Integration of eye health into primary care services in Tanzania: a qualitative investigation of experiences in two districts.

BACKGROUND: Visual impairment is a public health problem in sub-Saharan Africa, affecting nearly 5% of the population. Efforts to combat avoidable causes have been hampered by weak health systems and little evidence exists to suggest what interventions may be effective to improve the situation. Despite this, there are calls to promote some specific interventions, one of which being the closer integration of eye health services into health systems, often focusing on training primary health workers to deliver basic eye health services. This study seeks to understand how eye health services are delivered by primary health workers who have received training and what constraints remain to effective service provision. METHODS: This was a qualitative investigation into the experiences of 20 primary health workers trained in primary eye care and eight key informants working within specialist eye health services or regional and district health management positions in two districts in Tanzania. RESULTS: Despite feeling confident in their own eye care skills, most primary health workers felt constrained in the services they could provide to their communities by insufficient resources needed for diagnosis and treatment, and by lack of systematic supportive supervision to their work. Specialist ophthalmic staff were aware of this issue, although for the most part they felt it was not within their capacity to remedy and that it fell within the remit of general health managers. Many participants discussed the low support to eye health from the national government, evidenced through the lack of dedicated funding to the area and traditional reliance on outside funds including international charities. CONCLUSIONS: Although training of primary health workers is useful, it is recognised that is not sufficient to address the burden of eye health disease present in rural communities in Tanzania. It is likely that broader engagement with the general health system, and most likely with the private sector, will be necessary to improve the coverage of eye health care to remote and poor communities such as those in Morogoro. Further investment is needed to develop innovative approaches to delivering eye health services, including preventative, curative and rehabilitative services.

Laparoscopic Gastroepiploic Lymphovascular Pedicle Harvesting for the Treatment of Extremity Lymphedema: A Novel Technique.

Vascularized lymph node transfers have multiple donor sites with risk of iatrogenic lymphedema. We sought to describe in detail a surgical technique that is safe, reproducible, and efficient in harvesting gastroepiploic vascularized lymph nodes using real-time indocyanine green (ICG) fluorescent imaging. Photographs and video were acquired from a case to depict a step-by-step approach. ICG was endoscopically injected into the submucosa of the greater curvature of the stomach at the outset of the procedure. A laparoscopic harvest of the gastroepiploic vascular pedicle and lymph nodes ensued with the assistance of fluorescent imaging. Laparoscopic gastroepiploic lymph node harvesting aided by real-time ICG fluorescent mapping technique is safe, feasible, and effective at gathering vascularized lymphatic tissue for successful lymph node transfer in patients with severe lymphedema.

Mixed methods evaluation of a primary eye care training programme for primary health workers in Morogoro Tanzania.

BACKGROUND: There are 285 million people with visual impairment (VI) worldwide including 39 million who are blind; 15 % of those with VI live in Africa, and around 80 % of VI is preventable or treatable with the right equipment, information and skills. The scarcity of human resources for eye health, particularly in Sub-Saharan Africa, is a key challenge towards achieving this goal. Therefore training primary health workers (PHW) in providing eye-care services has been seen by some authors as a way to improve access to eye-care services in remote communities. However, the package of interventions which could be effectively delivered for eye-care at the primary-care level or the set of skills and competencies that PHWs need has not yet been delineated. The aim of the study was to evaluate the effectiveness of a four day training programme of PHWs in primary eye-care conducted in Morogoro, Tanzania in 2010/2011. METHODS: A mixed methods study using pre- and immediate post-training knowledge assessment of 60 trainees, and in-depth face to face interviews with 20 PHWs and 8 service managers 2 to 3 years after the training. RESULTS: Pre-and immediate post-training assessments indicated improvement in health worker knowledge about eye-care in the short term. Qualitative investigations 2 to 3 years after the training showed that although staff could make the correct management decisions when presented with eye-health problems, they often could not make a correct diagnosis. PHWs and managers reported satisfaction with the content of the training but some of the less well qualified staff found it overwhelming. Theoretical teaching was appreciated by most participants but almost all suggested increasing the time spent on acquiring skills. The training manual was accepted by many and some improvements were recommended. All interviewed PHWs were keen to improve their skills and knowledge. Acquired skills and knowledge were used for identification, referral of patients and for eye-health promotion. CONCLUSION: The training program in Morogoro was considered by PHWs as broadly successful and satisfying in terms of content, methods and duration of training. However, any future programme needs to be considered within the context of strengthening wider health systems.

Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals.

Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV(-)) and co-infected (HIV(+)) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV(-) individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV(+) individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV(-) TB, 0.95 for HIV(+) TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB.

Hereditary pancreatitis: endoscopic and surgical management.

INTRODUCTION: Hereditary pancreatitis is a rare cause of chronic pancreatitis. In recent years, genetic mutations have been characterized. The rarity of this disorder has resulted in a gap in clinical knowledge. The aims were to characterize patients with hereditary pancreatitis and establish clinical guidelines. METHODS: Pediatric and adult endoscopic, surgical, radiologic, and genetic databases from 1998 to 2012 were searched. Patients with recurrent acute or chronic pancreatitis and genetic mutation for either PRSS-1, SPINK-1, or CFTR or those who met the family history criteria were included. Patients with pancreatitis due to other causes, without a positive family history, familial pancreatic cancer, or cystic fibrosis, were excluded. RESULTS: Eighty-seven patients were identified. Genetic testing confirmed the diagnosis in 54 patients (62 %). Eighty-five patients (98 %) underwent 263 endoscopic procedures including sphincterotomy (72 %), stone removal (49 %), and pancreatic duct stenting (82 %). Twenty-eight patients (32 %) have undergone 37 operations which included 19 resections and 18 drainage procedures. The interval between procedures for recurrent pain was longer for surgery than for endoscopic therapy (9.1 vs. 3.4 years, p < 0.05). CONCLUSIONS: Most children and young adults with hereditary pancreatitis can be managed initially with endoscopic therapy. When surgery is undertaken, the procedure should be tailored to the pancreatic anatomy and cancer risk.

Protein biomarker quantification by mass spectrometry.

The protein biomarker field is becoming increasingly interested in multiple reaction monitoring mass spectrometry (MRM-MS) assays for biomarker tests. Originally developed years ago and used extensively to quantify small molecules, this technique is now being adapted to peptides. A summary is presented of MRM-MS techniques for quantification of protein biomarkers, including biomarker panels, and clinical applications of this approach. A survey of the current literature relating to the use of MRM-MS to quantify protein biomarker panels was conducted. Future directions for MRM-MS include qualification and verification of candidate protein biomarkers. Furthermore, the analytical characteristics of the MRM-MS approach make it ideally suited for the clinical laboratory as an assay for biomarker tests.

Intracellular adaptation of Brucella abortus.

Macrophages were infected with virulent Brucella abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration; adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strain's adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections.

Industrialized MS-based proteomics in the search for circulating biomarkers.

Proteomics is the study of the expression, structure and function of proteins under a range of cellular conditions. A rapidly evolving component of this field is clinical proteomics, which focuses on proteins involved in human disease and how they are affected by therapeutic intervention. MS is the main analytical technology for identifying and quantifying proteins whose expression is modulated across the normal to disease continuum. Applying this technology to clinical samples, however, is particularly challenging due to high biological variability in the population, a variety of disease stages, nonuniform response to therapy, multiple concomitant treatments and special requirements for handling samples from clinical trials. Given these challenges, an 'industrialized' approach is best suited to clinical biomarker development, with its standard operating procedures, process control and 'chain of custody'. This review will focus, therefore, on MS-based industrialized proteomics for the discovery and verification of circulating candidate clinical protein biomarkers.

PARPs database: a LIMS systems for protein-protein interaction data mining or laboratory information management system.

