Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep

Abstract Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

Resumo Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.

Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep / Mutações no gene da proteína morfogenética óssea 15 causadoras de defeitos na secreção proteica em ovelhas inférteis Cholistani

Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.

The conserved domain database in 2023.

NLM's conserved domain database (CDD) is a collection of protein domain and protein family models constructed as multiple sequence alignments. Its main purpose is to provide annotation for protein and translated nucleotide sequences with the location of domain footprints and associated functional sites, and to define protein domain architecture as a basis for assigning gene product names and putative/predicted function. CDD has been available publicly for over 20 years and has grown substantially during that time. Maintaining an archive of pre-computed annotation continues to be a challenge and has slowed down the cadence of CDD releases. CDD curation staff builds hierarchical classifications of large protein domain families, adds models for novel domain families via surveillance of the protein 'dark matter' that currently lacks annotation, and now spends considerable effort on providing names and attribution for conserved domain architectures. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

Comparison of Biochemical Parameters in Patients with Hepatitis B, C, and Dual Hepatitis B and C in Northwest Pakistan.

The leading causes of hepatitis are viral infections, Hepatitis B virus (HBV) and Hepatitis C virus (HCV). Millions of people have been infected with these deadly viral infections worldwide, and in Pakistan, every tenth person is infected with these viruses. Different populations respond with different rates to infectious diseases due to host genomic differences. To evaluate and compare the biochemical parameters in different types of hepatitis (Hepatitis B, C, and Co-infection) and different ethnic groups, a total of 200 pre-screened patients were recruited from District Headquarters Teaching Hospital Dera Ismail Khan and Tank. Blood samples (5ml) were taken from patients and were assayed for biochemical parameters, including four liver function tests (LFTs) and two renal function tests (RFTs). In 200 patients, the mean scores of Alanine transaminase (ALT) were 376±335, 315±265, and 478±519 IU/L in HBV, HCV, and co-infected patients, respectively. Moreover, the mean score of ALT was 31±7.2 IU/l in the normal control group. All other biochemical parameters demonstrated elevated levels in co-infection, HBV, and HCV, respectively, except total proteins. The RFTs showed a threshold or upper normal limit (UNL); nonetheless, when compared to normal control subjects, RFTs parameters were high in infected patients, as compared to normal control. Ethnicity wise comparison of parameters indicated that Pushtoon ethnic group indicated a high degree of severity of HBV infection and co-infection, as compared to Saraiki and Rajpoot ethnic groups, while Saraiki ethnic group showed a higher severity of HCV than both of Pushtoon and Rajpoot. Rajpoot ethnic group was least affected than both Pushtoon and Saraiki ethnic groups. Co-infected patients were more severely affected, as compared to HBV and HCV patients. The ethnicity-wise study provided evidence that different ethnic groups showed different degrees of severity. There may be some genetic background involved in hepatitis B and C viral infection due to which all three ethnic groups showed different degrees of severity. In gender-wise comparisons, male patients were more affected than female patients.

Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep.

Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) contains nearly 200 000 bacterial and archaeal genomes and 150 million proteins with up-to-date annotation. Changes in the Prokaryotic Genome Annotation Pipeline (PGAP) since 2018 have resulted in a substantial reduction in spurious annotation. The hierarchical collection of protein family models (PFMs) used by PGAP as evidence for structural and functional annotation was expanded to over 35 000 protein profile hidden Markov models (HMMs), 12 300 BlastRules and 36 000 curated CDD architectures. As a result, >122 million or 79% of RefSeq proteins are now named based on a match to a curated PFM. Gene symbols, Enzyme Commission numbers or supporting publication attributes are available on over 40% of the PFMs and are inherited by the proteins and features they name, facilitating multi-genome analyses and connections to the literature. In adherence with the principles of FAIR (findable, accessible, interoperable, reusable), the PFMs are available in the Protein Family Models Entrez database to any user. Finally, the reference and representative genome set, a taxonomically diverse subset of RefSeq prokaryotic genomes, is now recalculated regularly and available for download and homology searches with BLAST. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/.

