L. Tartar Trust to BJP, and the University of Puget definitely Sound Richard Bangs Collier scholarship to SMC. Declaration of Interests None declared. Supplementary Material Supplementary Data: Click here to view. Acknowledgments Special thanks to Jacob Raber, Ph.D., as well as Reid Olsen, Julie Mitchell, M.S., and Ruth Rowland for their technical support; Jean O��Malley, M.P.H., for statistical advice; Joel T. Nigg, Ph.D., for reading an earlier version of this manuscript; and the mothers that took part in this study.
The Family Smoking Prevention and Tobacco Control Act of 2009 (the ��Act��) represents an important landmark for tobacco control in the United States. The Act granted the U.S. Food and Drug Administration (FDA) authority to regulate tobacco products, including packaging and labeling regulations (Deyton, Sharfstein, & Hamburg, 2010).

Internationally, packaging and labeling regulations have emerged as an important component of tobacco control policy, both as a vehicle for governments to warn about the risks of tobacco use and to restrict misleading descriptors and design. Article 11 of the World Health Organization (WHO) Framework Convention on Tobacco Control (FCTC)��the world��s first public health treaty��has served as an impetus for the rapid uptake of packaging and labeling regulations among many of the 170 countries that have ratified the Framework (WHO, 2003). Article 11 includes recommendations for large pictorial health warnings and encourages more effective forms of disclosure for product constituents and emissions.

Article 11 also recognizes the importance of the package as a promotional vehicle for tobacco companies and requires the removal of potentially misleading packaging information, including the terms ��light�� and ��mild.�� Article 11 advises that Parties to consider broader restrictions on other descriptors and promotional elements of pack design (WHO, 2008). Packaging regulations have changed very little in the United States since 1984, when packages began displaying one of four mandatory text warnings printed on the side of packages (see Figure 1). However, the Act gives the FDA authority to regulate three primary areas of packaging and labeling: health warnings, the disclosure of product constituents or chemical Carfilzomib ��yields,�� and prohibitions on potentially misleading packaging information with respect to reduced health risk. The current paper will examine regulatory provisions in each of these areas, review evidence on best practices, and identify research priorities to help guide regulatory practice. Figure 1. U.S. health warnings implemented in 1984. The United States is a critical market for global tobacco control.

Furthermore, the presence of microalbuminuria is generally associated with a poorer glycometabolic control and a higher prevalence of chronic complications including diabetic retinopathy, peripheral vascular disease, sellectchem and diabetic neuropathy [7]. The association between microalbuminuria and Mg depletion is a controversial issue. A previous report showed that high doses of Mg reduce microalbuminuria in traumatic critically ill patients at 36 hour, after infusion [8]. Conversely, there were no significant differences between patients with hypomagnesemia and normal subjects with respect to microalbuminuria [9]. Therefore, the aim of the present study was to evaluate the association between serum Mg and microalbuminuria in diabetic patients in China. 2. Materials and Methods 2.1.

Research Design and Subjects This community-based cross-sectional study was conducted in Jiading district, Shanghai, China, from March to August, 2010. In brief, 10375 subjects, aged 40 years or above, were enrolled to participate in the survey. Among those subjects, there were 1872 diabetic patients, those with fasting plasma glucose (FPG) �� 7.0mmol/L and/or 2h plasma glucose (2h-PG) �� 11.1mmol/L or with a history of diabetes. The diagnosis of diabetes was defined according to the 1999 World Health Organization criteria [10]. Microalbuminuria was defined as 30mg/g �� urinary albumin-creatinine ratio (UACR) < 300mg/g [11]. For analysis, we excluded subjects who had missing data on serum Mg or urine albumin or urine creatinine (n = 5), those who had urinary tract infection, glomerulonephritis, nephritic syndrome, or kidney cancer (n = 17), and those who had UACR �� 300mg/g (n = 21).

Finally, a total of 1829 diabetic subjects (775 males and 1054 females) were included in the analysis. This study was conducted with the approval of the institutional review board of Ruijin Hospital affiliated to Shanghai Jiao-Tong University School of Medicine. All participants provided informed consent. 2.2. Clinical Data Collection and Biochemical Measurements The information about demographic characteristics, lifestyle, the history of chronic diseases, and current use of medication, including antihypertensive drugs and antidiabetic drugs, were obtained by a standard interview questionnaire.

