Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in  The advent of targetable DNA cleavage reagents has greatly enhanced the arsenal of tools for genetic and functional analysis. Zinc-finger nucleases (ZFNs) , homing The best of these reagents cleave their intended targets very efficiently, sometimes with frequencies exceeding 50% . ZFNs, i2His2 zinc finger, contacts primarily three base pairs, and fingers have been identified that recognize most of the 64 different triplets . New mutants in autosomal genes other than ry were collected by crossing to either 1118w ; Sco/S2CyO or TM2,rySC/MKRS and collecting flies carrying a targeted chromosome over a balancer chromosome. A variety of stocks were used to characterize TALEN-generated mutations. All were obtained from the Bloomington Stock Center. The deletion stock 1118w; Df(2L)BSC295/CyO was used to characterize mutations in Psf2 (CG18013). Mutations in Sld5 were characterized by crossing to 1118w; Df(3R)BSC495/TM6C, 1 cu1Sb. Mutations in y were scored by crossing to C(1)DX, 1 f1/FM6y. Potential ry mutants were scored by crossing to 1;ry506v. All stocks are described in FlyBase  and transcribed with MEGAscript T3 high yield transcription Kit (Ambion) and then capped with the ScriptCap m7G Capping System (CELLSCRIPT). RNAs were injected at concentrations from 0.2 to 0.4 mg/mL. Oligonucleotide donors generally were injected at 0.5 mg/mL, but in the case of the Psf2 phospho-oligo, injections were also done at 0.2 mg/mL. The ry donors used in this work were all single-stranded oligonucleotides obtained from the University of Utah DNA/peptide Core. The Psf2 donor was a single-stranded oligonucleotide obtained from Integrated DNA Technologies .RNAs were prepared and injected as described for ZFNs  and analyzed with Applied Biosystems GeneMapper software v 3.7. Donor-specific products were distinguished from wild-type based on a 1-bp deletion, then confirmed by sequencing, as described previously. Deletions generated between Try1 and Try3 were identified by PCR with ry-7100-F and ry-9532-R (Table S1). Wild type chromosomes generate a band of 3 kb, whereas deletions generate bands of ~490 bp. The latter were sequenced as described previously.Mutations in the Pcd  and CG12200 (X-linked) were screened by standard PCR and sequencing by selecting six F1 animals from each fertile G0 that had been previously crossed to an appropriate balancer. In brief, DNA was extracted from single flies (Table S1). Pcd and CG12200 heterozygous females were identified as those whose sequence lost coherence at the target site, whereas CG12200 mutant males were identified by sequence. When a mutation was identified in any of the six F1 progeny, 30 individual lines were established from the brothers and/or sisters of the original fly by standard methods and then screened as before to identify the individual lines that carried the mutation.Mutations in Figure S1) . F2 flies carrying balancers were collected from vials scored as heterozygotes and stocks established. Mutations were confirmed by sequencing at the University of Utah Sequencing Core, over a deletion if possible. All primers used in these assays are listed in Table S1.Mutations in all other genes were scored as follows: Injected (G0) flies were collected and crossed to an appropriate stock as described previously. In 5−7 d, the G0 flies were recovered and DNA extracted as described  and yellow (y). In each case, one site overlapped a sequence that had been successfully targeted with ZFNs . Both the proportion of G0 flies that yielded mutants and the number of mutants were much lower than seen in a parallel experiment with ZFNs for the For each pair, truncation of the linker led to substantially greater mutation frequencies . In the One TALEN pair, yT2, was ineffective at inducing new mutations with either linker . We founy G0 animals were fairly evenly divided between males and females. As the y gene is on the X chromosome, it is easy to account for the y males, but the y females required biallelic disruption in somatic cells. The yT1 TALEN pair is clearly very active.Interestingly, somatic mutations were observed directly in some G0 animals that were injected with the most active TALEN pairs. Approximately 45% of the yT1-63−injected G0 adults were predominately yellow throughout their cuticle, wings and bristles. These parents accounted for 69% of all the mutant progeny in the F1 generation; however, there were six such animals that produced no mutant progeny, so somatic phenotype is not a perfect indicator of germline mutagenesis. Surprisingly, the phenotypically ry gene is not expressed in the eye. Its product, xanthine dehydrogenase, is synthesized in other tissues and transported into the eye  completely eliminated the lethality seen with each ZFN alone. With a single substitution in yA and the wild type cleavage domain in its partner, yB, rather good mutant yields were obtained  are very effective in eliminating the toxicity of individual ZFNs. This comes with a substantial decrease in the efficiency of cleavage and mutagenesis, however. Efficacy is restored with the second generation modifications, DDD/RRR, at least in the context of TALENs, and likely for ZFNs as well.In summary, in Drosophila, the first generation obligate heterodimer modifications of the Mutations arising from cleavage by both TALENs and ZFNs are caused by inaccurate repair by NHEJ. Despite relying on the same process, the two sources yield somewhat different spectra of sequence changes . In contA very common mutation recovered after ryAB ZFN expression was a 4-bp insertion that we attribute to fill-in and blunt joining of the 4-base 5′ overhang generated by cleavage . InsertiFigure S2). Insertion sizes differed only slightly between the nucleases .The distribution of deletion sizes also differed between ZFNs and TALENs. While both distributions were broad, the median was significantly larger in the case of TALEN-induced deletions . This wary gene allowed us to test whether simultaneous expression of both pairs would induce deletions between them, as has been observed in cell lines with ZFNs  mutations were scored as deletions. Six of these were sequenced, including two pairs of siblings. Five of the six deletions were unique, and all ten endpoints were within or just outside the TALEN spacers . Althouglig4 mutant flies. The donors were 111-base single-stranded oligonucleotides originally constructed for use with ry ZFNs . These oligos carry a single G deletion creating a frameshift that inactivates the gene. The oligos also carry substitutions on either side of the cut site that are useful for determining how much donor sequence was incorporated into the target chromosome. We tested two oligos, one homologous to the forward strand, and the other the reverse complement . Both oligos served effectively as donors, with outcomes very similar to those seen for ZFN cleavage, including HR being greater in the lig4 background than in wild-type flies  on a number of G0 flies to identify ones with somatic mutations and therefore more likely to produce mutant offspring . Offspring of these flies were analyzed individually by HRMA and by sequence analysis of PCR products that included the target site . Details of these analyses are provided in Table S3.Mutations were readily obtained with all but two of the 17 TALEN pairs tested, including the ry and y TALENs . It is dPsf2 and Sld5 were able to generate mutations effectively, as revealed by HRMA, but many of the G0 individuals died as pupae. We attribute this to the production of biallelic mutations in enough somatic cells to cause lethality. Reducing the concentration of TALEN mRNAs injected allowed us to recover viable adults, although many of these were sterile. It is also possible that off-target cleavage is responsible for these effects, although this was typically revealed as much earlier lethality in embryos or larvae with toxic ZFNs. This again highlights the effectiveness of TALENs.TALENs for Table S4). Five of the 10 genes were successfully mutated, as previously reported (Table S5), particularly ones with very promising in vitro binding properties . The Psf2A pair that succeeded used exactly the same Right member of the pair but had a shorter spacer and a Left member that was not obviously a better design. The failure of the yT2 pair was quite surprising, because ZFNs for essentially the same target worked quite effectively . Because other TALENs for this gene produced useful mutants, the failed pair was not pursued. It does not appear that any of the failures were due to the toxicity we have seen due to off-target cleavage with ZFNs. Although in some cases we saw lethality late in development, this was most likely due to biallelic disruption of an essential target gene. We saw no reduction in viability that could not be tied to the phenotype of the gene being targeted. This is consistent with results seen by others working with TALENs (yellow gene that was successfully targeted with ZFNs (The most surprising failure was the yT2 TALEN pair. It was directed to the same site in the ith ZFNs . The ryTA feature of TALEN-induced mutations that has been described in other cell types as well is that they are biased toward deletions, in preference to insertions (FokI cleavage domain produces the same end configuration whether it is linked to zinc fingers or to TALE modules. The difference could again reflect the longer, more flexible spacer requirements for TALENs, either more rapid degradation or multiple cuts, as discussed above.We were also surprised not to find products that correspond to a simple 4-base fill-in and blunt join among any of the TALEN mutations we sequenced. This was quite a common product of ryAB ZFN cleavage (The generation of chromosomal deletions and, potentially, other rearrangements, is of particular interest to the fly community. Similar techniques have been used in cell lines to generate deletions, duplications, inversions and translocations (In conclusion, we have found TALENs to be very effective agents for germline mutagenesis in Drosophila. We were able to produce targeted mutations in eight different genes, six of which had no previously described alleles. The mutation frequency was high enough that straightforward molecular analysis of G0 and F1 flies was adequate to identify and isolate the desired mutants. The TALEN platform has clear advantages over ZFNs, including ease of construction and a higher success rate. The emerging CRISPR/Cas RNA-guided nuclease technology has its own favorable characteristics, including ease of generating reagents, simple multiplexing, and high efficiency; and it has been applied successfully to Drosophila (


