corals have been found to 160m depth in Raja Ampat, though those beyond the reaches of SCUBA remain uncharacterized (B. Robison, unpublished data). Similarly, intensive survey work around the BHS over the last decade has recorded 1638 species of coral reef fishes comprising 476 genera and 117 families (Allen and Erdmann, 2009 and Allen and Erdmann, 2012). Within the BHS, the highest diversities have been recorded in Raja Ampat (1437 spp.), the Fakfak-Kaimana coast (1005 spp.) and Cendrawasih Bay (965 spp.). Allen and Erdmann (2009) reported a total of 26 endemic reef fish species (from 14 families) in the BHS, though

more recent surveys have now increased this total to 41 (Dimara et al., 2010 and Allen and Erdmann, 2012). The factors that contribute to local endemism are 5-Fluoracil thought to be in part associated with the geological history of the region. For example, there is evidence that Cendrawasih Bay was isolated for a substantial period over the past 5 million years, resulting in high local endemism (11 endemic reef fishes and 18 endemic reef-building corals currently recognized), and significant genetic divergence of many marine invertebrate populations in the Bay (DeBoer et al., 2008, Crandall et al., 2008, Wallace et al., 2011 and Allen and Erdmann, 2012). The main reef types found in the region are fringing and patch reefs, and to a lesser extent seamounts, atolls and barrier reefs (Fig. 6; McKenna et al., 2002, WWF, 2003 and Donnelly et al.

Most of the published ultrasound studies have used the ESCT criteria and therefore it has to be kept in mind that the actual most widely accepted North American Symptomatic Carotid Endarterectomy Trial (NASCET) classification refers to the distal diameter reduction which leads to lower degrees of stenosis [3], [18] and [32]. In one of the largest patient series on 181 patients and 200 dissections of the ICA, stenoses of the ICA have been found according to the ESCT criteria in PD0325901 datasheet 88% of the patients (stenosis ≤50%

in 8%, stenosis 51–80% in 9%, stenosis >80% or occlusion in 71% of the cases) [17]. Due to the distal location of ICA dissection sometimes only indirect signs are detectable with ultrasound. These indirect signs comprise: (a) increased pulsatility upstream or decreased pulsatility downstream to the suspected lesion. This is detectable in about 77% of cases SCH 900776 order Taken the indirect and direct signs together, pathologic ultrasound findings suggestive for ICA dissection can be detected in 80–96% of all cases [18], [31] and [33]. However, clinical aspects are also very important. In patients with local symptoms only (new onset of so far unknown head and or neck ache (painful) Horner’s syndrome, pulsatile

tinnitus, palsies of the caudal cranial nerves (No IX–XII), or rarely palsies of the Nerves Nos. III, IV, VI), the ultrasound investigation is much less sensitive [3].

The initial duplex sonographical investigation in patients with isolated Nitroxoline Horner’s syndrome can be normal in up to 31% [34]. In summary the ultrasound investigation has a high sensitivity in detecting pathologic findings in patients with ICA dissection. However, it is not the sole investigation to verify the diagnosis of dissection especially in patients with local symptoms only. The ultrasound investigation of the vertebral artery (VA) should include all segments, the origin and pre-vertebral part of the artery (V0/V1 segment), the part between the foramina of the transverse processes (V2 segment), the atlas loop (V3 segment) and the intracranial part (V4 segment). The V1 and V2 segment is normally investigated with a linear probe. The origin of the VA is sometimes not accessible with the linear probe especially in obese patients, and an investigation with a sector probe is superior. This is also the case when the V3 segment with its curved course is investigated. The V4 segment should be investigated via the transnuchal approach with a phased array transducer. In analogy to the ICA dissection, the intramural hematoma of a VA dissection can cause an echolucent wall thickening and sometimes a double lumen. These signs can be found in 10–20% [31] (see Fig. 3).