Current smokers or drinkers were defined as subjects who smoked cigarettes or consumed alcohol regularly in the past 6 months, while subjects who never or formerly smoked cigarettes or consumed alcohol were defined as noncurrent smokers or noncurrent drinkers. Blood pressure was measured at the nondominant arm three times consecutively at 1min intervals after subjects had rested AV-951 for at least 5min in a sitting position, using an automated electronic device (OMRON Model HEM-752; Omron, Dalian, China). The average of the three measurements was used in the analysis.

, 2000). On the other figure 1 hand, nornicotine was shown to contain 70%�C96% (S)-nornicotine (Armstrong, Wang, Lee, & Liu, 1999; Carmella et al., 2000), and it has been previously noted that the enantiomeric composition of NNN in tobacco indicates that nornicotine, and not nicotine, is the major precursor of NNN in tobacco (Carmella et al., 2000). Our results further reinforce that hypothesis. Thus, removal of nornicotine from tobacco could be a potential strategy to reduce the levels of NNN in tobacco products (Gavilano et al., 2006). Even though the variation in percent (S)-NNN among brands and product types was not large, we found statistically significant differences among some product categories. This could be due to the differences in tobacco types and processing methods used.

For instance, the % contribution of (S)-NNN to NNN in novel tobacco products was found to be higher than that in conventional moist snuff (Table 1). Previous research showed that, compared with conventional U.S. moist snuff, Marlboro Snus and Camel Snus products are generally low in tobacco-specific nitrosamine content (Stepanov et al., 2008; Stepanov, Biener, et al., 2012). This is most likely due to differences in tobacco processing methods, with the tobacco used for the manufacturing of novel products undergoing pasteurization��a process known to inhibit TSNA formation in processed tobacco. However, due to the higher percentage of (S)-NNN, the absolute amount of this carcinogenic enantiomer in some novel products can be comparable to those found in conventional moist snuff (Table 1).

This is the first study to measure the enantiomeric composition of NNN in cigarette smoke. The measured percent contribution of (S)-NNN in smoke was similar to that in cigarette tobacco, indicating that no significant thermal racemization of NNN occurs during cigarette burning. The higher carcinogenic potential of (S)-NNN compared to (R)-NNN is indicated in both in vitro and in vivo studies. In vitro, cultured rat esophagus, a target tissue for NNN carcinogenicity, metabolizes (S)-NNN predominantly by 2��-hydroxylation, which is the major bioactivation pathway of NNN in rats (McIntee & Hecht, 2000). In vivo, the urine of rats treated with (S)-NNN contained higher levels of metabolites formed via 2��-hydroxylation than the urine of rats treated with (R)-NNN (McIntee & Hecht, 2000).

Furthermore, treatment of rats with (S)-NNN produced two to six times higher levels of DNA adducts in the esophagus and three to five higher levels of DNA adducts in oral tissue, compared with treatment with (R)-NNN (Lao et al., 2007; Zhang et al., 2009). In agreement with these results, the treatment of rats Dacomitinib with (S)-NNN in our recent study produced a 100% incidence of oral and esophageal tumors, compared with only 5 and 3 out of 24 rats developing oral and esophageal tumors, respectively, upon treatment with (R)-NNN (Balbo et al., 2012).

0001). No significant changes in the product pH occurred for either Camel Snus or Marlboro Snus. selleck compound Levels of Constituents per Gram Product Weight When expressed per gram wet weight of product, the mean levels of total nicotine in Camel Snus were lower in the latest large-pouch version of the product, as compared with the original one: 9.4 �� 0.4 mg/g wet weight versus 13 �� 3.2 mg/g wet weight, respectively (Table 1); however, this difference was not statistically significant. The opposite was true for the Marlboro Snus samples: the tobacco of larger pouches released in 2009 contained higher levels of total nicotine than the original ones (p < .0001, Table 1). The levels of unprotonated nicotine per gram product were not different among pouches of various sizes within the same product.

