Search

Reciprocating gait orthoses (RGOs) have long been used for orthotic management in patients with higher level spinal cord injuries, with one primary limitation in ambulation being attributable to the absence of knee flexion in swing phase. As a result, an individual is forced to use compensatory upper body motions to advance the legs. The authors report the use of stance-control knee-ankle-foot orthoses in combination with an isocentric RGO in a patient with a T10 spinal cord injury. A comparison is made between the scenarios of having the knees locked during the entire gait cycle to that of allowing the knees to flex freely during the swing phase, yet still be locked for stability during stance. Qualitative observation and kinematic three-dimensional gait data demonstrate that this subject ambulated with a faster, more efficient gait pattern with the stance-control feature activated. Despite having no voluntary control of his knees, this orthotic option afforded him the ability to walk safely and smoothly with both knees flexing during swing and with less upper body compensation. [ABSTRACT FROM AUTHOR]

Introduction: Constipation is a common and significant burden on individuals and healthcare systems. Accurate assessment of constipation severity and symptom improvement are vital aspects of caring for patients with constipation. Therefore, nurses and allied healthcare professionals should possess knowledge regarding the characteristics of constipation assessment tools (ie, aim, scope, definition of constipation, content, structure, mode, administration time and context of use). However, existing reviews summarising characteristics of tools have been restricted to chronic constipation and self-reported measures. Furthermore, they have not included literature published after 2011. This scoping review aims to identify and comprehensibly map the characteristics of available tools for screening and assessment of constipation in order to manage the nursing care need related to constipation within any healthcare or research context and any patient group., Methods and Analysis: This review will include primary research articles, methodological papers and clinical guidelines using tools for constipation screening and assessment, pertinent to nursing care management. It is not limited to a specific population or healthcare setting. Databases to be searched include PubMed, Embase, CINAHL, ProQuest, ClinicalKey and Google Scholar. To identify grey literature, national health services in selected countries will be searched. Papers written in English, Nordic language or German will be included. The reviewers will independently review the retrieved citations against the inclusion criteria, and data from included papers will be extracted using a data extraction form developed for this review. The scoping review will be conducted following the Joanna Briggs Institute Guidelines. The results will be presented in a table accompanied by a narrative summary., Ethics and Dissemination: Ethical approval is not required, as no individual patient data are included. Findings will be shared and discussed with relevant stakeholders and disseminated through peer-reviewed publications and conference presentations. The protocol is registered on Open Science Framework (registration number: osf.io/h2vzd)., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The β-coronavirus family, encompassing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Severe Acute Respiratory Syndrome Coronavirus (SARS), and Middle East Respiratory Syndrome Coronavirus (MERS), has triggered pandemics within the last two decades. With the possibility of future pandemics, studying the coronavirus family members is necessary to improve knowledge and treatment. These viruses possess 16 non-structural proteins, many of which play crucial roles in viral replication and in other vital functions. One such vital protein is non-structural protein 10 (nsp10), acting as a pivotal stimulator of nsp14 and nsp16, thereby influencing RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to unraveling its multifunctional roles. Our study involves the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. To elucidate their oligomeric state, we employed a combination of Multi-detection Size exclusion chromatography (Multi-detection SEC) with multi-angle static light scattering (MALS) and small angle X-ray scattering (SAXS) techniques. Our findings reveal that full-length nsp10s primarily exist as monomers in solution, while truncated versions tend to oligomerise. SAXS experiments reveal a globular shape for nsp10, a trait conserved in all three coronaviruses, although MERS nsp10, diverges most from SARS and SARS-CoV-2 nsp10s. In summary, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 exhibit a globular and predominantly monomeric state in solution.

Background: Cognitive development measured as intelligence quotient can predict socioeconomic markers in adulthood. It is therefore of interest to determine predictors of childhood intelligence quotient., Aim: To assess intelligence quotient scores based on standardised Danish age-appropriate scores and to evaluate potential predictors of intelligence quotient., Materials: At 7 years of age children in the Odense Child Cohort completed an abbreviated version of the Wechsler intelligence scale for children 5th edition consisting of four subtests (vocabulary, similarities, block design and matrix reasoning) from which the full scale intelligence quotient and verbal comprehension index were estimated. Potential predictors from pregnancy through childhood were collected from questionnaires, birth records and clinical examinations., Methods: Intelligence quotient scores were investigated through descriptive statistics and linear regression models., Results: The mean full scale intelligence quotient among 1375 children was 99.1 (95% confidence interval 98.5; 99.8) points. Higher full scale intelligence quotient scores were observed in girls 100.8 (95% confidence interval 100.0; 101.8) compared to boys 97.6 (96.7; 98.4), and in children of mothers with high and intermediate education 101.7 (100.4; 103.1) and 99.6 (98.7; 100.5), respectively, compared to low education 96.1 (94.9; 97.3). In linear regression analyses, longer maternal education and child sex (girls) remained strong predictors of intelligence quotient at age 7 years. In addition, paternal education, child head circumference and longer duration of breastfeeding were associated with higher intelligence quotient, whereas maternal overweight and obesity before pregnancy was associated with lower intelligence quotient., Conclusions: Mean intelligence quotient scores were comparable to the standardised mean intelligence quotient of 100 point of Danish peers. It is important to follow up these children to determine which predictors persist into adulthood.

