, 2004) injected into the cortex transduces almost exclusively

neurons locally near the injection site. The GFP is soluble and diffuses along the dendrites and axons of the transduced neurons, including long-range axonal projections. Lenti-GFP can therefore be used as an unequivocal anterograde anatomical tracer (Ferezou et al., 2007; Broser et al., 2008a). Whereas VSV-G pseudotyped lentivirus only transduces neurons with somata Panobinostat purchase within a few hundred microns of the cortical injection site, other viral vectors behave quite differently. Adeno-associated viruses (AAVs) are physically much smaller, so they can diffuse further, transducing neurons across larger brain regions. Different serotypes of AAV have different properties and, like adenovirus and rabies virus, some AAVs can be retrogradely transported after axonal uptake of vector (Taymans et al., 2007; Hollis et al., 2008). AAV serotype 6 (AAV6; Grimm et al., 2003) binds to heparin (like AAV serotype 2, but different from other serotypes) and probably because of this binding it diffuses less in the brain than many other AAV serotypes. Nonetheless, neurons transduced with AAV6 are found

far from the injection site, presumably because of retrograde transport (Kaspar et al., 2003; Towne et al., 2008, 2010). Injection of AAV6 encoding a ‘humanized’ cre-recombinase (AAV6-Cre; BMN 673 nmr Shimshek et al., 2002; Fig. 3F) into Rosa floxed-LacZ cre-reporter mice (Soriano, 1999), allows staining of transduced neurons with the blue XGal chromogenic substrate. If the AAV6-Cre vector is injected into the neocortex, it is taken up

DOK2 by axon boutons near the injection site (while also transducing neurons with somata near the injection site). The AAV6-Cre is then retrogradely transported to the nucleus of neurons with axonal projections to the injection site, and the subsequent expression of cre-recombinase can be monitored in cre-reporter mice. AAV6-Cre can therefore be used as a retrograde vector for anatomical labelling of neurons projecting to the injection site. Both the classical anatomical tracers and the viral vectors can be injected simultaneously to allow labelling of both anterograde and retrograde connectivity from a single well-defined injection site. Voltage-sensitive dye imaging reveals that activity within the C2 barrel column rapidly propagates to neighboring cortical columns (Fig. 2). This spread is likely to be mediated, at least in part, by the extensive local axonal projections of the pyramidal neurons located in the C2 barrel column. Injections into the C2 barrel column of the anterograde tracers Lenti-GFP (Fig. 4A and B; Dittgen et al., 2004) or BDA (Fig. 4C) indicate that C2 barrel cortex neurons extend axonal arborizations into layers 2/3 and layers 5/6, almost across the entire extent of S1 barrel cortex. The density of axons is highest close to the C2 barrel column and decreases across the neighboring cortical columns (Brecht et al.

pylori genes, including peptidyl-prolyl cis–trans isomerase (PPIase), which has been characterized as a virulent factor of Legionella pneumophila and Trypanosoma cruzi (Fischer et al., 1992; Pereira et al., 2002) and is implicated in the regulation of gastric epithelial cell growth and apoptosis (Basak et al., 2005). A total of 64 H. pylori strains were

cultured from gastric biopsies from adult patients. These included 22 cases from gastric cancer patients and 42 from superficial gastritis patients. All samples were obtained with informed consent under a protocol approved by the hospital ethical committee at the China Medical University. Helicobacter pylori were cultured at 5% O2/10% CO2/85% N2, 37oC on brain heart infusion agar (Difco) supplemented Tanespimycin cell line with 7% sheep blood, 0.4% IsoVitaleX, amphotericin B (8 g mL−1), trimethoprim (5 g mL−1) and vancomycin (6 g mL−1). Helicobacter pylori colonies were identified based on their typical morphology, characteristic appearance on Gram staining, a positive urease test and

gene-specific PCR tests. Bacterial DNA was extracted with phenol–chloroformisoamyl alcohol by standard procedures and precipitated by the addition of 1/10 volume of ammonium acetate and 2.5 volume of cold ethanol. After centrifugation, the DNA pellet was washed with 70% ethanol and dissolved in TE buffer [10 Mm Tris-HCl (pH 8.3), 0.1 mmol L−1 EDTA] as we have described previously (Gong et al., 2005). The differences in gene content between the gastric cancer-associated H. pylori strain and the superficial gastritis-associated strain was determined using p38 MAPK inhibitor the PCR-Select™ DNA Substraction Kit (Clontech). To detect gastric cancer-specific second genes, genomic DNA from gastric cancer strain, L301, was used as the tester DNA and genomic DNA from superficial gastritis strain, B975,