The sum of carcinogenic nitrosamines NNN and NNK in the tobacco of the large Camel Snus pouches was not significantly different than in the tobacco of original and medium pouches. The levels of NNN + NNK in the tobacco of large Marlboro Snus pouches were lower than in the tobacco of original pouches: 1.27 �� 0.7 ��g/g versus 0.50 �� 0.1 ��g/g wet weight, respectively (p < .0001; Table 1). Amounts of Constituents in Single Pouches Comparison of the amounts of analytes contained in a single pouch shows that the large Camel Snus pouches introduced in 2010 contain higher amount of total and unprotonated nicotine than the original small pouches released in 2006 (p < .0001 for both comparisons) and the medium-size pouches introduced in 2008 (p < .0001 for total nicotine and p < .

0002 for unprotonated nicotine; Table 1). In the case of Marlboro Snus, the larger pouches released in 2009 also contained higher amounts of total and unprotonated nicotine than those purchased prior to 2009 (p < .0001 for both comparisons). The amount of NNN + NNK present in large pouches of Camel Snus was 2.3 times higher than in the pouches of medium size (p < .0001) and 3.3 times higher than in the original small pouches (p < .002). Large Marlboro Snus pouches contained lower amounts of NNN + NNK than the earlier version: 0.21 ��g/pouch versus 0.31 ��g/pouch, respectively (Table 1); however, the difference was not statistically significant. Discussion The initial analyses of the new smokeless tobacco products Camel Snus and Marlboro Snus revealed that single pouches of these products contain relatively low amounts of nicotine and the tobacco carcinogens Drug_discovery NNN and NNK, as compared with traditional smokeless products. However, modifications in packaging, flavors, and pouch sizes occurred for both Camel Snus and Marlboro Snus since their first introduction to the market.

, 2002; Le Novere, Zoli, & Changeux, 1996; Quik et al., 2000), which plays a major role in supporting the positive reinforcing actions of nicotine. In the NAc and other domains of the striatum, dopaminergic terminals express ��6��2��3* and selleck chem inhibitor ��6��4��2��3* nAChR subtypes (Champtiaux et al., 2003; Salminen et al., 2004; Zoli et al., 2002), with considerable evidence suggesting that ��6��3* nAChRs regulate the stimulatory effects of nicotine on dopamine release in this region (Champtiaux et al., 2003). The ��6��4��2��3* nAChR subtype enriched in the NAc and striatum has the highest sensitivity to nicotine of any native nAChR so far identified (Grady et al., 2007). Gotti et al.

(2010) reported that infusion of ��-conotoxin MII (CntxMII), a ��3/��6��2* selective antagonist, or ��-conotoxin PIA (CntxPIA), a ��6��2* nAChR selective antagonist, directly into the VTA decreased nicotine-evoked increases in midbrain dopamine levels. Intra-VTA infusion of CntxMII also decreased responding for intravenous nicotine self-administration in rats (Gotti et al., 2010). Similarly, Brunzell and colleagues reported that intra-NAc infusion of CntxMII decreased the motivation of rats to self-administer nicotine, as measured using a progressive ratio schedule of reinforcement (Brunzell, Boschen, Hendrick, Beardsley, & McIntosh, 2010). More recently, it was shown that KO mice lacking protein kinase C epsilon (PKC�� KO mice) self-administered less nicotine and had attenuated place conditioning for nicotine than their wildtype counterparts (Lee & Messing, 2011).

The PKC�� KO mice also demonstrated decreased levels of ��6 and ��3 nAChR subunit mRNA transcripts in the midbrain and striatum, and deficits in cholinergic modulation of dopamine release in the NAc (Lee & Messing, 2011). Hence, it was hypothesized that PKC�� may regulate ��6 (and also ��3) nAChR signaling and thereby influence nicotine reinforcement (Lee & Messing, 2011). Using an ��acute�� nicotine self-administration procedure in which mice are restrained but can nose-poke for intravenous nicotine infusions via a catheter in the tail vein during a single session, it was shown that ��6 subunit KO mice had attenuated levels of nicotine intake compared with wildtype mice (Pons et al., 2008). Taken together, these findings suggest that ��6* nAChRs may be promising targets for medications development for tobacco dependence. However, it is important to note that ��6 KO mice were recently shown to respond for AV-951 nicotine infusions in a manner similar to wildtype mice, as measured using a self-administration procedure in which nicotine infusions were delivered directly into the VTA (Exley et al., 2011). Using this same procedure, ��4 KO mice had a marked deficit in responding for the drug (Exley et al., 2011).