Aims: Systematic use of patient-reported outcomes (PROs) have the potential to improve quality of care and reduce costs of health care services. We aimed to describe whether PROs in patients diagnosed with heart disease are directly associated with health care costs., Methods and Results: A national cross-sectional survey including PROs at discharge from a heart centre with 1-year follow-up using data from national registers. We included patients with either ischaemic heart disease (IHD), arrhythmia, heart failure (HF), or valvular heart disease (VHD). The Hospital Anxiety and Depression Scale, the heart-specific quality of life, the EuroQol five-dimensional questionnaire, and the Edmonton Symptom Assessment Scale were used. The economic analysis was based on direct costs including primary, secondary health care, and medical treatment. Patient-reported outcomes were available from 13 463 eligible patients out of 25.241 [IHD (n = 7179), arrhythmia (n = 4322), HF (n = 987), or VHD (n = 975)]. Mean annual total direct costs in all patients were €23 228 (patients with IHD: €19 479, patients with arrhythmia: €21 076, patients with HF: €34 747, patients with VDH: €48 677). Hospitalizations contributed overall to the highest part of direct costs. For patients discharged with IHD or arrhythmia, symptoms of anxiety or depression, worst heart-specific quality of life or health status, and the highest symptom burden were associated with increased economic expenditure. We found no associations in patients with HF or VHD., Conclusion: Patient-reported outcomes at discharge from a heart centre were associated with direct health care costs in patients with IHD and arrhythmia., Registration: ClinicalTrials.gov: NCT01926145., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The baculovirus expression vector system (BEVS) is one of the most popular eukaryotic systems for recombinant protein production. The focus of our protein production platform is the provision of recombinant proteins for research use, where generally only small quantities are required, in the range of tens of micrograms to a few hundred milligrams. Here, we present methods that reflect our standard operating procedures and setup to be able to frequently, and often repeatedly, produce many different types of proteins., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aims: Women report worse health-related patient-reported outcomes (PROs) compared with men following acute myocardial infarction (AMI). However, this association is not well established when accounting for demographic and clinical patient characteristics at discharge. This knowledge is essential for clinicians when planning individualised care for patients following AMI. The aim of this study is to examine whether gender is associated with health-related PROs at discharge from a Danish heart centre, combining PROs with data from the national health and administrative registries., Methods and Results: A cross-sectional study of 2131 patients with AMI discharged from a Danish heart centre responding to the following health-related PRO questionnaires: the Health-survey Short-Form-12 (SF-12), generating a physical component summary (PCS) and a mental component summary (MCS) score; the HeartQoL, providing a global, emotional, and physical score; the EuroQol five-dimensional questionnaire (EQ-5D-5L) and the EQ visual analogue scale (EQ VAS); the Hospital Anxiety and Depression Scale (HADS), generating an anxiety and depression score (HADS-A and HADS-D); the Edmonton Symptom Assessment Scale (ESAS); the Brief Illness Perception Questionnaire (B-IPQ). Patient-reported outcomes were linked to registry-based information adjusting for potential demographic and clinical confounding factors. In adjusted regression models, women reported worse health-related PROs compared with men in SF-12 PCS and SF-12 MCS, HeartQoL global, the HeartQoL emotional and HeartQoL physical score, EQ-5D-5L and EQ VAS, the HADS-A, ESAS, and in six out of eight B-IPQ items., Conclusions: Women reported worse health-related PROs compared with men. Health-related PROs have the potential to be further investigated to facilitate a more individualised healthcare follow-up after AMI., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Patient-reported outcome measures (PROMs) may predict poor clinical outcome in patients with heart failure (HF). It remains unclear whether PROMs are associated with subsequent adherence to HF medication. We aimed to determine whether health-related quality of life, anxiety, and depression were associated with long-term medication adherence in these patients., Methods and Results: A national cohort study of Danish patients with HF with 3-year follow-up (n = 1464). PROMs included the EuroQol five-dimensional, five-level questionnaire (EQ-5D-5L), the HeartQoL and the Hospital Anxiety and Depression Scale (HADS). Patient-reported outcomes (PRO) data were linked to demographic and clinical data at baseline, and data on all redeemed prescriptions for angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers/angiotensin receptor neprilysin inhibitors (ACEI/ARB/ARNI), β-blockers, and mineralocorticoid receptor antagonists during follow-up. Medication non-adherence was defined as <80% of proportion of days covered. In adjusted regression analyses, lower health-related quality of life (EQ-5D and HeartQoL) and symptoms of depression (HADS-D) at discharge were associated with non-adherence. After 3 years of follow-up, lower health-related quality of life (EQ-5D) was associated with non-adherence for ACEI/ARB/ARNI [adjusted OR 2.78, 95% confidence interval (CI): 1.19-6.49], β-blockers (adjusted OR 2.35, 95% CI: 1.04-5.29), whereas HADS-D was associated with non-adherence for ACEI/ARB/ARNI (adjusted OR 1.07, 95% CI: 1.03-1.11) and β-blockers (adjusted OR 1.06, 95% CI: 1.02-1.10)., Conclusion: Lower health-related quality of life and symptoms of depression were associated with non-adherence across HF medications at 1 and 3 years of follow-up. Person-centred care using PROMs may carry a potential for identifying patients at increased risk of future medication non-adherence., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Aims: An in-depth understanding of the prognostic value of patient-reported outcomes (PRO) is essential to facilitate person-centred care in heart failure (HF). This study aimed to clarify the prognostic role of subjective mental and physical health status in patients with HF., Methods and Results: Patients with HF were identified from the DenHeart Survey (n = 1499) and PRO data were obtained at hospital discharge, including the EuroQol five-dimensional questionnaire (EQ-5D), the HeartQoL, and the Hospital Anxiety and Depression Scale (HADS). Clinical baseline data were obtained from medical records and linked to nationwide registries with patient-level data on sociodemographics and healthcare contacts. Outcomes were all-cause and cardiovascular (CV) mortality, CV events, and HF hospitalization with 1- and 3-year follow-up. Analysing the PRO data on a continuous scale, a worse score in the following were associated with risk of all-cause and CV mortality after 1 year: the HeartQoL (adjusted hazard ratios (HRs) 1.91, 95% confidence interval (CI) 1.42-2.57 and 2.17, 95% CI 1.50-3.15, respectively), the EQ-5D (adjusted HRs 1.26, 95% CI 1.15-1.38 and 1.27, 95% CI 1.13-1.42, respectively), the HADS depression subscale (adjusted HRs 1.12, 95% CI 1.07-1.17 and 1.11, 95% CI 1.05-1.17, respectively), and the HADS anxiety subscale (adjusted HRs 1.08, 95% CI 1.03-1.13 and 1.09, 95% CI 1.04-1.15, respectively). Three-year results were overall in concordance with the 1-year results. A similar pattern was also observed for non-fatal outcomes., Conclusion: Health-related quality of life and symptoms of anxiety and depression at discharge were associated with all-cause and CV mortality at 1- and 3-year follow-up., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Background: It is well-established that heart failure has a negative impact on quality of life. However, little is known about patient-related predictors of health-related quality of life, anxiety and depression, symptoms and illness perception among patients with heart failure., Aim: To study the association between patient-related predictors and patient-reported outcome measures at discharge from hospital in a cohort of patients with heart failure., Methods: We used data from 1506 patients with heart failure, participating in the national DenHeart Survey of patient-reported outcome measures in patients with heart disease. The potential patient-related predictors included demographic, administrative, clinical and socioeconomic factors. The patient-reported outcome measures included six questionnaires: the Short Form-12, the Hospital Anxiety and Depression Scale, the EuroQol five-dimensional, five-level questionnaire, the HeartQoL, the Brief Illness Perception Questionnaire and the Edmonton Symptom Assessment Scale. Data were linked to national patient registry data and medical records. We performed multivariable linear and logistic regression analyses., Results: In adjusted linear regression analyses we found that a length of hospital stay of >2 days was associated with worse scores across questionnaires, except for the Brief Illness Perception Questionnaire. Higher comorbidity level was associated with worse scores across all questionnaires, whereas low social support was associated with worse scores across questionnaires, except for the physical domain of the Short Form-12 and the HeartQoL global score., Conclusions: This study identified length of hospital stay > 2 days, a higher comorbidity level and low social support to be associated with worse scores across questionnaires at discharge from a cardiac-related hospitalisation in patients with heart failure.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication.