The blood neutrophil to lymphocyte ratio (NLR) has been identified as a potentially useful marker of clinical outcome in disease states with an inflammatory component. The objective of this study was to evaluate the relationship between NLR and clinical status in children with cystic fibrosis.This was a retrospective chart review. Data collected included NLR, body mass index, and forced expiratory volume in 1 second (FEV1) while asymptomatic, and during hospitalizations for pulmonary exacerbation. An NLR breakpoint of 3 was used for comparisons of body mass index and FEV1.A total of 159 charts were reviewed. An NLR ≥ 3 was significantly associated with lower body mass index and lower FEV1. NLR during hospitalization was significantly higher than NLR while asymptomatic. NLR measured during the first 3 months of life was negatively correlated with FEV1 at age 12.NLR correlates with clinical status in children with cystic fibrosis and may be a useful biomarker in this population. Pseudomonas aeruginosa, and neutrophil-dominated inflammation plays a role in the pathophysiology [Cystic fibrosis (CF) is an autosomal recessive genetic disease that affects approximately 30,000 people in the United States . Patientysiology -5. Over ysiology , but othThe blood neutrophil to lymphocyte ratio (NLR) has been identified as a potentially useful marker of inflammation that is prognostic of outcome in various disease states, including cardiovascular disease and gastrointestinal cancers -11. As iA total of 159 charts were reviewed. Of these, 126 subjects had a clinic visit with a corresponding CBC while clinically stable. There were 37 subjects hospitalized for a pulmonary exacerbation with a CBC measured within 2 days of admission. 14 subjects had an NLR of ≥ 3 while clinically stable. The age range for subjects with an NLR ≥ 3 was 9-18 years, and so only subjects 9 years of age or older were included for this analysis. Clinical measures compared by NLR breakpoint are presented in Linear regression demonstrated a significant relationship between NLR and nutritional status  with eacAmong the 37 subjects (mean age of 11.3±5.0) who had NLR measured both while clinically stable and during the first 2 days of hospitalization for a pulmonary exacerbation, there were 51 hospital admissions for pulmonary exacerbation. NLR during a pulmonary exacerbation was significantly elevated compared to NLR measured while clinically stable .th [NLR is significantly correlated with clinical status in children with CF. Children with an NLR ≥ 3 have significantly worse lung function and nutritional status. According to lung function stratification referred to in the CF pulmonary guidelines  and commth . In thisIn several other disease states with an inflammatory component, an elevated NLR has been implicated as a marker of poorer outcome. Patients undergoing resection for non-small cell lung cancer had a worse 5-year survival rate if their preoperative NLR was ≥ 2.5 . Among aOther biomarkers of inflammation in peripheral blood have been evaluated in patients with CF, notably C-reactive protein (CRP) and neutrophilic chemokines such as IL-8. CRP has been associated with clinical status in people with CF, but it is a general biomarker of inflammation and does not differentiate between inflammation driven by neutrophils versus other origins, such as eosinophils as in allergic inflammation. It is also produced in response to inflammation and thus an elevation in CRP is likely to be a later event than elevation in NLR. Studies have been conducted evaluating the correlation of neutrophilic chemokines to clinical outcome in patients with CF and the results are conflicting. Biomarkers in expectorated or induced sputum have also been evaluated for their utility in predicting disease severity as measured by FEV1; most notably IL-8, neutrophil count, and neutrophil elastase. Coefficients of correlation vary between studies, with a range of -0.28 to -0.72 for IL-8, -0.29 to -0.57 for neutrophil count, and -0.35 to -0.75 for neutrophil elastase -25. NLR Future studies should evaluate whether the associations between NLR and clinical status persist into adulthood. It would also be important to establish whether the NLR changes in response to pharmacotherapy and if this results in a change in clinical status.This was a small retrospective study and thus has the associated limitations. The correlations with lung function and nutritional status are from cross-sectional data, and thus no conclusions can be drawn regarding how the timing of an elevated NLR is related to clinical status. Markers of inflammation are not routinely measured at our institution at clinic visits or during hospitalization, and thus were not available to compare to NLR. A larger prospective study is necessary to establish the utility of NLR as a biomarker in children with CF and the appropriate breakpoint. It is also challenging to evaluate its usefulness in young children with CF who are unable to perform pulmonary function testing. Finally, there has not been a methodical investigation to establish a reference range for NLR in healthy subjects and current studies evaluating the utility of this marker in various disease states have determined breakpoints based on a comparison of outcomes. It is possible that results would be different if compared to a healthy cohort.This retrospective study was approved by the University of Arkansas for Medical Sciences Institutional Review Board. Informed consent was waived by this same Institutional Review Board as this was a retrospective study and all data collected was preexisting.This was a retrospective chart review of all pediatric CF patients followed at the Arkansas CF Center. Subjects were eligible if they had a diagnosis of CF and at least one hospitalization for pulmonary exacerbation or one clinic visit between October 2010 and October 2011 with a corresponding complete blood count (CBC). Subjects had to be clinically stable at the clinic visit (i.e. no diagnosis of a pulmonary exacerbation). The CBC during the hospitalization had to be within 2 days of admission.Data collected included general demographics, CBC at the clinic visit and the first CBC measured during the first 3 years of life, lung function  at the clinic visit and at 7 and 12 years of age, nutritional status (as measured by body mass index (BMI) percentile) at the clinic visit, and CBC measured during the first 2 days of hospitalization for pulmonary exacerbations. If patients were diagnosed with a pulmonary exacerbation at the clinic visit, they were not considered “clinically stable” and the CBC with corresponding BMI and FEV1 were not collected.The first objective of the study was to evaluate the relationship of NLR measured while clinically stable (i.e. at the clinic visit) to clinical status. Clinical status was evaluated by two measures: nutritional status as measured by BMI percentile (weight-for-length percentile was used for subjects less than 2 years old) and FEV1. The second objective was to compare NLR while clinically stable to NLR upon hospitalization for a pulmonary exacerbation. The third objective was to explore the possible utility of NLR as a predictor of later outcome. For this objective, the relationship between NLR measured during early childhood (the first 3 years of life) was compared to FEV1 at 7 and 12 years of age. The age of 7 was chosen because younger children are frequently unable to reliably and consistently perform pulmonary function testing until approximately 6 years of age. The age of 12 was chosen because it provided the best likelihood of having access to both an early CBC and a later FEV1 in the electronic medical record.NLR was calculated from values collected from the CBC by dividing the absolute neutrophil count (both segs and bands) by the absolute lymphocyte count.Descriptive statistics were used to describe the study population. A breakpoint of 3 for the NLR was used to compare clinical measures FEV1, BMI/weight-for-length, and exacerbation frequency. As this was a pilot study, there was no preliminary data in CF subjects from which to choose an appropriate breakpoint for comparison of measures of clinical status. A breakpoint of 2.5 is the lowest published breakpoint that is significantly associated with clinical outcome -15. We aThe NLR is an easily collected and measured biomarker that correlates with clinical status in children with CF and may be indicative of conditions favorable to tissue damage in the CF lung. A breakpoint of 3 may be reasonable; however, larger prospective studies are needed to establish its utility as a biomarker that may be predictive of clinical outcome in this population.