was used as the driver DNA. To detect genes that were less abundant or absent in gastric cancer-associated H. pylori strain, genomic DNA from B975 was used as the tester DNA and genomic DNA from L301 was used as the driver DNA. Two micrograms of either tester or driver DNAs were digested to completion with 60 U of AluI (New England Biolabs) for 16 h in 200 μL reaction volumes. The digested products were extracted with phenol, precipitated with ethanol and resuspended in 10 mM Tris-HCl, pH 7.5, at a final concentration of 200 ng μL−1. Two aliquots of the digested tester DNAs were ligated separately to two different adaptors (Adaptor 1, 5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′ and 3′-GGCCCGTCCA-5′; Adaptor 2R, 5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3′ and 3′-GCCGGCTCCA-5′) using T4 DNA ligase (New England Biolabs). Then, 1 μL of each adaptor-ligated tester DNAs was mixed with 2.0 μL of digested driver DNAs and 1.0 μL of 4 × hybridization buffer. The DNA fragments were denatured at 98 °C for 1.5 min, and allowed to anneal at 63 °C for 1.5 h.

These results are consistent with the reduced levels of hippocampal endocannabinoids found after food restriction. Regarding the CB1 expression, AM251 induced specific changes focused in the CA1 stratum pyramidale of high-fat-diet-fed rats. These findings indicated that the cannabinoid antagonist AM251 modulates ECS-related proteins in the rat hippocampus in a

diet-specific click here manner. Overall, these results suggest that the hippocampal ECS participates in the physiological adaptations to different caloric diets. “
“The Rehabilitation Gaming System (RGS) has been designed as a flexible, virtual-reality (VR)-based device for rehabilitation of neurological patients. Recently, training of visuomotor processing with the RGS was shown to effectively improve arm function in acute and chronic stroke patients. It is assumed that the VR-based training protocol related to RGS creates conditions that aid recovery by virtue of the human mirror neuron system. Here, we provide evidence for this assumption by identifying the brain areas involved in controlling the catching of approaching colored balls in the virtual MEK inhibitor environment of the RGS. We used functional magnetic resonance imaging of 18 right-handed healthy subjects (24 ± 3 years) in both active and imagination conditions. We observed that the imagery

of target catching was related to activation of frontal, parietal, temporal, cingulate and cerebellar regions. We interpret these activations in relation to object processing, attention, mirror mechanisms, and motor intention. Active catching followed an anticipatory mode, and resulted in significantly less activity in the motor control areas. Our results provide preliminary support for the hypothesis underlying RGS that this novel neurorehabilitation approach engages human mirror mechanisms that can be employed for visuomotor training. Rehabilitation of neurological patients is a major challenge. Given that

stroke is a primary cause of permanent disability (Mukherjee & Patil, 2011), there is a wide demand for rehabilitation of neurological deficits after stroke. Neurological deficits resulting from stroke differ in severity, owing to different Buspirone HCl lesion locations, lesion volumes, and times elapsed since stroke (Seitz & Donnan, 2010). In this regard, a training program of basic arm–hand functions has been developed that scales in difficulty relative to the severity of the individual stroke survivor’s deficit on a session-by-session basis (Platz et al., 2009). Furthermore, it is well established that a dosing effect associated with more intense rehabilitative training leads to better neurological outcomes (Hummelsheim et al., 1995; Kwakkel et al., 1999).