Objective: The purpose of this study was to analyze the correlation between the genetic constitution and the phenotype in triploid pregnancies., Study Design: One hundred fifty-eight triploid pregnancies were identified in hospitals in Western Denmark from April 1986 to April 2010. Clinical data and karyotypes were collected retrospectively, and archived samples were retrieved. The parental origin of the genome, either double paternal contribution (PPM) or double maternal contribution (MMP) was determined by an analysis of methylation levels at imprinted sites., Results: There were significantly more PPM than MMP cases (P < .01). In MMP cases, the possible karyotypes had similar frequencies, whereas, in PPM cases, 43% had the karyotype 69,XXX, 51% had the karyotype 69,XXY, and 6% had the karyotype 69,XYY. Molar phenotype was seen only in PPM cases. However, PPM cases with a nonmolar phenotype were also seen. For both parental genotypes, various fetal phenotypes were seen at autopsy. Levels of human chorionic gonadotropin in maternal serum were low in MMP cases and varying in PPM cases, some being as low as in the MMP cases., Conclusion: In a triploid pregnancy, suspicion of hydatidiform mole at ultrasound scanning, by macroscopic inspection of the evacuated tissue, at histology, or because of a high human chorionic gonadotropin in maternal serum level each predict the parental type PPM with a very high specificity. In contrast, the sensitivity of these observations was <100%., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In Denmark the development of rehabilitation of patients with acquired brain injury (ABI) has differed from most parts of Europe. Denmark has no medical rehabilitation specialty. In 2000 two specialized hospital units were established to serve the population of 5.7 million inhabitants. They cover the needs of patients with the most severe ABIs. Only recently rehabilitation of patients with ABI has been anchored in the medical specialty of neurology. Since then the development has taken an up-going curve. This process has been supported by The Danish Health and Medicines Authority publishing several papers that highlights evidence-based organization and interventions. Current development aim to define: 1) skills of the medical doctor engaged in the rehabilitation of patients with ABI, 2) stratification to different levels of specialization at hospital and in the community, and 3) national guidelines that specify skills and collaboration between groups of professionals working with ABI rehabilitation.