Acute exacerbation of chronic bronchitis (AECB) is a commonly encountered problem and those suspected to be due to bacterial infections require antibiotic therapy. This randomized, controlled trial was designed to evaluate the effectiveness and safety of gemifloxacin, a new fluoroquinolone, versus cefpodoxime, an oral third-generation cephalosporin, for the treatment of mild to moderately severe cases of AECB.Adult subjects diagnosed with chronic bronchitis with clinical symptoms suggestive of an Anthonisen type II acute exacerbation  were eligible and those fulfilling the subject selection criteria were randomized to receive either gemifloxacin 320 mg once daily or cefpodoxime 200 mg twice daily orally for 7 days. The primary outcome measure was clinical success rate at day 14 visit and the secondary outcome measures were changes in Clinical Global impression (CGI) scales and incidence of adverse events (AEs). Fifty-two subjects were enrolled: 26 in gemifloxacin group and 24 in the other and 2 were lost to follow-up.The clinical success rates were comparable (84.6% in gemifloxacin group versus 83.3% in cefpodoxime group) and no statistically significant difference was observed between the groups. AEs were mild, self-limiting and few (two in gemifloxacin and three in cefpodoxime arm) and tolerability was also good.The results of this randomized, single-blind trial demonstrated that a 7-day course of gemifloxacin is therapeutically comparable to cefpodoxime in terms of both clinical effectiveness and safety for the treatment of type II Anthonisen category AECB patients. Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, and the less common ones include nonenteric, gram-negative organisms such as Pseudomonas aeruginosa.[Acute exacerbation of chronic bronchitis (AECB) is a common but serious respiratory tract ailment associated with significant morbidity and impact on healthcare costs. Bacterial infections are an important cause of AECB. Where bacterial infections are suspected, early institution of antimicrobial therapy and supportive measures ensure quicker recovery.–3 The orruginosa.2 The comruginosa.5 Mild toruginosa.–6in vitro activity against S. pneumoniae with lower minimal inhibitory concentration (MIC) values than the other respiratory fluoroquinolones.[H. influenzae, M. catarrhalis) and atypical pathogens, such as Chlamydia pneumoniae, Legionella pneumophila, and Mycoplasma pneumoniae, are however comparable.[Gemifloxacin, a newer generation fluoroquinolone, has pharmacokinetic and pharmacodynamic properties similar to other members of the class but has demonstrated better inolones.5 Antimicmparable.5S. pneumoniae, H. influenzae, M. catarrhalis and some action against anaerobic organisms as well. There are a few studies which have evaluated the effectiveness of cefpodoxime for various respiratory tract infections and results have shown that it can be considered as an effective agent for the treatment of AECB.[Cefpodoxime, an oral third-generation cephalosporin, has good antimicrobial activity against aerobic gram-positive and gram-negative bacteria like  of AECB.7Medline search revealed comparative clinical trials on gemifloxacin versus other fluoroquinolones or macrolides/amoxicillin–clauvulanic acid in patients of AECB and evaluated its effectiveness both clinically and bacteriologically,[ogically,–7 but tiThe primary objective of this trial was to demonstrate equivalence between gemifloxacin (test) and cefpodoxime (comparator) with respect to their efficacy  in mild to moderately severe cases of AECB.The secondary objective was to make a comparative assessment of their safety and tolerability profiles.This was a prospective, randomized, single-blind, controlled, phase IV (post-marketing) trial. Subject recruitment was done from the Respiratory Medicine outpatient department of the teaching hospital. All study-related documents were approved by the Institutional Ethics Committee and the trial was conducted in accordance with the ICMR guidelines for Biomedical Research on Human Subjects, 2006, and the Declaration of Helsinki.This clinical trial was designed to demonstrate equivalence in the efficacy  between the two treatment arms. The largest clinically acceptable effect for which equivalence could be declared was assumed as a difference in clinical cure rates of 10% . Considering the true mean difference between the treatments as zero and the expected standard deviation of 10% in the trial population, 90% power and α = 0.025, the number of evaluable subjects required in each treatment arm was 26. The sample size was calculated using standard formula for sample size calculation for clinical trials.Anthonisen criteria:[Adult subjects of either sex, in the age group 25 to 70 years and clinically documented cases of chronic bronchitis, presenting with clinical symptoms and signs suggestive of acute exacerbation of the disease (type II Anthonisen) due to bacterial pathogen, as evidenced by the presence of at least two of the following three criteria: exacerbaFemale patients who were pregnant or lactating or cases of AECB who had severe disease requiring hospitalization or parenteral antibiotic treatment, or suspected or proven cases of pneumonia, bronchial asthma, pulmonary tuberculosis or tubercular pleural effusion, lung cancer or lung metastasis, bronchiectasis, interstitial lung disease or patients who had a course (3 days or more) of antibiotic for respiratory ailments in the preceding 4 weeks of screening or chronic respiratory insufficiency associated with resting hypoxemia or baseline respiratory symptom score <6 and >12 or presence of comorbidities or hypersensitivity to penicillin or any of the study medications were excluded.Randomization of subjects who fulfilled the selection criteria was done using computerized random number list with recruitment rates in both arms in a 1:1 ratio. Allocation concealment was done by having the random number in an opaque sealed envelope.Although this was a single-blind trial, where the investigator who clinically assessed the subject was unaware of the treatment allocation and medication, dispensing was done by an independent member who was not part of the study evaluation team.Percentage of subjects achieving “treatment success” in each treatment arm was the primary efficacy parameter. Treatment success was defined on the basis of changes in the respiratory scores at day 14 visit and was subdivided as either (a) clinical cure if the respiratory symptom score was <5 at day 14 visit or (b) clinical improvement if the respiratory symptom score was at least one score less than the baseline score or between 6 and 10.Changes in clinical global impression (CGI) scales on a 5-point scale with 1 as the worsened state and 5 as the very much improved state were the secondary efficacy parameters. CGI denoted overall clinical assessment of patient’s condition by the physician and was noted during follow-up visits and at the end of the study. CGI was categorized on a 5-point scale with 1 as the worsened state, 2 as no improvement, 3 as mild improvement, 4 as moderate improvement and 5 as very good improvement state.Treatment failure was defined as failure to respond to the trial medication and thereby requiring modification of the antibiotic therapy or parenteral antibiotics. A subject was categorized as “treatment failure” if there was no change or increase in the baseline respiratory symptom score at day 14 visit.The total duration of the study was 2 weeks; the first 7 days were active treatment period, followed by 7 days of “treatment-free follow-up”. All subjects were evaluated at baseline and at all subsequent follow-up visits  clinically and the respiratory symptom score  was computed. Spirometry, serum creatinine, urea, plasma sugar, hemoglobin, total leukocyte count differential leukocyte count, erythrocyte sedimentation rate (ESR), platelet count and chest X-ray were performed at screening and at study end visit.Group A received gemifloxacin tablet (320 mg), while group B received cefpodoxime tablet (200 mg).Study medications were dispensed once at baseline visit for 3 days and subsequently at the first follow-up visit for the next 4 days. Patients were instructed to take one tablet of their respective study medication  after food for the first 7 days. Compliance was assessed by traditional “pill-count” method at each follow-up visit and at the end of the study.During the study period, subjects were not allowed to take any other systemic antibiotic or indigenous medicines for any medical or surgical cause. However, in cases of treatment failure or worsening of clinical condition, the subject was withdrawn from the trial prematurely. Bronchodilators like beta 2 agonists  by inhalational route, anticholinergics, theophylline derivatives and anti-inflammatory agents like inhalational corticosteroids were allowed. All the subjects participating in the trial were advised to stop smoking, and breathing exercises were advised for all subjects during the study period.Safety monitoring was done continuously throughout the study. All adverse events (AEs) spontaneously reported by the subjects or elicited by the investigators were recorded and causality analysis was done. Any significantly abnormal deviation of baseline investigation report values was considered as an AE and recorded as such.Efficacy analysis was done using modified intention-to-treat basis for subjects reporting for at least one post-baseline follow-up visit. All subjects who were randomized were considered for safety analysis. Missing values were dealt with by the last observation carried forward strategy. Parametric tests were used for comparison of normally distributed numerical variables. Categorical data were compared between groups by Chi-squared test or Fisher’s exact test, as appropriate. Repeated measure non-parametric data within group analysis was done by Friedman’s analysis of variance (ANOVA) and between group analysis was done by Mann Whitney U test.Out of 62 subjects screened, 52 fulfilled the selection criteria and were randomized – 26 to group A (gemifloxacin) arm and 26 to group B (cefpodoxime). However, two subjects were lost to follow-up in group B and did not attend the hospital after the first visit. The protocol specified sample size of 26 “evaluable” subjects could be achieved in group A only within the study period. The mean age of patients was 59.6 ± 6.7 years and 59.2 ± 8.5 years in the gemifloxacin and cefpodoxime groups, respectively. 