, 2008). This approach selleck products consisted of verbally asking about tobacco use, advising users to quit, assessing readiness to quit in the next month, assisting with the quitting process by offering a self-help guide and the opportunity to participate in three group cessation counseling sessions, and arranging follow-up with interested tobacco users. In addition, students with oral lesions were scheduled 1 week later for a follow-up exam by the nurse. Parents of students with persistent lesions were informed by the nurse who facilitated follow-up evaluation with a study dentist. The third component consisted of three noncompulsory, 1-hr nurse-led cessation sessions scheduled after school approximately 1 week apart. The first session focused on assessment, education, preparation to get ready to quit, and the importance of social support.

The second session focused on setting a quit date and skills to cope with cravings and temptation to use. The third session reviewed progress and focused on relapse prevention. Recruitment and training of interventionists School nurses Counties or individual schools provided study investigators with lists of county school nurses who visited high schools monthly in rural areas of CA. Investigators then recruited and trained 55 school nurses in 3-hr regional trainings that included a printed procedure manual, videos on how to perform oral assessment, screening, tobacco cessation counseling procedures, and role playing with feedback on student partners. Those nurses who were unable to attend regional trainings were provided an individual 3-hour training at a convenient consenting high school.

School nurses also were trained to complete and return an adverse events form after oral screenings and cessation counseling. Student peer leaders Student leaders were identified by their peers on the baseline questionnaire in response to the question ��Who in your class do you look up to and admire?�� Investigators then contacted identified-peer leaders and explained that, if they agreed AV-951 to help, they would receive credit toward community service and a letter from the study principal investigator indicating their contribution as a peer leader for use in applications for college and/or employment. One hundred and fifty-three peer leaders were recruited and trained in a 3-hr session at each high school that consisted of lecture/discussion, viewing videos and slides, and role playing with feedback. Students were allowed to select slides that they felt comfortable presenting. Data analysis We used survey analysis methods to estimate study retention adjusting for clustering within schools.

After fixation, the cells were then incubated with stain solution (40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2) selleck containing 1 mg/ml X-gal at 37��C for overnight. Fluorescence-activated cell sorting Isolated PT were cultured overnight in PT medium supplemented with 10 mIU/ml bTSH. On the next day, PT were collected and incubated with anti-STRO-1 or anti-TPO (Clone MoAb47, Dako Cytomation) antibody. After labeling with secondary antibody conjugated with Alexa 488 and 7-AAD (BD Biosciences, Franklin Lakes, NJ), the cells were analyzed and sorted using a FACS Vantage SE (BD Biosciences). Real-time RT-PCR Total RNA was extracted using ISOGEN reagent (Nippon gene, Tokyo, Japan) and reverse transcribed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) with random hexamers.

The following PCR amplifications were performed using QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, CA) for TTF1 and SYBR Premix Ex Taq Perfect Real Time kit (TaKaRa Bio, Ohtsu, Japan) for other genes in a Thermal Cycler Dice Real Time System (TaKaRa Bio). The cycle threshold value, which was determined using second derivative, was used to calculate the normalized expression of the indicated genes using Q-Gene software [15], using ribosomal RNA 18S as a reference. The following primer pairs were used: 18S, 5��-GTAACCCGTTGAACCCCATT-3�� and 5��-CCATCCAATCGGTAGTAGCG-3��; TG, 5��-CTGGTGTGTCATGGACAGCGGAGAA-3�� and 5��-CCCGAGATTGTCTCACACAGGAT-3��; TSH-R 5��-CTATAGATGTGACTCTGCAGCAGCT-3�� and 5��-GAGGGCATCAGGGTCTATGTAAGT-3��; PAX8 5��-CCCCCTACTCCTCCTACAGC-3�� and 5��-ACTGTCCCCATGGCAACTAC-3��; TTF1 5��-CCATGAGGAACAGCGCCTC-3�� and 5��-CTCACG TCCCCCAGCGA-3��.