Inversely, a connection between the reduction of intima–media progression with lipid-lowering therapies and a reduction of cardiovascular risk shown in clinical trials [7] and [8] has lead to considering cIMT a surrogate end point for the effect of anti-atherosclerotic therapy [9]. This is an important fact for risk evaluation since cIMT appears at an early stage of atherosclerosis when alterations in treatment can

substantially change the course of the disease more effectively. The advantage of measuring the cIMT by high resolution B-mode ultrasonography lies in its rapidly applicable and available, non-invasive and cost-effective nature [3]. Progression of cIMT is therefore an attractive method for use in research as it can be easily assessed to study vascular risk or the therapeutic effects of a specific treatment. Nevertheless, evidence considering cIMT as a surrogate marker for CVD is still a GW-572016 supplier matter of debate [2], [10], [11] and [12]. In order to

understand the distinctive nature of cIMT and carotid plaque in the risk of stroke and GDC-0199 clinical trial CVD the process of atherosclerosis has to be clearly understood. About 10–20% of ischemic strokes are due to large artery atherosclerosis, mainly located in the extracranial arteries [13]. Atherosclerotic process leads to luminal stenosis, flow restriction and plaque rupture and is therefore a strong predictor of ischemic stroke [14]. Atherosclerosis is a chronic inflammatory very process, involving endothelial injury, activation and recruitment of immune-inflammatory cells, smooth muscle cell proliferation, and influx of lipoprotein [15]. Various mediators like chemokines, cytokines, growth factors, proteases, adhesion molecules, hemostasis regulators, and their interactions are involved in the process of plaque growth. Proinflammatory signaling

is triggered by oxidized low-density lipoprotein (LDL) or through alterations and remodeling in the extracellular matrix [9] and [16]. This process leads to different plaque composition with variable vascular risk due to different susceptibility for plaque rupture resulting in artery-to-artery embolization. Depending on the stage of the atherosclerotic changes in the vessel wall there is a variety in plaque morphology. It differs from homogeneous thickening of the wall to hyperechogenic components consisting mainly of fibrous tissue and calcification, and hypoechogenic components representing areas with atheromatous material like lipid deposits, cell debris and necrotic material. Hypoechogenic components are considered more harmful due to their instability [17]. Atherosclerosis predominantly develops at specific sites in the vessel, mainly areas with altered blood flow, like bifurcations, branch points and areas of vessel curvature.

, 2007). As these adjacent brain areas have also been implicated in cognitive control tasks (particularly anterior cingulate), it is not possible to entirely disambiguate their possible contribution to the deficits observed in these studies. To our knowledge there has been no report of a patient whose lesion is entirely constrained within the borders of the

pre-SMA. Here we present a young patient with a highly focal, unilateral lesion of the caudal pre-SMA. Since pre-SMA has frequently been associated with cognitive control and executive function, we chose to investigate how this might have affected performance on three standard tasks, each of which indexes a different aspect of response selection or inhibition. The STOP-signal task assesses GPCR Compound Library manufacturer the ability to inhibit an on-going response, whereas the CHANGE-signal task requires the participant to rapidly switch to a different response plan. Finally the Eriksen flanker task measures how quickly an individual is able to select between conflicting response plans that are activated simultaneously. Together these tasks employ similar stimuli with different rules, to explore specific aspects of executive function. Surprisingly we learn more found that she did not display a significant

impairment when asked to stop an action (STOP task), but was significantly impaired when switching between response plans (CHANGE task). The patient also displayed Erlotinib no significant deficit when processing conflict at the level of the stimulus (Eriksen Flanker). Remarkably, it appears that this lesion of the caudal pre-SMA impaired the ability to rapidly switch between overt responses, whilst leaving stopping behaviour intact. We discuss these findings

in the context of the current literature and the implications for understanding the role of pre-SMA in voluntary action. Patient KP is a 28-year-old, right-handed woman who was diagnosed with epilepsy, following the onset of simple partial seizures. Following a subsequent grand mal seizure later in the year, further MRI investigations revealed a very small cavernoma (a blood vessel anomaly, also sometimes referred to as a cavernous haemangioma). This was located on the medial aspect of the right superior frontal gyrus. At the time, KP was experiencing complex partial seizures with secondary generalisations, and the cavernoma was subsequently resected. A follow-up structural scan 4 months after surgery demonstrates the focal nature of the lesion, which lies medial to the superior frontal sulcus and rostral to the paracentral sulcus. The paracentral sulcus has previously been demonstrated to be a useful landmark for the location of the supplementary eye field (SEF) (Grosbras, Lobel, Van de Moortele, LeBihan, & Berthoz, 1999), which lies at the caudal border of the pre-SMA; thus this lesion lies well within the pre-SMA. The sagittal sections in Fig.