80.7% were males in the gemifloxacin group and 87.5% were males in the other group. The duration of chronic bronchitis at screening was 7.92 ± 5.02 and 8.95 ± 4.4 years in the gemifloxacin and cefpodoxime groups, respectively.There was no statistically significant difference in the baseline demographic profile, smoking status and disease related profile (baseline symptom score and duration of chronic bronchitis at screening).P values included in table), denoting that there was a clinically significant improvement in the signs and symptoms of the acute exacerbation episode of the disease. Therefore, it can be concluded that both gemifloxacin and cefpodoxime are effective antibiotics for the management of AECB. A between group analysis of the symptom scores showed that there was no statistically significant difference in the baseline, first follow-up and end of study scores in the respiratory symptom score. A statistically significant difference was noted in the second follow-up visit (P = 0.04). Since in majority of the visits there was no difference, it can be concluded that both gemifloxacin and cefpodoxime are equally effective antibiotics for the management of AECB.The changes in the respiratory symptom score from baseline values are enlisted in P = 0.90). Changes in CGI assessed by the physician were noted in a 5-point Likert scale at each visit and the results at the study end visit showed that 84.6% of the subjects in gemifloxacin group and 83.3% in cefpodoxime group achieved a score of either 3 or 4 . There was no statistically significant difference (P = 0.85) of the study end CGI scores in between group comparison. The subject compliance of both the groups was comparable and majority of the subjects showed excellent compliance. There were no subjects who were categorized in the “poor” compliance group.There were four subjects in each group who were categorized as treatment failure and had to be put on other antibiotics after day 7 evaluation. The percentage of treatment failure rates in the two arms was not statistically significant  analysis. Only five AEs were noted during the entire study period - three AEs in cefpodoxime group, which were mild diarrhea, and two AEs in gemifloxacin group, one case each of rash and dizziness. These AEs were non-serious and mild in nature and did not require any dose reduction or withdrawal of the study medications. Causality analysis using the World Health Organization-Uppasala Monitoring Centre (WHO-UMC) criteria showed tP = 0.75).The use of concomitant medications in both the groups was comparable and no statistically significant difference was noted between groups. Most of the subjects had taken inhalational salbutamol or levo-salbutamol, ipratropium bromide, and few had taken oral doxophylline or theophyline. There were three diabetics and one hypothyroid patient in gemifloxacin group and two diabetics in cefpodoxime group. However, the percentage of subjects with concomitant illnesses in the two treatment arms was comparable  with macrolides  or with amoxicillin/clavulanate acid for the treatment of AECB,–16 but tThe results of this study were comparable to that of other published studies which evaluated gemifloxacin or cefpodoxime for the treatment of AECB using clinical improvement treatment outcomes. The clinical success rate with the newer quinolones was about 83–84%, which is comparable to our study results. Regarding cost effectiveness of a 7-day course of gemifloxacin versus cefpodoxime, we computed the current costs of both therapies by comparing the market prices of the test brand versus the control brand. Results revealed that gemifloxacin therapy was significantly less expensive  compared to cefpodoxime therapy.et al. conducted an randomised clinical trial (RCT) of gemifloxacin versus clarithromycin in Anthonisen type I AECB subjects and the reported clinical cure rate at 2-3 weeks was 85.4% with gemifloxacin.[et al.[Wilson floxacin. Another floxacin. The “Gemfloxacin. trial whn.[et al. comparedSome limitations of this study were as follows. As this was an investigator initiated academic project, a double-blind study could not be conducted due to financial constraints and logistic problems. The dosing schedule of cefpodoxime was twice daily while that of gemifloxacin was once daily. So, for conducting a double-blind study, supply of dummy medications was essential but it could not be arranged. However, to compensate for the bias, the investigator who assessed the subjects clinically and computed the respiratory symptom score was not aware of the subject allocation group. Secondly, we did not perform microbial efficacy of the cases since there are several reports which have stated that mere identification of organisms from the expectorated sputum is not representative of the organism causing AECB as the specimen gets contaminated by the upper airway and laryngeal commensals. Another reason is that very often clinicians start antimicrobial therapy at outpatient setting before the microbial culture report arrives which takes about 72 hours. Therefore, we conducted this study mainly to provide information to clinicians on the comparative effectiveness of these two antibiotics as initial antibiotics for AECB patients based on clinical assessment scores. Third, a prolonged follow-up of subjects to compute the relapse rates also was not done formally as part of the study but we have requested all subjects to come for monthly follow-ups for 6 months after study end visit.The results of this study demonstrated that a 7-day course of gemifloxacin is comparable to cefpodoxime in terms of both clinical effectiveness and safety for the treatment of AECB in an outpatient setting. Gemifloxacin has the additional advantage of a once daily dosing compared to a twice daily dosing for cefpodoxime and a lesser cost of therapy. However, a trial that also evaluated bacteriological cure and relapse rates would have provided supportive scientific evidence.


Arabidopsis thaliana roots growing on tilted hard agar surfaces. Under these conditions, the roots are exposed to both gravity and touch stimulation. Consequently, they tend to skew their growth away from the vertical and wave along the surface. This complex growth behavior is believed to help roots avoid obstacles in nature. Interestingly, A. thaliana accessions display distinct growth patterns under these conditions, suggesting the possibility of using this variation as a tool to identify the molecular mechanisms that modulate root behavior in response to their mechanical environment. We have used the Cvi/Ler recombinant inbred line population to identify quantitative trait loci that contribute to root skewing on tilted hard agar surfaces. A combination of fine mapping for one of these QTL and microarray analysis of expression differences between Cvi and Ler root tips identifies a region on chromosome 2 as contributing to root skewing on tilted surfaces, potentially by modulating cell wall composition.Plant root systems must grow in a manner that is dictated by endogenous genetic pathways, yet sensitive to environmental input. This allows them to provide the plant with water and nutrients while navigating a heterogeneous soil environment filled with obstacles, toxins, and pests. Gravity and touch, which constitute important cues for roots growing in soil, have been shown to modulate root architecture by altering growth patterns. This is illustrated by  Healthy roots take on an architecture that securely anchors the plant in soil and allows for dynamic growth toward adequate water and nutrients. The heterogeneous nature of soil leads to gradients in elements that the plant may need to acquire or avoid, and obstacles such as rocks must be bypassed to continue a downward trajectory. These environmental inputs combine with intrinsic developmental cues to determine root growth behavior. Understanding the genetics behind primary root elongation and how it is affected by various environmental stimuli may help in the breeding of crops better suited to their local soil conditions.A common way to observe root growth behavior is to germinate seedlings on a hard agar surface in a petri dish. The root is more easily observed than it would be in a soil environment, and the hard surface provides an obstacle as the roots grow along it, similar to that which a rock may present to a root in its natural environment. Also, if the plate is tilted back, the root tip will attempt to grow downward along the gravity vector. However, the hard medium is impenetrable, therefore, gravitropism will press the root tip against the agar causing stronger touch stimulus. The combined effect of gravity and touch stimuli on the root tip, along with other surface-derived stimuli and intrinsic cell machinery, cause characteristic root growth behaviors under these conditions , the degree of which correlates with the level of skewing. The handedness of the CFR also correlates with the direction of skewing: left- handed CFR for rightward skewing and right-handed CFR for leftward skewing . Rightward skewing is often accompanied by a left-handed rotation of the root tip about its axis, visible as a left-handed rotation of the epidermal cell files. The degree of skewing varies in wild-type roots of surfaces . Mutants skewing . HoweverA. thaliana interphase cells in the epidermal and lateral cap layers of the root elongation zone, cortical microtubules are arranged mostly in a transverse orientation. This arrangement correlates with the cells in this region expanding anisotropically, in a direction perpendicular to the transverse arrays  results in distinct phenotypes when introgressed into different genetic backgrounds  and Landsberg erecta (Ler)  population created from the er-2 (CS8581), and Cvi/Ler recombinant inbred lines (RILs) (CS22000). This population consists of 162 inbred lines that were genotyped at 293 marker loci (er plants were propagated side by side in a soil composed of Jiffy mix and vermiculite (4:1 volumetric ratio), using a 22°C growth room with a 16/8-hr light/dark cycle, to bulk seeds for analysis. Light was provided by cool-white fluorescent bulbs at a fluence rate of 35-50 µmol⋅m−2⋅s−1. Seed stocks used in fine mapping include near isogenic lines (NILs) HGI2.1, 0.2, 0.3, 0.4, and 0.5 provided by Julin Maloof , were provided by Leonie Bentsink : Cvi-1 (CS8580), LA. thaliana roots. Seeds were surface sterilized with 20% bleach + 0.01% SDS solution then rinsed four times with water. The seeds were sewn on the surface of the medium, and plates were sealed with micropore tape . Seeds were stratified on the plates for 4-6 days at 4°C, wrapped in two layers of aluminum foil, to overcome the dormancy phenotype of Cvi. Plates were then placed in a vertical orientation in either a Conviron TC16 growth chamber  for QTL trial 1, or in an Enconair AC60 growth chamber  for all other trials. The temperature was set at 22°C in both chambers. Light was provided by cool white fluorescent tubes at a fluence rate of 45-50 µmol⋅m−2⋅s−1 in the Conviron chamber , and 80-125 µmol⋅m−2⋅s−1 in the Enconair chamber , following a 16/8-hr light/dark cycle. Seedlings were grown under these conditions for 3 days. Then, the locations of the root tips of all germinated seedlings were marked with a black felt-tip marker on the back of the plate, and the plates were tilted back at a 30° angle. After 2 days of growth at the 30° tilt, images were taken of the roots through the medium from the back of the plates with a digital camera.A 1/2 strength LSPS medium  was supplemented with 1.5% agar type E  and poured into square petri dishes to form a hard medium mostly impenetrable to Image (http://rsb.info.nih.gov/nih-image/) version 1.62 was used to determine the following parameters as diagrammed in 2) was calculated by dividing the latter by the former [(variance within RILs)/(variance between RILs)].NIH er seeds on the other. The plates were positioned in the Conviron TC16 chamber and exposed to the wave-assay conditions described above. For QTL trials 2 and 3, nine seeds of the particular