Microarray analysis Total RNAs were extracted from PT from three different samples (PT0808, PT0811 and PT0812) on the next day of initial plating and corresponding SAGM-grown lines at two weeks after plating using ISOGEN reagent (Nippon gene) and subjected to Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray analysis service (Bio Matrix Research, Nagareyama, Japan). The array contained 54,675 probes for mRNAs. GeneSpring Software (Agilent Technologies, Santa Clara, CA) was used for data analyses. The data were deposited in NCBI GEO site (accession number: “type”:”entrez-geo”,”attrs”:”text”:”GSE24553″,”term_id”:”24553″GSE24553).

Results Isolation of proliferative cells from normal human thyroid tissues In an attempt to isolate primitive human thyroid epithelial cells, we tried several commercially available media containing growth factors but not serum which is generally thought to AV-951 enforce differentiation program. Those were basically developed for growing human primary cells or tissue stem cells. Among them, we found that SAGM was the most suitable for our purpose, which was developed for growing human primary small airway epithelial cells.

A study revealed that cyclosporiasis patients had a decreased level of CD3+ and CD4+, while CD8+ was normal [107]. 3.4.3. Recent Advances in Research Although the prevalence Src Bosutinib of cyclosporiasis among diarrheal patients can be considerable as documented in several provinces of China, few epidemiological surveys have been implemented and many techniques applied to cryptosporiasis have not been introduced to cyclosporiasis research. Consequently, one challenge in epidemiological surveys is the lack of sensitive techniques to differentiate C. cayetanensis from other intestinal protozoa. Indeed, misdiagnosis is common [108]. Humans currently are the only known host of C. cayetanensis. The lack of an animal model prevents studying this parasite [109]. To overcome this limitation, Ge and colleagues established a rat model for C.

cayetanensis. They first suppressed the immune system of rats using hydrocortisone or cyclophosphamide and then infected them with C. cayetanensis. The number of oocysts in stool gradually increased and reached a peak 5�C7 days after infection [110]. The susceptibility to C. cayetanensis infection is thought to be related to the status of the immune system. A series of relevant studies was performed in order to reveal the relationship between the likelihood of an infection and different immune status parameters [107,111,112]. It was found that among the infected, the titer of membrane interleukin-2 receptor, CD3+ and CD4+ was significantly decreased relative to non-infected individuals, while that of soluble interleukin-2 receptor as well as specific IgG and IgM were significantly elevated.

3.5. Blastocystosis 3.5.1. Parasite and Pathogenicity Blastocystis hominis is one of the most frequently diagnosed protozoan parasites in human faecal samples. It is found in both symptomatic and healthy individuals and therefore, its pathogenic potential is still debated [113]. Additionally, many aspects of this organism including its taxonomy, life cycle and mode of transmission continue to be controversial despite this parasite being first discovered in human faeces as early as 1912 [114]. The morphologic diversity of the organism and the low sensitivity of the generally used wet-mount detection technique add further difficulties to its study [113]. Most people carrying B.

hominis infections are asymptomatic while some show gastrointestinal symptoms including diarrhea, abdominal discomfort, abdominal pain or abdominal cramping and vomiting. Acute infections may cause watery diarrhea. In addition, fatigue, loss of appetite, bloating, and other non-specific gastrointestinal Cilengitide symptoms have been associated with B. hominis infections. 3.5.2. Epidemiology B. hominis is endemic across the World, with a focus in tropical and subtropical regions. The first national survey showed that the overall prevalence B. hominis in China was 1.3% [4].

As expected, cVac infection per se did not induce liver disease (Figure 9D,; red bar), nor did it suppress HBV gene expression (Figure 9E), Pacritinib suggesting that the induction of liver disease and the suppression of HBV gene expression in cVac infected HBV transgenic mice after COR93-specific CD8+ T cell adoptive transfer were mediated by the T cells. These results suggest that functional differentiation of HBV-specific CD8+ T cells in the HBV transgenic mouse liver is sufficiently restored in the context of a systemic virus infection to both cause hepatitis and inhibit viral gene expression. Activation of professional antigen presenting (pAPC) cells is believed to be essential for the induction of functional CD8+ T cell responses after virus infections, and several studies have demonstrated that ligation of CD40 induces pAPC activation, resulting in the induction of CD8+ T cell responses [40]�C[42].