7 Mks/input CD34+ cells using a 17 ± 2.5 FI-CD34+ and benefits from using UCB progenitors which are largely available and selleck inhibitor usually discarded after delivery involving a non-invasive

collection procedure. This work quantitatively demonstrates that the FI-CD34+, rather than expansion duration, can be used as a key parameter to maximize Mk cell generation from CD34+-enriched cells. When adapted to fully defined, GMP-compliant culture reagents and conditions, this protocol has the potential to impact cellular therapies within the hemato-oncological field. The authors declare no commercial or financial conflict of interest. This work was financially supported by the Fundação para a Ciência e Tecnologia (FCT), Portugal through Project PTDC/EQU-EQU/114231/2009, MIT-Portugal Program, PhD scholarship SFRH/BD/61450/2009 (J. Hatami) and the research contract IF/00442/2012 (F. Ferreira). The authors thank Isabel Nogueira

(MicroLab IST), Dr. Patrícia Carvalho (NanoLab IST) and Dr. António Pedro Matos (Hospital Curry Cabral, Lisbon, Portugal) for the contribution with electron microscopy analysis. The authors also thank Dr. Ana Paula Sousa (Instituto Português do Sangue, Lisbon, Portugal) for donation of PB-derived platelets. “
“Due to gradual depletion of world’s petroleum reserves and impact of environmental pollution of increasing exhaust emission, there is an urgent need to develop alternative energy resources such as biodiesel fuel. Vegetable Rigosertib supplier oil is a promising alternative because it has several advantages, viz it is renewable, environment friendly and produced easily in rural area, where Fludarabine there is acute need for modern forms of energy. Therefore in recent years several researches have been

studied to use vegetable oil as fuel in engine as biodiesel [27] and [28]. Various vegetable oils, palm oil, soybean oil, sunflower oil, rapeseed oil, canola oil, jatropa oil, pongamia oil have been used to produce biodiesel fuel and lubricants [29] and [30]. Biodiesel is usually produced by transesterification of vegetable oils or animal fats with methanol or ethanol [31]. Biodiesel producers are looking for alternative feed stock which are non-agricultural, high oil content seed and non-food crops. Simarouba species has the ability to substitute the requirement of low cost feed stock with the potential for high oil seed production and added benefit of an ability to grow on marginal land. This property supports the suitability of Simarouba species for the sustainable biodiesel industry [12]. Simarouba yields +3 t biofuel/ha/year at 5 m × 5 m plant spacing with proper nutrition and irrigation. Simarouba belongs to Simaroubaceae family, is indigenous to the Amazon rainforest and other tropical areas in Mexico, Cuba, Haiti, Jamaica, and Central America. It was brought to India from Latin America in 1960s.

, 1989, Cetinić et al., 2006 and Burić et al., 2007). Nevertheless, freshwater phytoplankton species such as Pediastrum spp. were occasionally observed in the samples, probably due to local freshwater input from small rivers and springs, which is greater mainly in the winter and spring. The dominance of the diatom S. marinoi in the spring and winter resulted in microphytoplankton dominance in total carbon biomass above the halocline. Skeletonema blooms were a distinct feature of the Bay, clearly distinguishing its

phytoplankton assemblages from those in adjacent waters. The species is reported to be one of the dominant species in the nutrient-richer areas ( Revelante and Gilmartin, 1976 and Viličić et al., 2009), where it usually exhibits