Objective: When a triploid pregnancy is diagnosed prenatally, gynaecologists have traditionally relied on the histopathological examination of the tissue from the terminated pregnancy to determine if the pregnancy is molar. However, reproducibility is poor and variability is high when diagnosing hydatidiform moles. Triploid pregnancies can have either the chromosomal constitution of two maternal and one paternal set, or two paternal and one maternal set, but only the conceptuses with two paternal sets have the potential to cause maternal complications. Therefore, it would be beneficial to introduce a method that gives the gynaecologist the parental origin of the genome of the triploid conceptus as early as possible, without delaying the process by first collecting parental samples., Methods: Using methylation-specific multiplex ligation-dependent probe amplification, we measured methylation levels at different imprinted sites., Results: We were able to correctly determine the parental origin of the genome in all 105 triploid pregnancies analysed., Conclusions: We present methylation-specific multiplex ligation-dependent probe amplification as a method capable of determining the parental origin of the genome of triploid conceptuses within 24 h; it is inexpensive, simple and easy to use, and parental samples are not needed., (© 2013 John Wiley & Sons, Ltd.)

Background: In colorectal cancer MLH1 deficiency causes microsatellite instability, which is relevant for the patient's prognosis and treatment, and its putative heredity. Dysfunction of MLH1 is caused by sporadic gene promoter hypermethylation or by hereditary mutations as seen in Lynch Syndrome. The aim of this study was to determine in detail how DNA methylation regulates MLH1 expression and impacts clinical management., Methods: Colorectal cancer samples were collected from 210 patients. The laboratory methods used to study these samples included methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), real-time quantitative PCR (qPCR), and immunohistochemistry (IHC)., Results: We found that the MLH1 mRNA and protein expression levels were highly related. MS-MLPA was successful in tumors from 195 patients. In these tumors, hypermethylation was observed in promoter regions A (n = 57), B (n = 30), C (n = 28), and D (n = 47), and in intron 1 (n = 25). The promoter region C and intron 1 methylation levels were found to be excellently suited for discriminating between low and high gene expression levels, whereas those of promoter regions A, B and D were less specific. Hypermethylation in any region (n = 77) served as an independent prognostic factor (hazard ratio 0.56, 95 % confidence interval 0.36-0.89, p = 0.01)., Conclusions: MLH1 inactivation through hypermethylation was found to be related to improved survival. Hypermethylation in promoter region C and intron 1 served as the most specific markers for this inactivation.

22q11.2 distal deletion syndrome is distinct from the common 22q11.2 deletion syndrome and caused by microdeletions localized adjacent to the common 22q11 deletion at its telomeric end. Most distal deletions of 22q11 extend from LCR22-4 to an LCR in the range LCR22-5 to LCR22-8. We present three patients with 22q11 distal deletions, of whom two have complex congenital heart malformation, thus broadening the phenotypic spectrum. We compare cardiac malformations reported in 22q11 distal deletion to those reported in the common 22q11 deletion syndrome. We also review the literature for patients with 22q11 distal deletions, and discuss the possible roles of haploinsufficiency of the MAPK1 gene. We find the most frequent features in 22q11 distal deletion to be developmental delay or learning disability, short stature, microcephalus, premature birth with low birth weight, and congenital heart malformation ranging from minor anomalies to complex malformations. Behavioral problems are also seen in a substantial portion of patients. The following dysmorphic features are relatively common: smooth philtrum, abnormally structured ears, cleft palate/bifid uvula, micro-/retrognathia, upslanting palpebral fissures, thin upper lip, and ear tags. Very distal deletions including region LCR22-6 to LCR22-7 encompassing the SMARCB1-gene are associated with an increased risk of malignant rhabdoid tumors., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