These results are consistent with the reduced levels of hippocampal endocannabinoids found after food restriction. Regarding the CB1 expression, AM251 induced specific changes focused in the CA1 stratum pyramidale of high-fat-diet-fed rats. These findings indicated that the cannabinoid antagonist AM251 modulates ECS-related proteins in the rat hippocampus in a

diet-specific FG 4592 manner. Overall, these results suggest that the hippocampal ECS participates in the physiological adaptations to different caloric diets. “
“The Rehabilitation Gaming System (RGS) has been designed as a flexible, virtual-reality (VR)-based device for rehabilitation of neurological patients. Recently, training of visuomotor processing with the RGS was shown to effectively improve arm function in acute and chronic stroke patients. It is assumed that the VR-based training protocol related to RGS creates conditions that aid recovery by virtue of the human mirror neuron system. Here, we provide evidence for this assumption by identifying the brain areas involved in controlling the catching of approaching colored balls in the virtual selleck screening library environment of the RGS. We used functional magnetic resonance imaging of 18 right-handed healthy subjects (24 ± 3 years) in both active and imagination conditions. We observed that the imagery

of target catching was related to activation of frontal, parietal, temporal, cingulate and cerebellar regions. We interpret these activations in relation to object processing, attention, mirror mechanisms, and motor intention. Active catching followed an anticipatory mode, and resulted in significantly less activity in the motor control areas. Our results provide preliminary support for the hypothesis underlying RGS that this novel neurorehabilitation approach engages human mirror mechanisms that can be employed for visuomotor training. Rehabilitation of neurological patients is a major challenge. Given that

stroke is a primary cause of permanent disability (Mukherjee & Patil, 2011), there is a wide demand for rehabilitation of neurological deficits after stroke. Neurological deficits resulting from stroke differ in severity, owing to different IMP dehydrogenase lesion locations, lesion volumes, and times elapsed since stroke (Seitz & Donnan, 2010). In this regard, a training program of basic arm–hand functions has been developed that scales in difficulty relative to the severity of the individual stroke survivor’s deficit on a session-by-session basis (Platz et al., 2009). Furthermore, it is well established that a dosing effect associated with more intense rehabilitative training leads to better neurological outcomes (Hummelsheim et al., 1995; Kwakkel et al., 1999).

Rather than relying on molecular diagnosis based on RNA detection, the point-of-care test www.selleckchem.com/products/MDV3100.html for dengue NS1 antigen would be appropriate for travelers’ screen. NS1 sensitivity is highest between the 2nd and 4th

day of illness and would be useful early in acute phase in non-endemic countries.3 Extreme utility of NS1 antigen assay was witnessed in travelers at airports in Taiwan. By NS1 antigen detection, 19 RT-PCR negative travelers could be labeled dengue positive. Two such travelers turned out to be IgM positive on day 17 or 18 of illness.4 Subhash C. Arya 1 and Nirmala Agarwal 1 “
“Cardiovascular disease is an increasing concern among HIV-infected persons and their providers. We determined if fatty liver disease is a marker for underlying coronary atherosclerosis among HIV-infected persons. We performed a cross-sectional study in HIV-infected adults to evaluate the prevalence of and factors, including fatty liver disease, associated with subclinical coronary atherosclerosis. All participants underwent computed tomography for determination of coronary artery calcium (CAC; positive defined as a score >0) and fatty liver disease (defined Selleck Dasatinib as a liver-to-spleen ratio <1.0). Factors associated with CAC were determined using multivariate logistic regression

models. We included in the study 223 HIV-infected adults with a median age of 43 years [interquartile range (IQR) 36–50 years]; 96% were male and 49% were Caucasian. The median CD4 count was 586 cells/μL and 83% were receiving antiretroviral medications. Seventy-five (34%) had a positive CAC score and 29 (13%) subjects had fatty liver disease. Among those with CAC scores of 0, 1–100 and >100, the percentage with concurrent fatty liver disease was 8, 18 and 41%, respectively (P=0.001). In the multivariate model, CAC was associated with increasing age [odds ratio (OR) 4.3 per 10 years; P<0.01], hypertension (OR 2.6; P<0.01) and fatty liver disease (OR 3.8; P<0.01). Coronary atherosclerosis as detected using CAC is prevalent among young HIV-infected persons. The detection of fatty

liver disease among HIV-infected adults should prompt consideration of assessment for underlying cardiovascular disease and risk factor reduction. As HIV-infected persons are experiencing longer life expectancies, there is increasing concern regarding non-AIDS-defining conditions, including cardiovascular Low-density-lipoprotein receptor kinase disease [1,2]. HIV-infected persons appear to have a higher risk of coronary artery atherosclerosis compared with the general population, which may be a result of HIV-induced inflammation, antiretroviral medications, or concurrent medical conditions, such as insulin resistance, dyslipidaemia, hypertension, visceral fat deposition and tobacco abuse [1–10]. Elevated prevalence rates of subclinical cardiovascular disease among HIV-infected persons have recently been demonstrated using computed tomography (CT) coronary artery calcium (CAC) scores [9,11–18].