marked seasonal behaviour, forming blooms PI3K inhibitor above the pycnocline in the late winter ( Totti et al., 2005, Bernardi et al., 2006 and Pugnetti et al., 2008). It is also found in other riverine water-influenced and nutrient-rich environments ( Blanc et al., 1975, Thompson and Ho, 1981, Spies and Parsons, 1985 and Morozova and Orlova, 2005). In the waters surrounding the investigated Bay its presence is detected sporadically, but even then in very low abundances ( Socal et al., 1999 and Rubino et al., 2009). It has recently been discovered that different strains of S. marinoi can tolerate a wide range of salinity ( Saravanan and Godhe, 2010 and Balzano et

al., 2011), which is in accordance with our findings of the species’ check details greatest abundance in surface samples (salinity < 5). Thus, its mass development in the surface waters of Boka Kotorska Bay can be attributed to the competitive advantages of this species over the other marine phytoplankton found in the water column in this period in view of its ability to flourish in conditions of low salinity and lower temperatures. In addition, bloom-forming species like S. marinoi are characterized by inherently high growth rates and can efficiently exploit nutrients, the levels of L-NAME HCl which are higher, especially in the layer above the halocline in the Bay ( Smayda 1998). The influence of the vertical salinity gradient in the phytoplankton distribution is also clearly perceptible in other phytoplankton groups. Cryptophytes and Dinobryon sp. correlated positively with nutrients and negatively with salinity, confirming their preference for the upper, nutrient-rich and less saline layer. The mixotrophic chrysophyte Dinobryon sp. ( McKenrie et al. 1995) and cryptophytes were found in high cell concentrations in the surface layer during spring. Their development was probably favoured by the higher inorganic nutrient concentrations as well by the release of organic matter by diatoms at this stage of the Skeletonema marinoi bloom.

6 and 3.76 h ( Table 2). Modeled flow compared reasonably well to observed flow at nine of the 10 gauges (Table 3 and Fig. 7). These values

improved during time periods when there was a rain gauge inside the watershed. For example, the Wappinger Creek NSE improved to 0.64 from 0.57 for daily flow after 2004, when a NOAA gauge is active inside the watershed. Using these measures, the model appears acceptable in nine of the 10 watersheds, although it selleck screening library fails in the Neshanic River, NJ in all three metrics. Interestingly, event flow analyses showed better performance relative to daily for the small watersheds and no change or worse performance for the larger watersheds. Over the 6-month period of observations in Town Brook watershed, the model predicted 16 occurrences of overland runoff. During 15 of those events, the median water table depth for locations estimated as being “wet” was less than 100 mm from the soil surface, while the median dry wells remained at or below a depth of 100 mm during all events (Fig. 8). This corroborates previous findings that overland runoff in the Northeast is initiated once the water table is within approximately 100 mm of the surface (Lyon et al., 2006 and Dahlke et al., 2012). Over the course of the 16 events, we compared 288 separate predictions

of wet or dry conditions to field measurements. In 18 cases (6%), we predicted a well to be wet when the water table at that location was below 100 mm and in 55 (19%) cases we predicted a well to be dry when the water table depth was within 100 mm of the PD-1/PD-L1 inhibitor 2 soil surface. The remaining 215 (75%) predictions correctly identified a location as wet or dry based on modeled results. On days when no runoff was predicted,

the average depth to the water table of all wells was 240 mm. At the Fall Creek site, four out of the 13 measurement dates were predicted to have DAPT datasheet saturated areas contributing to storm runoff. In three of the four dates, the median volumetric soil moisture reading in the modeled wet locations was above saturation (i.e., ≥53%), while dry locations had median values below saturation (Fig. 9, top). On the date that the “wet” wells were below the saturated value (June 26, 2013), the observed streamflow at the outlet did not show a discernible rise in the hydrograph, highlighting the difficulty in correctly modeling small storm runoff events. The Cascadilla Creek site only had one instance of measurements being taken in locations predicted to be wet, and on this date, the wet sites had a median soil moisture status above saturation (Fig. 9, bottom). The model presented here shows promise as a simple tool allowing for spatial prediction of saturation-excess runoff locations in the northeastern US. Areas that were predicted to generate overland runoff had higher average soil moisture status and an elevated water table compared to areas modeled to be dry within three watersheds.