The disease phenotype of transthyretin (TTR) is dramatically influenced by single point mutations in the TTR gene. Herein, we report on a novel mutation D99N (Asp99Asn) in TTR found in a Danish kindred. None of the family members carrying this mutation have so far shown any clinical signs of amyloidosis. One carrier found compound heterozygous for TTR D99N and L111M (Leu111Met) associated with cardiac amyloid is asymptomatic (42 years). Disease severity can often be linked to both the kinetics of fibril formation and the degree of destabilisation of the native state. In this study, we show that the thermodynamic stability and rate of tetramer dissociation of the variant TTR D99N is unchanged or slightly more stable than wild type (WT) TTR. Furthermore, the in vitro fibrillation kinetics of the variant reveals an unchanged or slightly suppressed tendency to form fibrils compared to WT. Thus, the in vitro experiments support the lack of clinical symptoms observed so far for the TTR D99N carriers. In line with this, studies on kinetic stability and fibrillation kinetics reveal indistinguishable stability of TTR heterotetramers D99N/L111M compared to the heterotetramers WT/L111M. In conclusion, TTR D99N is predicted to be a non-pathogenic benign mutation with WT properties.

Objective: It is believed that 24-40% of ovarian cancers have dysfunction in the BRCA1 or BRCA2 (BRCAness) genes, due to either inherited or somatic mutations or due to epigenetic inactivation. Demonstration of ovarian cancers with BRCAness is becoming important both due to the possibility of offering genetic counseling and due to beneficial effects of polyadenosine diphosphate ribose polymerase inhibitor treatment in this group. As DNA sequencing is expensive and time consuming, efforts have been devoted to develop more indirect methods for BRCA screening that can improve the selection of patients for sequence-based BRCA testing., Design: BRCA1 immunohistochemistry, fluorescence in situ hybridization (FISH) and methylation analyses were performed on formalin-fixed, paraffin-embedded ovarian cancer tissue., Sample: Fifty-four ovarian cancers; 15 BRCA1 cancers, four BRCA2 cancers, 10 cancers from patients with a family history but no mutation detected, and 25 ovarian cancers with unknown BRCA1 status., Results: Abnormal BRCA1 immunohistochemistry was found to indicate BRCA mutations with a sensitivity of 80%, a specificity of 93% and an estimated positive predictive value of 73%. The FISH analyses supported the diagnosis in most cases. Methylation analyses could indicate BRCA deficiency in combination with one of the other methods., Conclusions: BRCA1 immunohistochemistry is a promising screening method for BRCA1 mutation detection., (© 2011 The Authors Acta Obstetricia et Gynecologica Scandinavica© 2011 Nordic Federation of Societies of Obstetrics and Gynecology.)

In the past few years an increasing number of small non-coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the post-transcriptional level. These RNAs act under various growth and stress conditions, suggesting that one important physiological role of regulatory RNA molecules in Gram-negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP-sRNA network by showing that the expression of the OMP YbfM is silenced by a conserved sRNA, designated MicM (also known as RybC/SroB). The regulation is strictly dependent on the RNA chaperone Hfq, and mutational analysis indicates that MicM sequesters the ribosome binding site of ybfM mRNA by an antisense mechanism. Furthermore, we provide evidence that Hfq strongly enhances the on-rate of duplex formation between MicM and its target RNA in vitro, supporting the idea that a major cellular role of the RNA chaperone is to act as a catalyst in RNA-RNA duplex formation.

Background: The effect of anti-epidermal growth factor receptor (EGFR) antibodies (mAb) in metastatic colorectal cancer seems limited to KRAS wild-type (wt) tumours, but still a major fraction of KRASwt patients are nonresponders and supplementary selection criteria are needed. We investigated methodological aspects of KRAS testing and the predictive and prognostic value of KRAS status combined with three EGFR-related gene polymorphisms [single-nucleotide polymorphisms (SNPs)] in patients treated with cetuximab and irinotecan., Patients and Methods: The study included 71 patients referred to third-line cetuximab-irinotecan. Blood samples were analysed for SNPs. KRAS analysis was carried out by sequencing analysis and quantitative PCR (DxS kit) in primary tumour and distant metastases., Results: There was a clear correlation between KRAS status in primary tumours and metastasis. The DxS kit presented the highest sensitivity. Response was confined to KRASwt patients (40% response rate versus 0%, P < 0.1(-3)), which translated into a significant difference in PFS. The EGF61A>G polymorphism showed relation to clinical outcome. A combined biomarker analysis showed a 19% progression rate in KRASwt-EGF61 homozygote patients and 60% in the EGF61A/G patients (P = 0.006) and a significant increase in overall survival (17.1 versus 5.9 months, log-rank, P = 0.002)., Conclusion: The combined biomarker analysis maybe an attractive approach to selection of patients for third-line treatment including anti-EGFR mAbs.