RIL or parental control line were placed in one row near the top of the plate. These trials were performed in the Enconair AC60 chamber, also as described above. Plate arrangement was randomized in the growth chamber. For all trials, photographs were taken on day 5, and the roots were measured with NIH Image version 1.62. It should be noted that only the top two rows of seedlings were used for QTL analysis in trial 1 as the growth parameters of Cvi and Ler were found to be significantly different for the top and middle rows vs. the bottom row (P < 0.01), possibly due to lower light intensity on the bottoms of the plate. Means for each trait along with genotypic data . For WinQTL Cartographer, maps were created using the Kosambi map function  was based on multiple sources including sequences provided by Julin Maloof , polymorphisms listed on TAIR (http://www.arabidopsis.org), polymorphisms described by Monsanto/CEREON between Columbia and Landsberg erecta  per tube, shaken for 30 sec at 20 beats per second. RNA extraction of the ground tissue was carried out with the RNAeasy kit (Qiagen). RNA yields were 19–32.5 μg, quantified on a Nanodrop spectrophotometer .Three biological repeats with two processing replicates each were plated for the wave assay for the following lines: Cvi, Ler, and HGI2.1, was used for microarray hybridization. The hybridization was carried out by the Gene Expression facility at the University of Wisconsin–Madison. RNA integrity was verified with the Agilent 2100 BioAnalyzer . Biotin-labeled cDNA was generated using the MessageAmp II kit . For each sample, the labeled cDNA was hybridized overnight on an ATH1 GeneChip  in an AFX HybOven 480 (Affymetrix). Post processing was done on an automated Fluidics 450 Station (Affymetrix), and the chips were scanned with the GC 3000 G7 scanner (Affymetrix). The scanned images were preprocessed with the AFX Expression Console software. Primary data are publicly available at Gene Expression Omnibus .One of the above technical replicates, consisting of three biological repeats each of Cvi, Lt-test option was employed with a FDR (Benjamini-Hochberg correction) multiple testing correction algorithm. From the output, expression differences between the samples being compared were requested at 90%, 95%, or 99% confidence levels.Quality of microarray hybridization was determined using the Affymetrix MAS5 algorithm. Expression differences were analyzed using ArrayStar 3 . The data were preprocessed using RMA and a quantile normalization method. For statistical comparisons of expression levels, the Student’s Materials and Methods), different A. thaliana accessions show a variety of root skewing and waving phenotypes (erecta (Ler) were chosen for the QTL mapping study because of their distinctive root morphologies on the wave assay, and because a genotyped RIL population was available for them at the time we initiated these experiments. Cvi has a strong skew to the right (viewed from the back of the plate) and no structured pattern of waving, whereas Ler has a weaker right skew and a more regular pattern of waving.When grown on tilted hard agar during the wave assay  . The greater the angle or the greater the HGI, the greater the skew of the root to the right. We found these two measurements to be highly correlated (R2 = 0.93), and in this study we will predominantly use HGI as the indicator of root skew. Along with length and HGI, we also calculated vertical growth index (VGI) and the straightness of the root along the chord of growth (Lc/L) . Straightness is a rough measure of root waving since the more a root waves, the less direct its path along the axis of growth, Lc, which lowers the Lc/L ratio.Although the skewing and waving differences between Cvi and Ler as indicated by HGI, and they grow straighter along their paths as indicated by a higher Lc/L value. To get an idea of the dominance of the alleles contributing to skewing and waving in Cvi and Ler, crosses were made to create heterozygotes. For skewing, the F1 seedlings from Ler × Cvi display a mean root HGI that is intermediate to both parent lines and significantly different from both  population was subjected to the wave assay for three trials. Of the available 162 RILs, 149, 153, and 156 individuals were used in trials 1, 2, and 3, respectively, due to poor germination of some of the lines. Measurements made on the seedling roots include root length, HGI, VGI, angle B, and straightness. For each of these phenotypes, RIL means showed transgression with respect to parental means, and broad sense heritabilities ranged from 0.29 to 0.62 . Both of these factors indicate the population is well-suited to QTL mapping for these traits.Differences between Cvi and LFigure S1, Table S2). Many of the QTL were consistently significant in all three trials. Two-dimensional (2D) scans were also performed on the traits for all three trials. As a whole, the 2D scans were relatively consistent from trial to trial for the additive portion, and consistent with the composite interval maps. The epistatic interaction portions of the graphs were more variable among the three trials. 2D scans for trial 2 for all the traits are presented here  as well as the 2D scans for trials 1 and 3 .The phenotype information we compiled from these measurements was paired with the genotype information already generated by er, we also evaluated the means of the traits for each of the three trials with respect to RIL cytoplasm donor. 117 of the RILs of the Cvi/Ler population were created using Cvi as the maternal parent, and 45 RILs have Ler as the maternal parent . It is surprising that HGI showed no significant differences with respect to cytoplasm in the RILs considering Cvi and Ler reciprocal heterozygotes had shown a possible maternal effect for this trait , and the fact that no known tubulin or microtubule-associated proteins resided in the predicted 2-LOD interval. The additive effects map shown at the bottom of the QTL-map graph .We decided to focus on fine mapping the QTL for HGI due to our interest in skewing and the insights mutant studies have given us into the involvement of microtubule dynamics and cell wall composition, among others, in modulating this behavior. For HGI, all three trials predicted significant QTL in the same positions on chromosomes 2, 4, and 5 . In partap graph  indicateer background. For the QTL at the end of chromosome 4, LCN4-6 is weakly significant for greater skew than Ler (P = 0.05) as would be predicted if its Cvi introgression overlaps with the causative locus. However, LCN4-7 . This result may narrow the candidate interval for this QTL. However, it will be difficult to further map the causative locus because its effect on skewing is barely above significance threshold.A series of NILs was used to analyze the effects of Cvi segments on skewing in the three HGI QTL regions on chromosomes 2, 4, and 5. The NILs have Cvi segments of variable lengths introgressed into the area of interest in an otherwise Ler for HGI and several of the other measured traits (Table S3). This would be predicted as all of the traits measured over three trials had significant QTL peaks for this region of chromosome 5 , and for some epistatic interactions are predicted to affect some of these traits . This region seems to be a very complex contributor to root growth behavior, and further dissection is needed to narrow the regions contributing to the phenotypes and their relative additive and interactive effects.For the QTL centered around position 78-80 on chromosome 5, some the DOG17 NILs .For the chromosome 2 HGI QTL, NILs HGI2.1, 2.2, 2.3, 2.4, and 2.5  and used them to define the breakpoints between introgressed Cvi and Ler segments in these NILs. The combination of this genotypic data with the phenotypic observations for HGI of the NILs indicate the causative QTL is located between 9.3 and 11.2 Mb . Of these sets, nine fell in the interval on chromosome 2 between 9.3 and 11.2 Mb, with a twofold or more change in expression level between the accessions  documents that this accession was first collected growing on a “rocky wall with moss.” It may be that the skewing pattern its root takes along the hard agar surface reflects an adaptation to this type of soil environment where the root must grow over long spans of rocks for periods of time.We observed a great deal of variation in the root growth behaviors of Arabidopsis thaliana. The accessions Cvi and Ler differ in both skewing and waving, and so were good candidates for use in a QTL study to try and determine the genetic contributors to these differences. Through QTL mapping with the Cvi/Ler RIL population, we have pinpointed several chromosomal regions predicted to modify root skewing and waving behaviors, as well as root length. A QTL for skewing on chromosome 2 is being pursued by fine mapping and expression analysis of the candidate interval.Our goal was to use natural genetic variation to find regions of the genome that contribute to root growth behaviors on hard surfaces in er. Using Cvi as the female led to significantly greater skewing than that seen in crosses with Ler as the female parent. In the QTL trials, only trial 3 showed any significant differences for trait means with respect to RIL cytoplasm donor. Because three traits were affected by maternal lineage in this trial, but not in the others, it may be that the experimental conditions of trial 3 were such that they exposed environmentally responsive maternal contributors to root behaviors.For the traits we studied, many of the QTL were repeatable over three trials. QTL that did not appear in all studies are more likely to be subject to environmental and experimental variances, or are simply false positives. The differences could also be due to maternal effects that are affected by environmental conditions. Our results indicate there may be a cytoplasmic component to some of the traits we evaluated. Root skewing differed between reciprocal heterozygotes of Cvi and Ler cytoplasm, so there should be no maternal effects on skewing between them and Ler. This was seen in reciprocal crosses between Ler and HGI2.1.We focused our efforts on characterizing the loci contributing to the difference in root skewing, in particular a region centered near 40 cM on chromosome 2. With the help of two sets of NILs, the candidate region has been narrowed to the area between 9.3 and 11.2 Mb. All of the NILs have LTable S4) and should help us further locate genes that contribute to this QTL. Preliminary studies of this region indicate the possibility of multiple loci contributing to the QTL, acting in an additive fashion. We have looked at the wave assay phenotypes of several mapping population lines with Cvi introgressions of various lengths, broken down from the HGI2.1 introgression. Lines with nonoverlapping segments of Cvi in both the proximal and distal portions of the introgression have shown significantly greater HGI than Ler. Also, the lines with a proximal or distal segment allowing for skew greater than Ler usually skew significantly less the HGI2.1, indicating both segments are needed for the greater HGI seen in HGI2.1. The hypothesis that multiple regions on chromosome 2 are contributing to greater skew over Ler is also supported by the data from NIL2-7 and 