Factors affecting remission to salvage chemotherapy with EtoposideCisplatin/Etoposide-MethotrexateActinomycin D (EP-EMA regimen) among chemoresistant high-risk Gestational Trophoblastic Neoplasia patients admitted in a tertiary institution: A 10-year retrospective descriptive study / Philippine Journal of Obstetrics and Gynecology

Background@#Approximately 20%–25% of high-risk gestational trophoblastic neoplasia (GTN) patients initially treated with first-line chemotherapy regimen develop resistance to the regimen. The EP-EMA (Etoposide-cisplatin and etoposide, methotrexate and actinomycin D) regimen is the most commonly utilized second-line agent. @*Objective@#This study aimed to identify factors leading to remission using etoposide and cisplatin-etoposide, methotrexate, and Actinomycin D (EP-EMA) as salvage chemotherapy among resistant high-risk GTN.@*Methods@#This is a retrospective descriptive study that reviewed the medical records of patients admitted in the section of trophoblastic diseases diagnosed with high-risk GTN from January 2006 to December 2015. @*Results@#The medical records of 20 patients were retrieved and reviewed. The complete remission rate with EP-EMA is 60% (12/20). The overall survival rate for 1 year is 70% (14/20). Only 20% of the patients went home against advice and did not complete treatment. This regimen reported toxicities ranging from Grade 2–4 myelosuppression and electrolyte imbalance. Forty-five percent had Grade 4 neutropenia and Grade 2 anemia and 20% had Grade 2 thrombocytopenia. Hypokalemia and hypomagnesemia were noted in 8 patients (40%). Although not statistically significant, a trend showed that those in the remission group mostly had Stage III diseases with metastasis only in the lungs, prognostic score of between 7 and 12, and with beta-human chorionic gonadotropin (β-hCG) levels <10,000 mIu/ml at the start of EP-EMA treatment.@*Conclusion@#There is an improved response with EP-EMA chemotherapy across the years in our institution. Factors such as stage of disease, pulmonary metastasis, and low β-hCG at the start EP-EMA chemotherapy denote a possible good response and may contribute to patients' complete remission with EP-EMA chemotherapy. However, further studies with larger patient sample size are recommended to support the latter.

CDD/SPARCLE: the conserved domain database in 2020.

As NLM's Conserved Domain Database (CDD) enters its 20th year of operations as a publicly available resource, CDD curation staff continues to develop hierarchical classifications of widely distributed protein domain families, and to record conserved sites associated with molecular function, so that they can be mapped onto user queries in support of hypothesis-driven biomolecular research. CDD offers both an archive of pre-computed domain annotations as well as live search services for both single protein or nucleotide queries and larger sets of protein query sequences. CDD staff has continued to characterize protein families via conserved domain architectures and has built up a significant corpus of curated domain architectures in support of naming bacterial proteins in RefSeq. These architecture definitions are available via SPARCLE, the Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

PubMed Text Similarity Model and its application to curation efforts in the Conserved Domain Database.

This study proposes a text similarity model to help biocuration efforts of the Conserved Domain Database (CDD). CDD is a curated resource that catalogs annotated multiple sequence alignment models for ancient domains and full-length proteins. These models allow for fast searching and quick identification of conserved motifs in protein sequences via Reverse PSI-BLAST. In addition, CDD curators prepare summaries detailing the function of these conserved domains and specific protein families, based on published peer-reviewed articles. To facilitate information access for database users, it is desirable to specifically identify the referenced articles that support the assertions of curator-composed sentences. Moreover, CDD curators desire an alert system that scans the newly published literature and proposes related articles of relevance to the existing CDD records. Our approach to address these needs is a text similarity method that automatically maps a curator-written statement to candidate sentences extracted from the list of referenced articles, as well as the articles in the PubMed Central database. To evaluate this proposal, we paired CDD description sentences with the top 10 matching sentences from the literature, which were given to curators for review. Through this exercise, we discovered that we were able to map the articles in the reference list to the CDD description statements with an accuracy of 77%. In the dataset that was reviewed by curators, we were able to successfully provide references for 86% of the curator statements. In addition, we suggested new articles for curator review, which were accepted by curators to be added into the reference list at an acceptance rate of 50%. Through this process, we developed a substantial corpus of similar sentences from biomedical articles on protein sequence, structure and function research, which constitute the CDD text similarity corpus. This corpus contains 5159 sentence pairs judged for their similarity on a scale from 1 (low) to 5 (high) doubly annotated by four CDD curators. Curator-assigned similarity scores have a Pearson correlation coefficient of 0.70 and an inter-annotator agreement of 85%. To date, this is the largest biomedical text similarity resource that has been manually judged, evaluated and made publicly available to the community to foster research and development of text similarity algorithms.