Gene regulation by small non-coding RNAs has been recognized as an important post-transcriptional regulatory mechanism for several years. In Gram-negative bacteria such as Escherichia coli and Salmonella, these RNAs control stress response and translation of outer membrane proteins and therefore are key regulators of environmental stress. Recent work has revealed an intimate interplay between small RNA regulation of outer membrane proteins and the stress-induced sigmaE-signalling system, which has an essential role in the maintenance of the integrity of the outer membrane.

Enteric bacteria respond to misfolded proteins by activating the transcription of "heat shock" genes. These genes are arranged in two major regulons controlled by the alternative sigma factors sigmaH and sigmaE. The two transcription factors coordinate the stress response in different cellular compartments; the sigmaH regulon is induced by stress in the cytoplasm whereas the sigmaE regulon is activated by stress signals in the cell envelope. In Escherichia coli sigmaE plays a central role in maintaining cell envelope integrity both under stress conditions and during normal growth. Previous work established that sigmaE is essential for viability of the bacterium and up-regulates expression of approximately 100 protein-encoding genes that influences nearly every aspect of the cell envelope. Moreover, the expression of several outer membrane proteins is down-regulated upon sigmaE activation. Here, we show that two Hfq-binding small RNAs, MicA and RybB, are under positive control of sigmaE. Transient induction of RybB resulted in decreased levels of the mRNAs encoding OmpC and OmpW. sigmaE -mediated regulation of ompC and ompW expression was abolished in strains lacking RybB or Hfq. Recently MicA was shown to act in destabilizing the ompA transcript when rapidly grown cells entered the stationary phase of growth. Also, the alternative sigma factor down-regulates this message in a small non-coding RNA-dependent fashion. These findings add the sigmaE regulon to the growing list of stress induced regulatory circuits that include small regulatory RNAs and provide insight in a homeostatic loop that prevent a build-up of unassembled outer membrane proteins in the envelope.

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.

The Escherichia coli ompA mRNA, encoding a highly abundant outer membrane protein, has served as a model for regulated mRNA decay in bacteria. The half-life of this transcript correlates inversely with the bacterial growth rate and is growth stage-dependent. The stability of the messenger is determined by the 5'-untranslated region which possesses cleavage sites for RNase E. Hfq binds to this region, is essential for controlling the stability and has been suggested to directly regulate ompA mRNA decay. Here we report that the 78 nucleotide SraD RNA, which is highly conserved among Enterobacteriaceae, acts in destabilizing the ompA transcript when rapidly grown cells enter the stationary phase of growth. During this growth-stage the expression of SraD RNA becomes strongly increased. The SraD-mediated decay of ompA mRNA depends on Hfq and in vitro studies revealed that Hfq facilitates binding of the regulatory RNA to the translational initiation region of the messenger. Deletion of sraD, however, does not significantly affect the stability of the ompA mRNA in slowly growing cells. Our results indicate that distinct regulatory circuits are responsible for growth phase- and growth rate-dependent control of the ompA mRNA stability.

We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.

In response to starvation, Myxococcus xanthus initiates a developmental programme that results in the formation of spore-filled multicellular fruiting bodies. Fruiting body formation depends on the temporal and spatial coordination of aggregation and sporulation and involves temporally and spatially coordinated changes in gene expression. This paper reports the identification of two genes, hthA and hthB, that are important for fruiting body formation. hthA and hthB are co-transcribed, and transcription of the two genes decreases strongly during development. Loss of HthA and HthB function results in delayed aggregation, a reduction in the level of sporulation, and abnormal developmental gene expression. Extracellular complementation experiments showed that the developmental defects caused by loss of HthA and HthB function are not due to the inability to synthesize an intercellular signal required for fruiting body formation. HthA, independent of HthB, is required for aggregation. HthB, alone or in combination with HthA, is required for sporulation. HthA is predicted to contain a C-terminal helix-turn-helix DNA-binding domain. Intriguingly, the N-terminal part of HthA does not exhibit significant amino acid similarity to proteins in the databases. The HthB protein lacks homologues in the databases. The results suggest that HthA is a novel DNA-binding protein, which regulates transcription of genes important for aggregation, and that HthB, alone or in combination with HthA, stimulates sporulation.

In response to starvation, Myxococcus xanthus initiates a developmental program that results in the formation of spore-filled multicellular fruiting bodies. Fruiting body formation depends on the temporal and spatial coordination of aggregation and sporulation. These two processes are induced by the cell surface-associated C signal, with aggregation being induced after 6 h and sporulation being induced once cells have completed the aggregation process. We report the identification of TodK, a putative histidine protein kinase of two-component regulatory systems that is important for the correct timing of aggregation and sporulation. Loss of TodK function results in early aggregation and early, as well as increased levels of, sporulation. Transcription of todK decreases 10-fold in response to starvation independently of the stringent response. Loss of TodK function specifically results in increased expression of a subset of C-signal-dependent genes. Accelerated development in a todK mutant depends on the known components in the C-signal transduction pathway. TodK is not important for synthesis of the C signal. From these results we suggest that TodK is part of a signal transduction system which converges on the C-signal transduction pathway to negatively regulate aggregation, sporulation, and the expression of a subset of C-signal-dependent genes. TodK and the SdeK histidine protein kinase, which is part of a signal transduction system that converges on the C-signal transduction pathway to stimulate aggregation, sporulation, and C-signal-dependent gene expression, act in independent genetic pathways. We suggest that the signal transduction pathways defined by TodK and SdeK act in concert with the C-signal transduction pathway to control the timing of aggregation and sporulation.