To examine whether CD40 activation could induce functional differentiation of HBV-specific CD8+ T cells in this model, we adoptively transferred na?ve COR93-specfic CD8+ T cells into HBV transgenic mice and nontransgenic controls that had been injected intravenously either with 100 ��g/mouse of an agonistic anti-CD40 antibody (��CD40) [43], [44] or with saline (NaCl) 16 hours before transfer. Seven days later, the mice were sacrificed, and intrahepatic COR93-specific CD8+ T cells were analyzed for expansion, IFN�� producing ability and Granzyme B (GrB) expression. The results were correlated with the degree of liver damage and HBV gene expression monitored by serum ALT activity and Northern Blot analysis, respectively.

As shown in Figure 10A, by day 7, ��CD40 treatment increased the intrahepatic expansion of COR93-specific CD8+ T cells in HBV transgenic mice by 5 fold compared to the saline treated transgenic animals. Furthermore, by day 7, approximately 40% of intrahepatic COR93-specific CD8+ T cells in the ��CD40 treated animals produced IFN�� in response to 5 hours in vitro peptide stimulation (Figure 10B), and almost all the COR93-specific CD8+ T cells expressed GrB directly ex vivo (Figure 10C), contrasting strikingly to their counterparts in the saline treated animals. Interestingly the induction of T cell effector functions coincided with PD-1 downregulation in intrahepatic COR93-specific CD8+ T cells (Figure 10D), suggesting that activation of CD40 signaling counteracted the PD-1 mediated negative signaling.

In contrast to these observations, neither the expansion nor the functional differentiation of the COR93-specific CD8+ T cells were enhanced in ��CD40 treated nontransgenic recipients (not shown). Importantly, ��CD40 treated HBV transgenic recipients displayed higher serum ALT activity (Figure 10E) and very strong suppression of intrahepatic Batimastat HBV gene expression (Figure 10F), after adoptive transfer of na?ve COR93-specific CD8+ T cells compared to saline treated animals.

At present, only interferon-based therapies are licensed for chronic HDV hepatitis treatment, but these approaches achieve the objective of curing the infection (namely, HDV clearance) in only a very few cases (5). Chronic hepatitis D is thus considered a nearly incurable liver disease. Moreover, since HDV does not produce selleck inhibitor its own enzymatic proteins (21, 30), there is no room for typical antiviral therapeutic approaches based on the use of specific inhibitors of viral enzymes (20). Natural recovery from HDV infection is a rare event, usually occurring only in the event of HBsAg seroclearance (21, 35), thus confirming that studying the mechanisms of HBV and HDV interplay may have great importance not only from the virological point of view but also in identifying new methods for the cure of HDV-related diseases.

Very few data are currently available on intrahepatic HBV and HDV molecular status for coinfected patients (2, 12), and most of the existing evidence derives from experiments with animal models (1, 8, 15, 29). In this study, we applied sensitive molecular assays to evaluate serologic and intrahepatic quantitative profiles of HBV and HDV in chronic infections. In accordance with previous studies (9, 10, 23, 40), our results seem to confirm that HDV exerts a dominant role over HBV, as indicated by its higher levels of replication and by the lower levels of serum and intrahepatic HBV DNA, cccDNA, and HBV transcripts detected in the majority of coinfected patients than those in HBV-monoinfected individuals.

However, in agreement with previously reported data, we found that serum HBsAg levels were comparable between HDV-positive and HDV-negative patients (8) and that amounts of pre-S/S RNAs and HBsAg produced per cccDNA molecule were higher in HDV-positive patients. Considering that HDV depends on HBV only to acquire HBsAg and that HDV nucleoproteins may be considered competitors of replicating HBV genomes (21, 35), our findings support the hypothesis that HDV might be able to induce an opposing effect on HBV replication and on HBV transcription (3, 13). In fact, our results showing a significantly smaller proportion of pgRNA among HBV transcripts imply that the reduction of virion production might be due mainly to a selective suppression of pgRNA transcription, thus suggesting a kind of dissociation between pgRNA and pre-S/S RNA production in cases of HDV coinfection.

This hypothesis is biologically plausible, since the transcription of pgRNA and that of pre-S/S RNAs Batimastat are under the control of two different HBV genomic regulatory regions (14, 26). However, the molecular mechanisms possibly implicated in the discrepancy between pgRNA and pre-S/S RNA steady-state levels are unknown, and one cannot exclude that the discrepancy might be dependent on differences in transcript stabilities.