RefSeq: an update on prokaryotic genome annotation and curation.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) provides annotation for over 95 000 prokaryotic genomes that meet standards for sequence quality, completeness, and freedom from contamination. Genomes are annotated by a single Prokaryotic Genome Annotation Pipeline (PGAP) to provide users with a resource that is as consistent and accurate as possible. Notable recent changes include the development of a hierarchical evidence scheme, a new focus on curating annotation evidence sources, the addition and curation of protein profile hidden Markov models (HMMs), release of an updated pipeline (PGAP-4), and comprehensive re-annotation of RefSeq prokaryotic genomes. Antimicrobial resistance proteins have been reannotated comprehensively, improved structural annotation of insertion sequence transposases and selenoproteins is provided, curated complex domain architectures have given upgraded names to millions of multidomain proteins, and we introduce a new kind of annotation rule-BlastRules. Continual curation of supporting evidence, and propagation of improved names onto RefSeq proteins ensures that the functional annotation of genomes is kept current. An increasing share of our annotation now derives from HMMs and other sets of annotation rules that are portable by nature, and available for download and for reuse by other investigators. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/.

CDD/SPARCLE: functional classification of proteins via subfamily domain architectures.

NCBI's Conserved Domain Database (CDD) aims at annotating biomolecular sequences with the location of evolutionarily conserved protein domain footprints, and functional sites inferred from such footprints. An archive of pre-computed domain annotation is maintained for proteins tracked by NCBI's Entrez database, and live search services are offered as well. CDD curation staff supplements a comprehensive collection of protein domain and protein family models, which have been imported from external providers, with representations of selected domain families that are curated in-house and organized into hierarchical classifications of functionally distinct families and sub-families. CDD also supports comparative analyses of protein families via conserved domain architectures, and a recent curation effort focuses on providing functional characterizations of distinct subfamily architectures using SPARCLE: Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

Analysis of Workflow and Time to Treatment on Thrombectomy Outcome in the Endovascular Treatment for Small Core and Proximal Occlusion Ischemic Stroke (ESCAPE) Randomized, Controlled Trial.

BACKGROUND: The Endovascular Treatment for Small Core and Proximal Occlusion Ischemic Stroke (ESCAPE) trial used innovative imaging and aggressive target time metrics to demonstrate the benefit of endovascular treatment in patients with acute ischemic stroke. We analyze the impact of time on clinical outcome and the effect of patient, hospital, and health system characteristics on workflow within the trial. METHODS AND RESULTS: Relationship between outcome (modified Rankin Scale) and interval times was modeled by using logistic regression. Association between time intervals (stroke onset to arrival in endovascular-capable hospital, to qualifying computed tomography, to groin puncture, and to reperfusion) and patient, hospital, and health system characteristics were modeled by using negative binomial regression. Every 30-minute increase in computed tomography-to-reperfusion time reduced the probability of achieving a functionally independent outcome (90-day modified Rankin Scale 0-2) by 8.3% (P=0.006). Symptom onset-to-imaging time was not associated with outcome (P>0.05). Onset-to-endovascular hospital arrival time was 42% (34 minutes) longer among patients receiving intravenous alteplase at the referring hospital (drip and ship) versus direct transfer (mothership). Computed tomography-to-groin puncture time was 15% (8 minutes) shorter among patients presenting during work hours versus off hours, 41% (24 minutes) shorter in drip-ship patients versus mothership, and 43% (22 minutes) longer when general anesthesia was administered. The use of a balloon guide catheter during endovascular procedures shortened puncture-to-reperfusion time by 21% (8 minutes). CONCLUSIONS: Imaging-to-reperfusion time is a significant predictor of outcome in the ESCAPE trial. Inefficiencies in triaging, off-hour presentation, intravenous alteplase administration, use of general anesthesia, and endovascular techniques offer major opportunities for improvement in workflow. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01778335.

Improving the consistency of domain annotation within the Conserved Domain Database.