The wet weight (WW) of R. harrisii inhabiting the Gulf of Gdańsk was sexually dimorphic and differed significantly between the sexes; this has been shown for other crab species ( Fransozo et al. 2003, Czerniejewski & Wawrzyniak 2006, Pinheiro & Hattori 2006). According to Fransozo et al. (2003), this could be due to a difference in energy allocation resulting from reproductive differences (i.e. females cannot attain the larger sizes or heavier weights of males owing to the larger energy requirements of egg

production). In some crab species, this weight difference is due to the males’ positive allometric growth of chelipeds ( Pinheiro Selleckchem Atezolizumab & Hattori 2006). According to Turoboyski (1973), R. harrisii male claw weight accounts for up to 64.0% of the total body weight, whereas female claws contribute only 11.1 to 28.0% to the total body weight. This may also explain why males of R. harrisii were heavier than females of the same carapace width. However, a few individual females were outliers and exhibited either higher or lower wet weights in regard to the power function fitted to the empirical points of this CW: WW relationship. A greater wet weight might be observed prior to the female laying eggs when the gonads

are heavy; a lower wet weight could indicate that the female had already produced an egg mass and the eggs had hatched. Moreover, individual variation Sitaxentan in wet weight could also be influenced by differential stomach fullness. According to Le Cren (1951), the condition factor can selleck chemical provide important information about the ‘well-being’ of a species and can indicate such aspects as recent feeding conditions and the degree of adjustment to the environment. Based on the condition factor, R. harrisii females from Gulf of Gdańsk are in better condition than males even though the males in this population grow faster than females of the same carapace width as a consequence of isometric

weight gain. While most studies show a higher condition factor for males ( Emmanuel 2008, Mohapatra et al. 2010, Patil & Patil 2012), the condition factor is known to be species-specific and can also vary between populations with female gonadal development and time of year ( Branco & Masunari 2000, Pinheiro & Fiscarelli 2009). Some crustacean females increase the weight/volume of the hepatopancreas, the gland responsible for the storage and transport of energy reserves to the ovaries during vitellogenesis ( Hæfner & Spaargaren 1993). Therefore, in some crab populations like swimming crabs (Callinectes danae, Dilocarcinus pagei) or West African blue crabs (Callinectes pallidus), the condition factor for females was higher than males ( Branco & Masunari 2000, Pinheiro & Taddei 2005, Oluwatoyin et al. 2013). R.

Respiratory ventilation within one cycle consisted of one or several successions of single abdominal pumping movements. These successions were counted as single ventilatory events. The durations of these ventilatory events were determined, and related to the whole cycle as well as the cycle phases (open, closed or flutter). As we tested two species of vespine wasps, Vespula vulgaris and V. germanica, we had to analyze our data regarding the possibility of inter-species differences in respiration parameters. ANOVA revealed no influence of the tested wasp species on respiration cycle duration Gefitinib price (P = 0.5449, F-quotient = 0.39,

DF = 1) and CO2 release per cycle (P = 0.9239, F-quotient = 0.01, DF = 1; see Supplementary material, Table S1; data for the two species in Table S2). Therefore, species was not considered for the further analysis. Over the entire temperature range, spiracle control was functioning well for Vespula sp. At the lowest experimental temperatures (Ta = 2.9 °C) yellow jackets showed discontinuous gas exchange resembling an “interburst–burst” pattern similarly to that described by Marais and Chown (2003) for Perisphaeria sp. cockroaches. Interburst phases with LY294002 a minimum of 0.6 and a maximum of 81.73 min duration (mean: 11.86 ± 12.05 min) were followed by 0.42–14.57 min long burst phases (mean: 6.19 ± 4.91 min) consisting

of 1–5 initial higher peaks and several subsequent lower ones (see Fig. 1A). A flutter phase could not be observed at this Ta. Sporadic single CO2 spikes with similar peak height and duration as the initial peaks of the burst phases were counted as separate open phases. They caused the rather high SD in duration of GPX6 closed as well as open phases ( Fig. 3). With increasing Ta DGC appeared in a more common fashion with a closed phase followed by a distinct flutter phase and the main peak or open phase ( Fig. 2A). The open phase oscillations of the CO2 signal merged (but remained detectable), and flutter became visible ( Fig. 1B and C). At temperatures of 15–25 °C Vespula sp. showed typical DGC patterns ( Hetz and Bradley, 2005 and Lighton, 1996)