BACKGROUND: In the "post-genome" era, mass spectrometry (MS) has become an important method for the analysis of proteins and the rapid advancement of this technique, in combination with other proteomics methods, results in an increasing amount of proteome data. This data must be archived and analysed using specialized bioinformatics tools. DESCRIPTION: We herein describe "PARPs database," a data analysis and management pipeline for liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. PARPs database is a web-based tool whose features include experiment annotation, protein database searching, protein sequence management, as well as data-mining of the peptides and proteins identified. CONCLUSION: Using this pipeline, we have successfully identified several interactions of biological significance between PARP-1 and other proteins, namely RFC-1, 2, 3, 4 and 5.

Peptide Shifter: enhancing separation reproducibility using correlated expression profiles.

Chromatographic protein and peptide separation technologies enable comprehensive proteomic analysis of plasma and other complex biological samples by mass spectrometry. However, as the number of separations and/or fractions increases, so does the number of peptides split across fraction boundaries. Irreproducibility of peptide chromatographic separation results in peptides on or near the boundary moving partially or entirely into adjacent fractions. Peptide shifting across fraction boundaries increases the variability of measured peptide abundance, and so there is a trade-off between proteomic comprehensiveness using separation technologies and accurate quantitative proteomic measurements. In this paper, a method for detecting and correcting split peptides, called Peptide Shifter, is introduced and evaluated. An essential component of Peptide Shifter is a global peptide expression profile analysis that allows the inference of the underlying peptide shift pattern without the use of peptide labeling or internal standards. A controlled proteomic analysis of plasma samples demonstrates a 34% decrease in peptide intensity variability after the application of Peptide Shifter.

Extensive cell envelope modulation is associated with virulence in Brucella abortus.

Brucella virulence is linked to components of the cell envelope and tightly connected to the function of the BvrR/BvrS sensory-regulatory system. To quantify the impact of BvrR/BvrS on cell envelope proteins, we performed a label-free mass spectrometry-based proteomic analysis of spontaneously released outer membrane fragments from four strains of Brucella abortus (wild type virulent, avirulent bvrR- and bvrS- mutants as well as reconstituted virulent bvrR+ (bvrR-/pbvrR+)). We identified 167 differentially expressed proteins, of which 25 were assigned to the outer membrane. Approximately half of the outer membrane proteins decreased in abundance, whereas half increased. Notably, expression of five Omp3 family proteins decreased whereas five lipoproteins increased in the mutant strains. In the periplasmic space, by contrast, approximately 80% of the 60 differentially expressed proteins were increased in at least one avirulent mutant. Periplasmic proteins are primarily involved in substrate uptake and transport, and a uniform increase in this class may indicate a nutritional stress response, possibly a consequence of defective outer membrane function. Virtually all proteins reverted to wild type levels in the reconstituted virulent bvrR+ strain. We propose that the wide changes in cell envelope protein expression relate to the markedly avirulent phenotype of bvrR- and bvrS- mutants and that Brucella virulence depends on regulatory networks involving cell envelope and metabolism rather than on discrete virulence factors. This model may be relevant to other alpha-Proteobacteria harboring BvrR/BvrS orthologous systems known to be essential for parasitism or endosymbiosis.

An evaluation of multidimensional fingerprinting in the context of clinical proteomics.

Multidimensional fingerprinting (MDF) utilizes measurable peptide characteristics to identify proteins. In this study, 3-D fingerprinting, namely, parent protein molecular weight, peptide mass, and peptide retention time on RPLC, is used to identify 331 differentially expressed proteins between normal and human colon cancer plasma membrane samples. A false discovery rate (FDR) procedure is introduced to evaluate the performance of MDF on the colon cancer dataset. This evaluation establishes a false protein identification rate below 15% for this dataset. Western blot analysis is performed to validate the differential expression of the MDF-identified protein VDAC1 on the original tissue samples. The limits of MDF are further assessed by a simulation study where key parameters such as database size, query size, and mass accuracy are varied. The results of this simulation study demonstrate that fingerprinting with three dimensions yields low FDR values even for large queries on the complete human proteome without the need for prior peptide sequencing by tandem mass spectrometry. Specifically, when mass accuracy is 10 ppm or lower, full human proteome searches can achieve FDR values of 10% or less.