A recurring theme in morphogenesis is the coupling of the expression of genes that drive morphogenesis and the morphogenetic process per se. This coupling ensures that gene expression and morphogenesis are carried out in synchrony. Morphogenesis of the spore-filled fruiting bodies in Myxococcus xanthus illustrates this coupling in the construction of a multicellular structure. Fruiting body formation involves two stages: aggregation of cells into mounds and the position-specific sporulation of cells that have accumulated inside mounds. Developmental gene expression propels these two processes. In addition, gene expression in individual cells is adjusted according to their spatial position. Progress in the understanding of the cell surface-associated C-signal is beginning to reveal the framework of an intercellular signalling system that allows the coupling of gene expression and multicellular morphogenesis. Accumulation of the C-signal is tightly regulated and involves transcriptional activation of the csgA gene and proteolysis of the full-length CsgA protein to produce the shorter cell surface-associated 17 kDa C-signal protein. The C-signal induces aggregation, sporulation and developmental gene expression at specific thresholds. The ordered increase in C-signalling levels, in combination with the specific thresholds, allows the C-signal to induce these three processes in the correct temporal order. The contact-dependent C-signal transmission mechanism, in turn, guarantees that C-signalling levels reflect the spatial position of individual cells relative to other cells and, thus, allows the cells to decode their spatial position during morphogenesis. By this mechanism, individual cells can tailor their gene expression profile to one that matches their spatial position. In this scheme, the molecular device that keeps gene expression in individual cells in register with morphogenesis is the C-signalling system, and the morphological structure, which is assessed, is the spatial position of individual cells relative to that of other cells.

Background: Chemotherapy is widely used in patients with recurrent head and neck cancer, but no clear markers are available that can predict response to treatment or survival in these patients., Methods: Twenty-nine patients evaluated in this study had recurrent head and neck squamous carcinomas, previously treated with surgery and/or radiotherapy. Patients received either cisplatin and 5-fluorouracil (5-FU) (n = 15) or cisplatin and paclitaxel (Taxol) (n = 14). Expression of c-erbB2, p53, glutathione S-transferase pi, multidrug resistance-associated protein, thymidylate synthase, and glutathione synthetase were evaluated in biopsy tissues., Results: Response to chemotherapy was significantly correlated with improved survival (progression-free survival, p =.0005; overall survival, p =. 007). Of the factors examined, expression of c-erbB2 was associated with significantly decreased progression-free survival (p =.023) and overall survival (p =.029). This was seen in patients treated with cisplatin/taxol but not in patients treated with cisplatin/5-FU., Conclusion: Expression of c-erbB2 may be a clinically useful predictor of survival in this group of patients.

Purpose: To evaluate the correlation between cisplatin sensitivity, intracellular glutathione, and platinum/DNA adduct formation (measured by atomic absorption spectroscopy) in a series of seven head and neck cancer cell lines, and to evaluate the effect of biochemical modulation of glutathione on platinum/DNA adduct formation and repair., Methods: Cisplatin/DNA adducts were measured by atomic absorption spectroscopy. Glutathione content was measured by enzymatic assay and was modulated with buthionine sulfoximine. Apoptosis was measured by double-labeled flow cytometry., Results: Intracellular glutathione concentration was strongly correlated with cisplatin resistance (P = 0.002, R2 = 0.7). There was also a statistically significant inverse correlation between cisplatin/DNA adduct formation and the IC50 for cisplatin in these cell lines. (P = 0.0004, R2 = 0.67). In addition, resistant cells were able to repair approximately 70% of cisplatin/DNA adducts at 24 h, while sensitive cells repaired less than 28% of adducts in the same period. However, despite the positive correlation between cellular glutathione and cisplatin resistance, there was no direct correlation between intracellular glutathione concentration and platinum/DNA adduct formation. Further, depletion of intracellular glutathione by buthionine sulfoximine did not dramatically alter formation of cisplatin/DNA adducts even though it resulted in marked increase in cisplatin cytotoxicity and was associated with increased apoptosis., Conclusions: These results suggest that glutathione has multiple effects not directly related to formation of cisplatin/DNA adducts, but may also be an important determinant of the cell's ability to repair cisplatin-induced DNA damage and resist apoptosis.