When annotating protein sequences with the footprints of evolutionarily conserved domains, conservative score or E-value thresholds need to be applied for RPS-BLAST hits, to avoid many false positives. We notice that manual inspection and classification of hits gathered at a higher threshold can add a significant amount of valuable domain annotation. We report an automated algorithm that 'rescues' valuable borderline-scoring domain hits that are well-supported by domain architecture (DA, the sequential order of conserved domains in a protein query), including tandem repeats of domain hits reported at a more conservative threshold. This algorithm is now available as a selectable option on the public conserved domain search (CD-Search) pages. We also report on the possibility to 'suppress' domain hits close to the threshold based on a lack of well-supported DA and to implement this conservatively as an option in live conserved domain searches and for pre-computed results. Improving domain annotation consistency will in turn reduce the fraction of NR sequences with incomplete DAs.

Randomized assessment of rapid endovascular treatment of ischemic stroke.

BACKGROUND: Among patients with a proximal vessel occlusion in the anterior circulation, 60 to 80% of patients die within 90 days after stroke onset or do not regain functional independence despite alteplase treatment. We evaluated rapid endovascular treatment in addition to standard care in patients with acute ischemic stroke with a small infarct core, a proximal intracranial arterial occlusion, and moderate-to-good collateral circulation. METHODS: We randomly assigned participants to receive standard care (control group) or standard care plus endovascular treatment with the use of available thrombectomy devices (intervention group). Patients with a proximal intracranial occlusion in the anterior circulation were included up to 12 hours after symptom onset. Patients with a large infarct core or poor collateral circulation on computed tomography (CT) and CT angiography were excluded. Workflow times were measured against predetermined targets. The primary outcome was the score on the modified Rankin scale (range, 0 [no symptoms] to 6 [death]) at 90 days. A proportional odds model was used to calculate the common odds ratio as a measure of the likelihood that the intervention would lead to lower scores on the modified Rankin scale than would control care (shift analysis). RESULTS: The trial was stopped early because of efficacy. At 22 centers worldwide, 316 participants were enrolled, of whom 238 received intravenous alteplase (120 in the intervention group and 118 in the control group). In the intervention group, the median time from study CT of the head to first reperfusion was 84 minutes. The rate of functional independence (90-day modified Rankin score of 0 to 2) was increased with the intervention (53.0%, vs. 29.3% in the control group; P<0.001). The primary outcome favored the intervention (common odds ratio, 2.6; 95% confidence interval, 1.7 to 3.8; P<0.001), and the intervention was associated with reduced mortality (10.4%, vs. 19.0% in the control group; P=0.04). Symptomatic intracerebral hemorrhage occurred in 3.6% of participants in intervention group and 2.7% of participants in control group (P=0.75). CONCLUSIONS: Among patients with acute ischemic stroke with a proximal vessel occlusion, a small infarct core, and moderate-to-good collateral circulation, rapid endovascular treatment improved functional outcomes and reduced mortality. (Funded by Covidien and others; ESCAPE ClinicalTrials.gov number, NCT01778335.).

CDD: NCBI's conserved domain database.

NCBI's CDD, the Conserved Domain Database, enters its 15(th) year as a public resource for the annotation of proteins with the location of conserved domain footprints. Going forward, we strive to improve the coverage and consistency of domain annotation provided by CDD. We maintain a live search system as well as an archive of pre-computed domain annotation for sequences tracked in NCBI's Entrez protein database, which can be retrieved for single sequences or in bulk. We also maintain import procedures so that CDD contains domain models and domain definitions provided by several collections available in the public domain, as well as those produced by an in-house curation effort. The curation effort aims at increasing coverage and providing finer-grained classifications of common protein domains, for which a wealth of functional and structural data has become available. CDD curation generates alignment models of representative sequence fragments, which are in agreement with domain boundaries as observed in protein 3D structure, and which model the structurally conserved cores of domain families as well as annotate conserved features. CDD can be accessed at http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

Inhaled aztreonam for chronic Burkholderia infection in cystic fibrosis: a placebo-controlled trial.

BACKGROUND: Individuals with Burkholderia spp. infection have historically been excluded from efficacy trials of inhaled antibiotics, including aztreonam for inhalation solution (AZLI). METHODS: A double-blind, placebo-controlled, 24-week trial of continuous AZLI/placebo treatment was undertaken in individuals with cystic fibrosis (CF) and chronic Burkholderia spp. infection. All subjects also received usual medical care (determined by their physicians). Additional antibiotic use was not restricted. RESULTS: Baseline FEV1% predicted values ranged from 15.8% to 114.6%. No significant treatment differences (AZLI vs. placebo) were observed at week 24 for any endpoints, including FEV1% predicted, number of respiratory exacerbations requiring systemic/inhaled antibiotics, or hospitalizations. Continuous AZLI administration was well tolerated. Burkholderia spp. susceptibility to antibiotics commonly used in CF therapy showed little change. CONCLUSIONS: 24-weeks of continuous AZLI treatment did not significantly improve lung function in CF subjects with chronic Burkholderia spp. infection. Non-study antibiotic use may have confounded any potential AZLI effects.