with closed, flutter and open phase ( Fig. 2B). Exceptional body movements, e.g. when the wasp lost and regained hold with a leg or flipped the wings ( Fig. 1B–D, arrows) were clearly distinguishable from the “normal” respiration pattern. At Ta = 26.2 °C the CO2 level inside the measurement chamber did not always reach zero between two respiration cycles. However, CO2 emission before the open phase resembled a flutter pattern consisting of merging single peaks. In certain individuals, residues of this particular pattern could be observed in some cycles as slight increases in the CO2 signal prior to the main respiratory peak even at Ta = 31.4 and 36.4 °C ( Fig. 2B, D; see large triangles in Fig. 3). At Ta > 31.4 °C all individuals showed cyclic respiration ( Fig. 2C). At the highest experimental temperatures (Ta = 39.7 and 42.

Primary antibodies were obtained Carfilzomib from Santa Cruz (Santa Cruz, CA, US: COX-2), Millipore (activated caspase-3: Temecula, CA, USA), Peprotech (London, UK: IL-1β), Abcam (Cambridge UK: IBA-1), Invitrogen (NY, USA: IRF3), Promega (TUNEL, Southampton, UK). Biotinylated secondary antibodies, normal sera, and avidin–biotin

complex were from Vector Laboratories (Peterborough, UK). Avidin-horseradish peroxidase was obtained from DAKO (Cambridge, UK). Immunohistochemistry for all antigens was carried out by the Avidin–Biotin-Complex (ABC) method with minor modifications, depending on the antibody used, and has been described in detail in previous publications (Cunningham et al., 2005a). Cell counting was performed for IL-1b, TUNEL and IBA-1. For IL-1b and TUNEL, positive cells were identified and counted throughout the hippocampus and thalamus of all animal groups. For IBA-1, a 0.62 × 0.47 mm section of the centre of the dorsal hippocampus, (containing the CA1 pyramidal layer and stratum oriens and radiatum, but not the dentate gyrus granule layer) was photographed and used for microglial counts in NBH and ME7 animals treated with poly I:C. Positively stained cells were identified and counted using the

analyse particles function in Image J software (rsbweb.nih.gov). Animals challenged intraperitoneally with poly I:C (12 mg/kg) or saline were terminally anaesthetised at 4 and 6 h after poly I:C and then transcardially perfused with heparinised saline. Brains were rapidly removed, hippocampi and hypothalami dissected out, placed in eppendorf tubes, snap frozen on liquid nitrogen and stored at −80 °C until further selleckchem use. Total RNA was extracted from

brain samples using Qiagen RNeasy® Plus mini kits (Qiagen, Crawley, UK) according to the manufacturer’s instructions. Contaminating genomic DNA was eliminated via degradation during extraction using the Qiagen of RNase-free DNase1 enzyme. Approximate yields were determined by spectrophotometry at 260 and 280 nm. RNA was stored at −80 °C until cDNA synthesis and PCR assay. All equipment and reagents were supplied by Applied Biosystems (Warrington, UK) unless otherwise stated. Assays for IFN-α, IFN-β, IL-10, IP-10, IRF-7, TLR3, RIG-I, PKR, OAS, Mx1, Bax, Fas, IFNγ, and all T cell transcripts were designed using the published sequences for these genes, applied to Primes Express™ software. Where possible, probes were designed to cross an intron such that they were cDNA specific. All primer pairs were checked for specificity by standard reverse transcription (RT)-PCR followed by gel electrophoresis. Each primer pair produced a discrete band of expected amplicon size. We subsequently learned that C57 mice have a non-functional Mx1 protein due to a deletion in exons 9 though 11 in the Mx1 gene ( Staeheli and Sutcliffe, 1988 and Jin et al., 1998). Our primers for this gene were designed such that they are specific to an unaffected region of the gene and span the boundary of exons 2 and 3.