2-9. As mentioned in results, LCN2-7 consistently showed a greater skew than Ler over five trials, and LCN2-9 had a significantly greater skew than Ler in two of these trials. So, along with the region between 9.3 and 11.2 Mb, a more distal segment on chromosome 2 that is contained in the Cvi introgression of LCN2-9, but not LCN2-8, appears to be contributing additively to root skewing on surfaces.Further fine mapping of the candidate region is underway. From the over 1300-line mapping population, we have identified 143 F3 lines that have a crossover in this interval  are based on Columbia sequence, so probe-binding differences could also be due to polymorphisms changing hybridization efficiency. This may be helpful to identify polymorphisms within regions between the accessions. In the candidate region for the chromosome 2 QTL, nine genes are predicted to be differentially expressed.A microarray study using root tips from Cvi, LSKU5-similar gene, SKS16, and an extra large G protein XLG1. Both sku5 and the G protein mutants xlg3 and agb1 have skewing phenotypes (sks16 T-DNA insertion lines did not show any discernable skewing phenotype (data not shown), and xlg1 does not have a skewing phenotype as a single mutant . One branch of this pathway produces dolichyl-phosphate-mannose, which is used for the GPI-anchoring of proteins, among other things , is predicted to glycosylate SKU5 .To begin the analysis of changes that may exist between Cvi and Ler root tips, including additional potentially interesting candidates for this QTL. Further mapping and transgenic-rescue experiments will be needed to address this possibility.It should be cautioned here that the Affymetrix ATH1 microarray contains probes for only 376 of the 591 annotated genes present on the QTL interval we mapped on chromosome 2. Therefore, it is also possible that other, yet unidentified genes within that interval are differentially expressed between Cvi an LA. thaliana root skewing and waving behaviors. A region on chromosome 2 between 9.3 and 11.2 Mb, which has no characterized skewing mutants, contributes to greater rightward skewing on tilted hard agar plates in Cvi compared to Ler. Further experiments are underway to characterize the causative locus or loci, and preliminary analyses indicate two genes involved in dolichol/dolichyl biosynthesis may be contributing to the skewing difference, possibly by changing cell wall composition. The use of natural genetic variation to gain a greater understanding of how roots respond to gravity, touch, and other tropic stimuli may benefit us in the breeding of plants more suited to particular soil environments. As we try to produce high-yielding crops in diverse conditions, our knowledge of root growth behaviors and their modulation by external cues can help us in the endeavor.This study has shown that natural variation can be harnessed to give us new information on


Infection of healthcare workers with the severe acute respiratory syndrome–associated coronavirus (SARS-CoV) is thought to occur primarily by either contact or large respiratory droplet transmission. However, infrequent healthcare worker infections occurred despite the use of contact and droplet precautions, particularly during certain aerosol-generating medical procedures. We investigated a possible cluster of SARS-CoV infections in healthcare workers who used contact and droplet precautions during attempted cardiopulmonary resuscitation of a SARS patient. Unlike previously reported instances of transmission during aerosol-generating procedures, the index case-patient was unresponsive, and the intubation procedure was performed quickly and without difficulty. However, before intubation, the patient was ventilated with a bag-valve-mask that may have contributed to aerosolization of SARS-CoV. On the basis of the results of this investigation and previous reports of SARS transmission during aerosol-generating procedures, a systematic approach to the problem is outlined, including the use of the following: 1) administrative controls, 2) environmental engineering controls, 3) personal protective equipment, and 4) quality control. During the global spread of severe acute respiratory syndrome (SARS)  with primers specific for SARS-CoV , worn by some of the healthcare workers involved in the resuscitation attempt, was conducted to estimate the operating parameters, including particle removal efficiency and air-flow rate. A Met One Model 227B Hand-Held Particle Counter  was used to count ambient particles outside and inside the hood; five replicates were collected for each condition over a 1-minute sampling period. All information was obtained as part of an ongoing joint investigation into the cause of the second phase of the Toronto SARS outbreak conducted by Toronto Public Health, Health Canada, and the Centers for Disease Control and Prevention . She was admitted to the hospital and placed in respiratory isolation on the SARS unit. Progressive respiratory failure later developed in the patient, and within 72 hours of admission, she required 100% supplemental oxygen. On May 28, 2003, she was found to have no vital signs and cardiopulmonary resuscitation was attempted.Nine healthcare workers participated in the resuscitation attempt. Three ward nurses (RN1–3) were the initial responders . RN1 perAll nurses in the room during the resuscitation were wearing protection equipment that was considered standard for routine SARS patient care at this hospital. This equipment consisted of two gowns, two sets of gloves, goggles, a full-face shield (with the exception of RN1 and RN2), shoe covers, hair cover, and NIOSH-approved N95 disposable respirators that were not fit-tested. In addition, all nurses involved in the resuscitation were experienced in working on SARS units and thus familiar with the recommended infection control policies and procedures. In contrast to the nurses, both RTs and the MD were wearing T4 Personal Protection Systems during the resuscitation. All nurses left the room immediately after the intubation and removed their protection equipment following the standard hospital protocol. Approximate exposure times are outlined in Table.On May 31, 2003, both ICU-RN1 and ICU-RN2 had a temperature >38.0°C, myalgia, and malaise. In addition, ICU-RN1 complained of headache and nausea, and ICU-RN2 reported dyspnea. ICU-RN1 had a normal chest radiograph results, but the radiograph of ICU-RN2 showed a left lower lobe infiltrate that persisted for several days. Both RNs were admitted to the hospital for observation; their condition remained stable. RN3 reported a headache and myalgia on June 1, 2003, but her maximum temperature reached only 37.8°C. She remained in home quarantine, and her symptoms resolved without further progression. Results of RT-PCR performed on nasopharyngeal swabs from ICU-RN1 and ICU-RN2 were negative .Evaluation of the Stryker T4 Personal Protection System indicated an average removal efficiency of 68% for particles >0.5 μm in diameter and 54% for particles >5 μm. This equates to a reduction factor  of 3.1 and 2.2, respectively.This report describes the apparent transmission of SARS-CoV from a patient to healthcare workers during an attempted resuscitation. The similar symptom onset dates suggest a point source of exposure. In this case, SARS-CoV was transmitted despite healthcare workers’ wearing protection equipment designed to protect against contact and droplet transmission; no breaches in droplet protection equipment were identified, and exposure times were fairly brief. Although SARS transmission that involved intubation and BiPAP  can, in addition to contaminating both animate and inanimate surfaces in close range of the patient, travel short distances through the air and make direct contact with the exposed mucous membranes of healthcare workers or other patients.In contrast, airborne transmission is mediated by respiratory aerosols. These aerosols of infectious organisms contain droplet nuclei <10 μm in size and, depending upon their size within this range as well as ambient environmental conditions, can float on air currents and remain airborne for many hours (Two explanations may account for the transmission observed in this case: 1) an unrecognized breach in contact and droplet precautions occurred, or 2) an airborne viral load was great enough to overwhelm the protection offered by droplet precautions, including non–fit-tested N95 disposable respirators. If the last form of transmission was responsible, airborne virus may have been generated by the coughing patient  administrative controls, 2) environmental engineering, 3) protection equipment, and 4) quality control.Policies and protocols for emergency resuscitation involving patients known to have or suspected of having SARS should include 1) description of the roles and responsibilities of healthcare workers responding to the emergency, 2) mechanisms to alert responders that the emergency involves a potentially contagious patient , 3) steps to limit the number of healthcare workers involved to minimize potential exposures, 4) plans for having auxiliary staff staged in a safe area where they can be easily called on if needed but otherwise preventing unnecessary exposure, 5) plans for safe disposal and cleaning of equipment used during the emergency response, and 6) procedures for disposition of the patient after the emergency, either to the ICU if resuscitation is successful or the morgue if unsuccessful.Policies must be developed that consider all high-risk exposures or emergency situations and not just individual procedures. Policies that are too focused are of little value in dealing with the hundreds of unforeseeable possible situations that may arise. Conversely, policies that educate healthcare workers to assess the risks of a task and empower them to take appropriate protective action will be more effective. These policies should be crafted at each healthcare facility by a team that involves key stakeholders, including persons involved in the clinical response along with infection control practitioners and infectious disease experts.It is also important to minimize the chance that a patient will suffer unwitnessed cardiopulmonary arrest or require emergency intubation on a SARS unit. Prevention of these events will involve two changes in policy. The first is to recognize that isolation wards cannot be staffed with the same nurse-to-patient ratio as a regular ward. Care of patients in isolation is more time intensive due to both the physical barriers  and the required use of protection equipment. The nurse-to-patient ratio on the SARS ward at the time of the arrest was between 1:4 and 1:5; a more ideal ratio might be 1:2 or 1:3. It is also necessary to have a lower threshold for transferring patients to a higher acuity setting  when they first begin to show signs of a clinical deterioration. To enable this, all patients on a SARS unit should have measurement of vital signs along with pulse oximetry at a minimum of every 4 hours. Should their oxygen saturation drop below 92% on room air one should administer oxygen through nasal prongs 1–4 L per minute to maintain saturation >92%, and increase vital signs/pulse oximetry to every 2 hours. If the patient subsequently requires oxygen through nasal prongs