The different frequencies of HLA-B*5701 found in patients from different African countries strongly suggest greater utility of HLA-B*5701 screening in some African groups compared with others. Although ethnic-based

pharmacogenetic screening in UK clinics with diverse populations is unlikely to be practical and also likely to be ethically unacceptable, our figures add to the need for caution when considering screening in diverse African settings. Despite an overall sample size of 1502, only two sub-divisions had a sufficiently large sample to allow meaningful interpretation of the prevalence rate [White/Eurasian and Niger-Congo with prevalence rates of 7.95% (CI 5.88%–10.02%) and 0.52% (CI 0.18%–1.52%), respectively]. On the basis of previous Wnt activity work, we sub-divided our African patients according to linguistic index that language groups might reflect genetic structure [9]. However, more recent data suggest that this

may not necessarily be true in all settings and particularly not African ones [14]. In contrast, a recent, well-publicized study of 121 African populations demonstrated genetic clustering across the Niger-Congo ITF2357 supplier (Niger-Kordofanian) linguistic populations [15]. Our data, where both Ugandan and Zimbabwean populations were classified as Niger-Congo (Bantu) but had very different HLA-B*5701 prevalences, demonstrate the difficulties in using such classifications to distinguish populations Thymidylate synthase in genetic studies of specific, non-neutral alleles. Our study was not able to distinguish northern (Nilotic) Ugandans from Bantu Ugandans, so it is possible that the different rates among

Zimbabweans and Ugandans were because of cross-classification. However, a significant majority of Ugandans in the United Kingdom are thought to be Bantu and HLA-B*5701 frequency among Nilotics has been reported to be lower than among Bantu [4]. The 244 unclassifiable subjects underline the difficulties in basing decisions on these self-reported measures of ethnicity. Only one of the three HLA-B*5701 positive subjects in the Niger-Congo sub-division self-reported as genetically homogenous reflecting the potential of genetic admixture to complicate analysis. Future studies may consider the use of ancestry information bio-markers to define population groups more accurately. The single false-positive result in a local laboratory reinforces the need for robust laboratory quality assurance. It is reassuring that Sequence specific primer (SSP) methodology failed safe by identifying the patient as HLA-B*5701 positive; by doing so it ensured patient safety was maintained. In the present study, HLA-B*5701 prevalence in the UK was similar to previously reported rates in White HIV-infected subjects but considerably lower than those reported in Black HIV-infected subjects, probably as a result of the large proportion of Black subjects that were of African origin. In addition, 99.

44, p = 0.001). The increases in the maximum temperature had no significant effect on the attack rate. In contrast to the rates for ETEC, the rates of EAEC-associated diarrhea remained relatively constant despite seasonal temperature variations (p = 0.1). TD is caused by a variety of bacterial agents of which ETEC and EAEC are the most common identifiable pathogens.1

In agreement with the previously published studies on TD acquired in Guadalajara, Mexico from 1986 to 19899, this DAPT clinical trial study found that the rates of TD were higher during summertime when compared to wintertime in central Mexico. This second study was conducted in Cuernavaca, Mexico, which is called “the city of eternal springtime” where temperature variations are milder. The warmer and wetter summer months are associated with an increased occurrence of diarrhea.12 Warmer climates may encourage propagation of enteric bacterial pathogens in food13 and water14 explaining the increase in bacterial diarrhea during this website the summertime. Furthermore, in the case of ETEC, seasonality also appears to influence the rates of identified toxin phenotypes. It has previously been suggested that in Egypt, ST (heat-stable toxin)-producing ETEC strains are more commonly identified in the stools of children with diarrhea in the summer, whereas LT