Advancing clinical trial design in pulmonary hypertension.

In pulmonary hypertension, as in many other diseases, there is a need for a smarter approach to evaluating new treatments. The traditional randomized controlled trial has served medical science well, but constrains the development of treatments for rare diseases. A workshop was established to consider alternative clinical trial designs in pulmonary hypertension and here discusses their merits, limitations and challenges to implementation of novel approaches.

Eradication of Pseudomonas aeruginosa biofilms on cultured airway cells by a fosfomycin/tobramycin antibiotic combination.

Chronic biofilm formation by Pseudomonas aeruginosa in cystic fibrosis (CF) lungs is a major cause of morbidity and mortality for patients with CF. To gain insights into effectiveness of novel anti-infective therapies, the inhibitory effects of fosfomycin, tobramycin, and a 4:1 (wt/wt) fosfomycin/tobramycin combination (FTI) on Pseudomonas aeruginosa biofilms grown on cultured human CF-derived airway cells (CFBE41o-) were investigated. In preformed biofilms treated for 16 h with antibiotics, P. aeruginosa CFU per mL were reduced 4 log10 units by both FTI and tobramycin at 256 mg L(-1) , while fosfomycin alone had no effect. Importantly, the FTI treatment contained five times less tobramycin than the tobramycin-alone treatment. Inhibition of initial biofilm formation was achieved at 64 mg L(-1) FTI and 16 mg L(-1) tobramycin. Fosfomycin (1024 mg L(-1)) did not inhibit biofilm formation. Cytotoxicity was also determined by measuring lactate dehydrogenase (LDH). Intriguingly, sub-inhibitory concentrations of FTI (16 mg L(-1)) and tobramycin (4 mg L(-1)) and high concentrations of fosfomycin (1024 mg L(-1)) prevented bacterially mediated airway cell toxicity without a corresponding reduction in CFU. Overall, it was observed that FTI and tobramycin demonstrated comparable activity on biofilm formation and disruption. Decreased administration of tobramycin upon treatment with FTI might lead to a decrease in negative side effects of aminoglycosides.

CDD: conserved domains and protein three-dimensional structure.

CDD, the Conserved Domain Database, is part of NCBI's Entrez query and retrieval system and is also accessible via http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml. CDD provides annotation of protein sequences with the location of conserved domain footprints and functional sites inferred from these footprints. Pre-computed annotation is available via Entrez, and interactive search services accept single protein or nucleotide queries, as well as batch submissions of protein query sequences, utilizing RPS-BLAST to rapidly identify putative matches. CDD incorporates several protein domain and full-length protein model collections, and maintains an active curation effort that aims at providing fine grained classifications for major and well-characterized protein domain families, as supported by available protein three-dimensional (3D) structure and the published literature. To this date, the majority of protein 3D structures are represented by models tracked by CDD, and CDD curators are characterizing novel families that emerge from protein structure determination efforts.

CDD: a Conserved Domain Database for the functional annotation of proteins.

NCBI's Conserved Domain Database (CDD) is a resource for the annotation of protein sequences with the location of conserved domain footprints, and functional sites inferred from these footprints. CDD includes manually curated domain models that make use of protein 3D structure to refine domain models and provide insights into sequence/structure/function relationships. Manually curated models are organized hierarchically if they describe domain families that are clearly related by common descent. As CDD also imports domain family models from a variety of external sources, it is a partially redundant collection. To simplify protein annotation, redundant models and models describing homologous families are clustered into superfamilies. By default, domain footprints are annotated with the corresponding superfamily designation, on top of which specific annotation may indicate high-confidence assignment of family membership. Pre-computed domain annotation is available for proteins in the Entrez/Protein dataset, and a novel interface, Batch CD-Search, allows the computation and download of annotation for large sets of protein queries. CDD can be accessed via http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.