at >4 L per minute the responsible physician should be notified and increase vital signs or pulse oximetry to every 1 hour. Finally, if the patient requires supplemental oxygen of >40% to maintain saturation >92%, the patient should be transferred to the intensive care unit and undergo elective intubation in a controlled manner. This later policy has worked well in other SARS units, as well as in hospital A after it was implemented by one of the authors (M.L.) after this cluster.Finally, policies should be developed to address the appropriateness and application of advanced cardiac life support for patients suffering cardiopulmonary arrest on a SARS ward. Many considerations must enter into any such discussion, including the usefulness and outcome of resuscitation efforts, particularly in unwitnessed arrests  . This syAs a form of protection equipment, this system has both advantages and disadvantages. The primary advantage is that the entire body of the healthcare worker is covered, providing a high level of droplet protection. The primary disadvantage of the T4 is the length of time required to put one on during an emergency. In the emergency resuscitation described in this report, the delay in certain rescuers responding was due to the time required to put on the T4. This resulted in the need for a second code blue to be announced for the same patient, which drew additional personnel to the code and thus increased the number of healthcare workers exposed to SARS.The healthcare worker must also be attentive to avoid contamination when removing the T4. Moreover, the airborne reduction factors of 3.1, for particles >0.5 μm in diameter, and 2.2 for particles >5 μm were less than the protection factor of 10 that is assigned  for a fit-tested, disposable N95 respirator. However, a disposable N95 is commonly worn under the T4 used in Ontario hospitals, suggesting the respiratory protection afforded healthcare workers using the T4 would be greater.The powered air-purifying respirators (PAPRs) most commonly used in healthcare settings have a disposable full hood with face shield covering the healthcare worker’s upper body . This deRegardless of what device (T4 versus PAPR) is used in an institution for potentially aerosol generating procedures, it is essential that they are distributed throughout the hospital in areas where they are most likely to be required by primary responders in an emergency situation as opposed to a central area where teams must wait for them to be brought to the emergency. In addition, extra protection equipment should be included as part of any “crash cart” used by the responding code team.Although there is a tendency to focus only on high-tech forms of protection equipment, it is important not to forget the basics of infection control procedures such as glove changing and hand hygiene. Healthcare workers must remain vigilant about not only protecting themselves from SARS transmission but also protecting against patient-to-patient transmission. As was found in the second phase of the SARS outbreak in Toronto (After developing good policies and training staff who are rehearsed for emergencies and provided with appropriate protection equipment, the last step is to ensure ongoing adherence to the standards set. This adherence is achieved through quality control. Without an effective quality control program in place, lapses in infection control procedures will occur, particularly as healthcare workers become fatigued during a prolonged outbreak.A variety of quality control methods can be implemented, including administrative checks to ensure equipment is in good repair, policies are current, and training materials are up to date. Another quality control practice often used by emergency services personnel dealing with hazardous situations is the “buddy system.” In this system, healthcare workers always work in teams on SARS units with each person being responsible for double checking to make sure that their partner is wearing appropriate equipment and following correct infection control practices before entering a patient’s room. Finally, a process should be in place to review responses to emergencies after they have occurred to learn from the experience and facilitate continuous quality improvement.SARS has increased the medical community’s awareness of issues related to occupational health and safety. It has also highlighted the importance of infection control programs and practices. A systematic approach, including administrative controls, environmental engineering, protection equipment, and quality control, is advocated to prevent future SARS-CoV transmission to healthcare workers.


The transplants were placed into full-thickness skin wounds and were fully connected to the surrounding tissue when explanted after 11 days. The printed keratinocytes formed a multi-layered epidermis with beginning differentiation and stratum corneum. Proliferation of the keratinocytes was mainly detected in the suprabasal layers. In vitro controls, which were cultivated at the air-liquid-interface, also exhibited proliferative cells, but they were rather located in the whole epidermis. E-cadherin as a hint for adherens junctions and therefore tissue formation could be found in the epidermis in vivo as well as in vitro. In both conditions, the printed fibroblasts partly stayed on top of the underlying Matriderm® where they produced collagen, while part of them migrated into the Matriderm®. In the mice, some blood vessels could be found to grow from the wound bed and the wound edges in direction of the printed cells. In conclusion, we could show the successful 3D printing of a cell construct via LaBP and the subsequent tissue formation in vivo. These findings represent the prerequisite for the creation of a complex tissue like skin, consisting of different cell types in an intricate 3D pattern.Tissue engineering plays an important role in the production of skin equivalents for the therapy of chronic and especially burn wounds. Actually, there exists no (cellularized) skin equivalent which might be able to satisfactorily mimic native skin. Here, we utilized a laser-assisted bioprinting (LaBP) technique to create a fully cellularized skin substitute. The unique feature of LaBP is the possibility to position different cell types in an exact three-dimensional (3D) spatial pattern. For the creation of the skin substitutes, we positioned fibroblasts and keratinocytes on top of a stabilizing matrix (Matriderm®). These skin constructs were subsequently tested  Major burn injuries often prove difficult in therapy due to their complexity, the high risk of infection, the large area which might be affected and the potential destruction of deeper skin layers including the dermis. Often, the availability of autologous split-thickness skin grafts and keratinocytes for wound coverage is limited, especially in case of large burned areas. Therefore, the need of skin substitutes for temporary or permanent wound coverage is high. Several skin substitutes like Integra® and Matriderm® are already employed in the clinical application, being complemented by the use of autologous split-thickness skin grafts in vitro is thought to be dependent on the direct interaction of endothelial cells with fibroblasts and their secreted extracellular matrix proteins and growth factors in vitro cultivation of complex tissues offering the possibility to mimic and control the desired microenvironment Tissue engineering promises to have high potential in the production of new skin. In this context, it remains a challenge to create a precise and complex new tissue comprising several cell types which are arranged in a specific 3D pattern. Furthermore, the different tissue functions strongly depend on its specific structure and on the cells which are influenced by their distinct microenvironment in vivo testing of the here produced skin substitutes we could already show tissue formation and functional cell-cell contacts in corresponding 3D tissue constructs in vitroOne solution for the problem of creating complex 3D tissues might be the use of LaBP. It offers the possibility to produce specific high resolution two-dimensional (2D) as well as 3D patterns, incorporating different cell types like human osteosarcoma and mouse endothelial cells via the use of LaBP, we created a multi-layered, fully cellularized skin equivalent for the future treatment of burn patients. The transplanted skin equivalent was tested in vivo for its ability to form tissue as well as cellular behaviour of the printed cells, the differentiation of the keratinocytes and potential neovascularisation using the dorsal skin fold chamber in mice. In vitro controls supplemented the in vivo experiments.In this study, NIH3T3 fibroblasts  and HaCaT keratinocytes  have previously been labelled by stable transduction with lentiviral or gammaretroviral vectors encoding for either eGFP or mCherry Cells were arranged in 3D skin constructs using LaBP , 5 µl phosphate buffered saline  and 0.85±0.5 µl sodium hydroxide  for neutralization (pH = 7.1±0.3) prior to the transfer. For the skin substitutes 20 layers of fibroblast-containing collagen and 20 layers of keratinocyte-containing collagen were printed subsequently onto a sheet of Matriderm® , used as a stabilization matrix.in vivo (one per mouse). As four independent printing processes were conducted, altogether 12 animals were used. The remaining six pieces of each printing process served as in vitro controls. Two of them were directly fixed on day 0 to depict the situation at the beginning of the experiments, whereas the remaining four pieces were raised to the air-liquid-interface. In vitro controls were then fixed on days 5 and 11 (duplicates per time point) and in vivo specimen on day 11.The printed cells were kept in the incubator under submerged conditions over night. The next day (defined as day 0), nine round pieces (diameter 6.0 mm) were removed from the large construct with a biopsy punch, three of which were implanted into the skin fold chambers −7 mM isoprenaline hydrochloride , 10−7 mM hydrocortisone (Sigma-Aldrich) and 10−7 mM insulin . The resulting calcium chloride concentration of the culture medium is 158.3 mg/L.Constructs were raised to the air-liquid-interface and cultivated with differentiation medium on top of plastic platforms. The latter consisted of cell strainers  turned upside down. The medium was composed of DMEM high glucose medium (PAA) mixed with the same amount of DMEM/Ham’s F12 medium (PAA), supplemented with 1% FBS, 1% penicillin/streptomycin, 10All animal experiments were evaluated and approved by the responsible animal care committee (Nds. Landesamt für Verbraucherschutz und Lebensmittelsicherheit) and the Hannover Medical School (Institut für Versuchstierkunde). The animals  were purchased from Charles River and kept in the local animal care facility according to the institution guidelines. They received standardized food and water, living with a day-night cycle of 12 hours each. Animals were used for the experiments when they were at least 12 weeks old.in vivo as published previously in vivoThe dorsal skin fold chamber was used for the evaluation of tissue engineered skin Samples were fixed in 4% paraformaldehyde, embedded in paraffin and subsequently