(heat-labile toxin)-producing ETEC strains are identified all year around.15 In our study, the rates of LT- and ST-producing ETEC did not appear to vary according to seasonality. In this study, we found that minimum and average temperatures are positively associated to higher rates of ETEC-associated diarrhea. We hypothesize that since weekly maximum temperatures do not fluctuate as much as minimum temperatures, Non-specific serine/threonine protein kinase the analysis failed to show a statistical correlation with maximum temperatures. When studied in the univariate analysis, the identification of STEC as defined by the

presence of stx1 or stx2 in stools also showed a positive correlation with warmer temperature and summertime diarrhea, however only ETEC showed a significant correlation when an adjusted multivariate analysis was performed. An important observation in this study is that in contrast to ETEC, the rates for EAEC, the second most common bacterial cause of TD, remained similar in both seasons. This is consistent with a previous study carried out in Korea that failed to find a seasonal pattern for EAEC infection16 and contrasts with a 12-month study in a US pediatric population, where Cohen and colleagues reported a seasonal peak of EAEC in children during March to April months; However, a confounding variable in that study was that many of the EAEC cases were coinfected with Rotavirus.4 Although EIEC was only identified in the summer, additional studies are needed to determine if the occurrence of EIEC infection is also seasonal.

2%), wearing a face mask by 91 (48.9%), cough etiquette by 86 (46.2%), social distancing by 64 (34.4%), and contact Rapamycin in vitro avoidance by 45 (24.2%). Seasonal influenza vaccination in the previous 12 months was reported by 138 (63.0%) respondents. Influenza A(H1N1) vaccinations were reported by 72 (38.7%) respondents. Respiratory illness during the Hajj and/or in the first 7 days post-Hajj was reported by 76 (41.3%) respondents (respiratory illness during Hajj = 32 (17.3%) respondents and post-Hajj =53 (29.0%) respondents). Among the 76 respondents who reported respiratory symptoms,

coughing was reported by 56 (73.7%), sneezing by 48 (63.2%), sore throat by 29 (38.2%), fever by 25 (31.1%), congestion by 16 (32.9%), breathing problems by 4 (5.3%), and “bronchitis” by 2 (2.6%). Of the 76 respondents who reported respiratory illness, 18 (23.7%) met criteria for self-reported influenza-like illness (ILI), defined as fever plus sore throat and/or coughing.11 Three protective behaviors were associated with reduced risk of respiratory illness: social distancing, hand hygiene, and contact avoidance (Table 2). When the number of protective practices was analyzed as a continuous variable, reduced risk of Caspase activity respiratory illness was associated with engaging in more protective behaviors during the

Hajj (F = 3.13,p = 0.03) (Figure 1). Engaging in more protective measures was associated with noticing influenza A(H1N1) health messages during the Hajj (F = 6.93,p = 0.01). Respiratory illness mild enough that the respondents did not need to see a doctor or nurse was reported by 47 (65.3%) respondents, 23 (31.9%) were ill enough to see a doctor or nurse, and 2 (2.8%) needed to be hospitalized. No protective behaviors during Hajj

were associated with less severe respiratory illness. Reduced severity of respiratory illness during Hajj was associated with fewer years lived in the United States (F = 4.72,p = 0.01). The mean duration of respiratory illness reported during Hajj was 7 days (range = 1–21d). Practicing contact avoidance during Hajj was associated with shorter duration of respiratory illness (F = 3.54,p = 0.06). Shorter duration of respiratory illness during Hajj was also DOCK10 associated with younger age (r2 = 0.361,p = 0.002), fewer health risks (F = 3.99,p = 0.02), and higher levels of perceived influenza A(H1N1) severity (F = 8.02,p < 0.001). A multivariable model contained two significant predictors of reduced duration of respiratory illness: practicing contact avoidance (β = −0.38,p = 0.01) and noticing influenza A(H1N1) health messages during Hajj (β = 0.25,p = 0.06). These factors also explained a significant proportion of variance in the duration of respiratory illness (r2 = 0.13,F6,45 = 2.29,p = 0.05). When the number of protective practices was analyzed as a continuous variable, engaging in more protective measures during Hajj was correlated with shorter duration of respiratory illness (r2 = −0.307,p = 0.02 ) (Figure 2).