Neoadjuvant cisplatin-based chemotherapy has been widely used in the last decade for organ preservation or unresectable disease in advanced stage head and neck cancer. We examined the expression of a series of tumor markers that have been associated with chemotherapy resistance in pretreatment biopsies from 68 patients who received cisplatin-based neoadjuvant chemotherapy at either of two institutions. Patients received either cisplatin/5-fluorouracil (n = 49) or cisplatin/paclitaxel (n = 19). Expression of p53, glutathione S-transferase pi (GSTpi), thymidylate synthase (TS), c-erbB2, and multidrug resistance-associated protein was examined by immunohistochemistry. Expression of glutathione synthetase mRNA was measured by in situ hybridization. The overall response rate for cisplatin-based neoadjuvant treatment was 79%. The expression of several of the tumor markers was associated with resistance to neoadjuvant treatment, but none reached statistical significance. Overall survival (OS) was strongly correlated with the absence of p53 expression. The OS at 3 years was 81% in the p53-negative group, whereas it was 30% in the p53-positive group for patients treated with neoadjuvant chemotherapy (P < 0.0001). Expression of GST pi and TS was also significantly correlated with decreased OS after neoadjuvant treatment. At 3 years, the OS rate was 82% in the low GSTpi score group, compared to 46% in the high GSTpi score group (P = 0.0018). In the TS-negative group, the 3-year OS rate was 71% compared with 40% in the TS-positive group (P = 0.0071). We conclude that p53, GSTpi, and TS may be clinically important predictors of survival in patients receiving neoadjuvant chemotherapy for head and neck cancer.

Local environmental signals regulate the growth and development of both normal and malignant breast epithelium. Members of the insulin-like growth factor (IGF) family likely influence both of these processes. The localization of IGF2 to stroma specifically surrounding malignant breast epithelium indicates that this growth factor may play a critical role in the genesis or maintenance of this transformed phenotype. Recent studies have sought to understand the mechanism by which IGF2 expressing fibroblasts are localized to the periphery of malignant breast cancer cells. In addition, the consequences of the expression of IGF-signaling components likely expand beyond their direct effects on mitogenesis. Indirect effects predominantly associated with the IGF2 receptor could also influence the invasive potential of breast tumor cells.

Purpose: To correlate cellular glutathione content and gamma-glutamyl cysteine synthetase (gamma GCS) mRNA expression with cisplatin sensitivity in a panel of seven head and neck squamous cancer cell lines., Methods: Cisplatin IC50 was determined for each cell line using a sodium tetreazolium (XTT) assay. Cellular glutathione content was measured by using a previously reported enzymic method. gamma GCS mRNA expression was measured using an RNase protection assay., Results: Total cellular glutathione was an excellent predictor of cisplatin sensitivity in this series of cell lines. The IC50 for cisplatin in the cell line with the highest glutathione concentration was approximately 90 times higher than in the cell line with the lowest glutathione concentration. Regression analysis showed a highly statistically significant positive correlation between cisplatin IC50 and cellular glutathione (coefficient of determination R2 = 0.81, P = 0.0012). Some-what surprisingly, in contrast to previous studies in ovarian cancer, gamma GCS mRNA expression in these cell lines was not significantly predictive of either total cellular glutathione or cisplatin sensitivity (R2 = 0.005, P = 0.84). As expected, treatment of resistant cell lines with buthionine sulfoximine resulted in decreased cellular glutathione and enhanced cisplatin sensitivity., Conclusions: Our results suggest that glutathione may be an important determinant of cisplatin sensitivity in clinical head and neck cancer. Since cisplatin is the most active chemotherapy drug for the treatment of this disease, this correlation may have important clinical relevance. The lack of correlation between glutathione level and gamma GCS expression suggests that salvage or alternate synthetic pathways may be critical in these cells.

The glutathione S-transferases (GSTs) play an important role in the cell's defense against toxic substances. The GSTs are a family of enzymes produced by several genes that interact with distinct but overlapping substrates and that may play a role in resistance of tumor cells to several chemotherapeutic agents. We examined the correlation between expression of GSTs determined by immunohistochemistry and clinical response to platinum-based chemotherapy in 51 patients with head and neck cancer, who received a total of 56 courses of chemotherapy. The overall response rate for the 56 chemotherapy treatment courses was 48%. The overall response rate (complete response + partial response) for patients with low GST scores was 88% (21 of 24), whereas among the patients with high GST scores, the overall response rate was 19% (6 of 32; P = 0.001). Patients with a low GST score were 4.7 times more likely to respond to chemotherapy than patients with high GST scores. GST scores corresponded to response in 84% of cases. Among 23 patients treated with neoadjuvant chemotherapy, the overall response rate for patients with low GST scores was 100% (14 of 14), whereas among the patients with high GST scores, the overall response rate was 44% (4 of 9; P = 0.002). Among 33 patients treated with chemotherapy for relapsed disease, the overall response rate for patients with low GST scores was 70% (7 of 10), whereas among the patients with high GST scores, the overall response rate was 8.6% (2 of 23; P < 0.001). We conclude that GST expression correlates well with response to platinum-based chemotherapy in head and neck cancer.