cut to sections of 5 µm thickness. Masson’s trichrome stainings were conducted following standard procedures.In order to detect the presence of e-cadherin, collagen IV, cytokeratin 14 and Ki67, immunohistochemistry was carried out. For Ki67  and e-cadherin staining  the deparaffinised and rehydrated paraffin sections were incubated in a 99°C heated water bath for 25 min (Ki67) and 15 min (e-cadherin), respectively, before blocking. For cytokeratin 14  and collagen IV  stainings the sections were incubated in a 37°C warm water bath for 10 min, using 100 ml 0.2 N HCl solution with 100 mg pepsin. All sections were blocked with 2% FBS in PBS at room temperature for 30 min, followed by incubation with first antibody in 1% FBS in PBS at 4°C over night. After washing with PBS for all but the collagen IV staining a goat anti rabbit secondary antibody  coupled to an alkaline phosphatase was employed. The samples were incubated at 37°C for 1 h. As a substrate nitro-blue tetrazolium-5-bromo-4-chloro-3'-indolyphosphate  was used. In case of the collagen IV primary antibody, a biotin coupled secondary anti rabbit antibody  was used for 90 min at room temperature. Subsequently, the Vectastain ABC Kit  was employed for 60 min at room temperature to enhance the signal. The latter was then visualised with diaminobenzidine tetrahydro chloride (ICN98068). Staining of all antibodies was detected with a light microscope (Olympus).in vivo generated via LaBP, we printed 20 layers of a keratinocyte cell line (HaCaT) on top of 20 layers of a fibroblast cell line (NIH3T3) by an established laser-assisted bioprinting procedure. Using stable transduction, cell lines were labelled with genes encoding for green or red fluorescent proteins, respectively: NIH3T3-eGFP, NIH3T3-mCherry, HaCaT-eGFP and HaCaT-mCherry. Matriderm® was used as a carrier matrix to enhance stability of the constructs for transplantation  developed a dense stratified tissue , similarFurthermore, the migration pattern of the printed fibroblasts was assessed. As can be seen in the histological sections, the fibroblasts partly migrated into the Matriderm®, closely following the fibres of the latter . Some fiOne important issue in respect of the use of skin substitutes is their vascularisation. Here, small blood vessels could be found in the skin constructs which seem to grow in from the depth of the wound bed as well as from wound edges into the Matriderm® in the direction of the printed cell layers  and 5.Adherens junctions – containing especially e-cadherin - are essential for stable cell-cell contact and can abundantly be found in epithelia like skin. Therefore, e-cadherin can be used as a hint for epithelia formation and consequently for tissue development. By means of immunostaining, e-cadherin could be detected between the keratinocytes of the skin constructs inserted into the wounds. Here, the pattern of the e-cadherin localisation is the same as in native skin and can be found in the whole epidermis .One important characteristic of an epidermis is the differentiation of the keratinocytes. Cytokeratin 14 is a marker for undifferentiated keratinocytes. The corresponding immunostaining revealed the presence of cytokeratin 14 in the whole epidermis of the skin constructs . In natiIn contrast, Ki67 as a proliferation marker, showed a signal mainly in the suprabasal layer of the skin constructs , indicatin vitro with the addition of differentiation medium. Samples were taken for histology on day 0-at the same time point as the corresponding transplants were inserted into the chambers of the mice – to demonstrate the starting conditions of the experiments. Further on, samples were also secured on day 11, corresponding to the end of the in vivo experiments and in between (on day 5).As a control, printed skin constructs were also cultivated On day 0 the two multi-layers of different cell types on top of the Matriderm® could clearly be seen . The kerA thickening of the epidermis-like tissue formed by the keratinocytes could be observed comparing day 11 to day 5 . Surprisin vitro cultures, immunostaining was carried out. The presence of e-cadherin increased during time from none on day 0 to its detection in the whole epidermis-like tissue on day 11,  mainly in the suprabasal layers hints at the beginning differentiation of the keratinocytes. In native skin, only the keratinocytes in the stratum basale maintain proliferation, whereas the differentiating keratinocytes in the other skin layers cease proliferating. Furthermore, the Matriderm® carrier becomes populated by the printed fibroblasts (presented in this work) as well as murine host fibroblasts  and consisting of different cell types  has already been analysed in vivo as a technical prerequisite to develop vascularised bone tissue in the future. The implanted cells could be detected several weeks after implantation indicating good survival rates. Interestingly, the used stem cells were able to differentiate into bone in vivo and endothelial cells formed a network of blood vessels in the implants after six weeks As skin is a complex organ consisting of different cell types and substructures arranged in defined spatial patterns, LaBP is suited for the production of tissue engineered skin substitutes. It offers many possibilities and is a very promising technique for the fabrication of other kinds of tissues as e.g. bone or cartilage ex vivo. They maintained their deposited positions due to simultaneous photopolymerization of a surrounding biomaterial scaffold, and attached firmly to the enclosing cartilage tissue Concerning maintenance of cellular phenotype, human chondrocytes were found to express cartilage specific genes after being printed into cartilage lesions by inkjet printing and being cultivated in vitro samples, a horizontal stripe of collagen without cells could be observed in the lower part of the epidermis-like structure. Obviously, the fibroblasts did not degrade the circumjacent collagen - which has been used as a hydrogel during the printing process - but started to migrate into the Matriderm® right away. During the printing, the cells are mixed with a hydrogel  to serve four different purposes. Firstly, the matrix is necessary to achieve a uniform coverage of the donor glass slide with the biomaterial to be transferred. This is important as only then a consistency and uniformity of the printing process is possible. Secondly, the matrix helps the cells to survive, providing a moist environment preventing drying. Thirdly, it presents a specific surrounding for the printed cells and thereby acts as a biomimetic gel to create the desired micro-environment. In our case, collagen is already present in physiological skin and therefore is very suited as a printing matrix. Fourthly, a matrix like collagen enables the formation of a 3D construct due to its gelling effect. While in the in vitro controls the collagen is left by the fibroblasts, in the in vivo situation outstretched fibroblasts can be found in the collagen, probably also expressing and producing collagen by themselves. As this is similar to the situation in physiological skin, this process is much desired.In the in vitro, resulting in small vessels similar to capillaries present in the skin’s microcirculation Very important for the take of a grafted skin or skin substitute is its fast vascularisation. It is a major prerequisite for the successful clinical use of a skin substitute. In our experiments, blood vessels could be found to start growing into the Matriderm® from the wound bed and the wound edge mostly in the direction of the transplanted cells. In a previous study, in skin constructs consisting of Matriderm® covered with collagen type I but without cells et al. integrated human umbilical vein endothelial cells (HUVEC) into tissue engineered skin equivalents containing fibroblasts and keratinocytes in combination with a collagen chitosan scaffold in vitro. In a similar approach, skin equivalents constructed by seeding acellular dermis with keratinocytes and Bcl-2-transduced HUVEC showed perfusion through HUVEC-lined microvessels two weeks after implantation into mice In our experiments no complete vascularisation of the printed skin equivalents could be achieved under the current conditions. Probably, the period was too short for complete vascularisation of the skin equivalent. Following the idea of improving graft vascularisation, Black in vivo situation, our in vitro controls formed a multi-layered tissue with collagen producing fibroblasts, but did not show any differentiation of the keratinocytes (HaCaT). This might be due to the culturing method in vitro. Although differentiation aiding supplements were added to the culture medium and the skin constructs were raised to the air-liquid-interface, this might not have been appropriate enough to trigger the differentiation. Also, the culturing period of 11 days is quite short and induction of differentiation in vitro might have been observed at a later time point. However, the beginning differentiation of the keratinocytes in vivo could be due to the growth factors present in mice but absent in vitro.In contrast to the Summing up, LaBP offers the possibility to place cells of a specific type wherever in the tissue they are needed. This is a unique feature of bioprinting techniques and it may be used to print skin supplemented with endothelial cells, hair follicle cells, peripheral nerve cells, Schwann cells, melanocytes or cells present in perspiratory and sebaceous glands. Therefore, we hope to be able to produce a much more similar skin construct to native skin compared to other current approaches. This is especially important for the future patients as they gain a much more functional and aesthetic skin substitute. This in turn would lead to a major increase of their quality of life, on the physical as well as on the mental level.As an alternative to LaBP, a similar technique called inkjet printing is available, which has already been used to print 2D protein arrays To further improve the use of LaBP for the creation of (skin) tissue, an adaptation to automation would be of advantage. Our setup is not suited for the high throughput production of skin substitutes yet, but in principal an automation of the whole process - including the printing process as such as well as the cultivation of the skin substitutes - is conceivable.in vivo, forming a tissue similar to simple skin within the time frame of 11 days. As a further limitation, compared to the common approach of simple full-thickness wounds in the dorso-lateral region of mice with an area of 2 cm×2 cm to 2 cm×3 cm We used the dorsal skin fold chamber in mice for the assessment of the tissue engineered skin constructs. This approach exhibits different advantages as well as drawbacks. The common approach of dorso-lateral full-thickness wounds without a chamber allows for the cultivation of a tissue engineered skin constructs for a long period of time. While two to eight weeks are the most frequently used time intervals in vivo formation of a 3D tissue like skin. Therefore, LaBP represents a major promise for the improvement of burn therapy and thus for a raised quality of life for the patients.In conclusion, we could show LaBP to be an adequate technique for the creation and