Answers with a recommendation level of A or B represent current standard care practices in Japan. All 104 Clinical Questions and Answers items, with the omission of the Discussion, List of References, and Tables and Figures, are presented herein to promote a better understanding among English readers of the current standard care practices for pregnant women in Japan. “
“Transplantation of gynecological organs is a medical field where considerable advancements have been made in research during the last 25 years

and with some procedures already introduced as clinical treatments. These types of transplantations aim at curing permanent infertility. Uterus transplantation has been proven to be a feasible procedure in different experimentation animal models with proof of concept concerning selleck chemical surgery, control of rejection and fertility. There has already been one human transplantation INK 128 research buy attempt, which, however, was unsuccessful. Based on the progress in this area, we predict that the first successful uterus transplantation attempt will come within 2–3 years. Orthotopic ovarian cortex transplantation has overcome the status of an experimental procedure since more than 20 pregnancies have been reported. Its main field of application is fertility preservation in oncologic patients undergoing

high gonadotoxic risk therapies. The role of heterotopic ovarian cortex transplantation still remains at the research level, although co-transplantation with an orthotopic cortex might facilitate a more accurate endocrine environment. The major drawback of ovarian cortex transplantation Fossariinae remains the long ischemic interval between re-implantation and the establishment of neovascularization. Whole ovary cryopreservation followed by transplantation through vascular anastomosis may emerge as an important procedure in this field, because the warm ischemic time would be reduced from several days to less than 1 h, which will most likely improve follicle

survival. In summary, transplantation surgery is also entering the field of gynecology and in the future several types of transplantations of organs/tissues of the female reproductive tract may become established clinical procedures. “
“Aim:  Career satisfaction level, degree of mental distress associated with certain work-related factors, and demographics were examined for the first time in obstetricians and gynecologists in Japan. Material and Methods:  Associations between the score on Kessler 6 screening scale, or the job satisfaction level, and the scores on the job content questionnaire, Social Support Questionnaire (SSQ), working conditions and demographics were examined in 1301 members of the Japan Society of Obstetrics and Gynecology. Results:  8.4% of respondents were speculated to suffer from depression or anxiety disorder.

Consequently, Tn916 insertion in pCY186 may lead to the instability of this nonessential replicon in vitro, leading to the observed insertion frequencies. Analysis of the complete genome sequence from B. proteoclasticus B316T indicates that approximately 90.0% of the genome is made up of ORFs, but annotation

of the full sequence indicated find more that only (18 of 53, 34.0%) of the Tn916 insertion sites were in ORFs (Fig. 1, Table 1). The association of transposon integration within intergenic regions in the B316T genome may be inter-related with the analysis of the coding vs. noncoding regions: intergenic regions have an overall GC ratio of 34.7%, compared with 39.3% for the genome overall, and thus are comparatively more AT-rich. Similarly, the intergenic regions of H. influenzae Rd KW20 were 5% more AT-rich than the coding regions (Nelson et al., 1997). These data indicate that the integration of Tn916 in B316T is likely to be site-specific with hot spots for insertion, despite it having a relatively AT-rich/GC-poor genome. Identification of the

integration sites in the isolates generated from this study enabled the modelling of a consensus sequence for Tn916 integration in B316T (Fig. 2), which consisted of transposon target sites that were characteristically AT-rich, with a more variable 6-bp spacer sequence contained within the AT-rich target regions Ipilimumab in vivo (Fig. 2). Comparison of the modelled B316T-derived consensus with those derived from the insertion sites of Tn916 transposon mutants in other bacterial strains indicates conservation of the core TTTTTnnnnnnAAAAA sequence across all mutants that were examined (Scott et al., 1994; Nelson et al., 1997). Modelled consensus

target sequences for Tn916 insertions in ORFs, intergenic regions and sites where more than five separate Tn916 insertions occurred were also determined, but no specific characteristics were observed that differentiated these modelled consensus sequences from those that represented all insertion sites (data not shown). However, when the modelled 16-bp consensus target sequence (Fig. 2), with up to two mismatches, was used to search the complete B316T genome, 39 theoretical insertion FAD sites were identified, 19 of which were in coding regions, 20 of which were in noncoding regions and included nine where the insertion site had been identified from purified B316T tetracycline-resistant mutants (six noncoding: insertion numbers 13, 23, 28, 30, 32 and 48 and three coding: insertion numbers 21, 46 and 52, Table 2) during conjugation experiments. We were unable to categorically deduce whether any theoretical transposon insertion sites in any of the specific genes was lethal, but based on our assessments on the likely gene function of the mutated gene and the adjacent gene, four of the 16 theoretical Tn916 insertion sites were likely to be essential and could be lethal.