Death with dignity: devising a withdrawal of treatment process.

In 2002, a study was undertaken at St Thomas' Hospital to ascertain whether nurses felt adequately prepared in caring for patients in the intensive care unit (ICU) during the withdrawal of treatment (WoT) process (Dean, 2002). The study concluded that nurses on the ICU were unclear of the process and lacked confidence and knowledge of WoT. This study inspired the establishment of a WoT steering group to address the many issues involved in this process.

Protein depletion from blood plasma using a volatile buffer.

Removal of high abundance proteins is widely used in sample processing for proteomics studies of blood plasma. Immunoaffinity (IA) depletion is currently the most specific method for performing this step. Historically, IA depletion matrices have been designed to be used with inorganic buffers. However, the presence of salts in depleted samples presents a particular problem, and these must be removed in order to make samples compatible with post-depletion processing. Desalting (dialysis, ultrafiltration, size-exclusion, etc.) usually diminishes sample integrity due to labware associated losses. Moreover, these steps require additional labor, increasing the processing time and cost of analysis. In order to avoid these problems, we have developed an IA method using a volatile buffer that can be removed from depleted samples by lyophilization. This method allows the execution of reproducible and efficient depletion of blood plasma in a semi-automated manner.

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D.

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.

Experimental and bioinformatic approaches for interrogating protein-protein interactions to determine protein function.

An ambitious goal of proteomics is to elucidate the structure, interactions and functions of all proteins within cells and organisms. One strategy to determine protein function is to identify the protein-protein interactions. The increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. A challenge for bioinformatics is to explore this disparate data and to uncover biologically relevant interactions and pathways. In parallel, there is clearly a need for the development of approaches that can predict novel protein-protein interaction networks in silico. Here, we present an overview of different experimental and bioinformatic methods to elucidate protein-protein interactions.

A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins.

A new class of poly(ADP-ribose) (pADPr)-binding proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), has been identified by a proteomic approach using matrix-assisted laser-desorption-ionization time-of-flight ('MALDI-TOF') MS. Liquid-phase isoelectric focusing with a Rotofor cell (Bio-Rad) allowed pre-fractionation of proteins extracted from HeLa cells. Rotofor protein fractions were further separated by SDS/PAGE and then transferred to a PVDF membrane. pADPr-binding proteins were analysed by autoradiography of the protein blot after incubation with (32)P-labelled automodified pADPr polymerase-1 (PARP-1). Peptide mass fingerprinting of selected bands identified the most abundant pADPr-binding proteins as hnRNPs, a family of proteins that bind pre-mRNA into functional complexes involved in mRNA maturation and transport to the cytoplasm. Sequence homology database searching against a previously reported pADPr-binding sequence motif revealed that the hnRNPs contain a putative pADPr-binding sequence pattern [Pleschke, Kleczkowska, Strohm and Althaus (2000) J. Biol. Chem. 275, 40974-40980]. pADPr-binding assays performed with synthetic peptides by the dot-blot technique and with nitrocellulose-transferred recombinant hnRNPs confirmed the pADPr-binding protein identification and the specificity of the interaction. These results could establish a link between increased levels of pADPr in DNA damaged cells and the modified protein expression pattern resulting from altered mRNA trafficking.

Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 methyltransferase.

Yeast disruptor of telomeric silencing-1 (DOT1) is involved in gene silencing and in the pachytene checkpoint during meiotic cell cycle. Here we show that the Dot1 protein possesses intrinsic histone methyltransferase (HMT) activity. When compared with Rmt1, another putative yeast HMT, Dot1 shows very distinct substrate specificity. While Rmt1 methylates histone H4, Dot1 targets histone H3. In contrast to Rmt1, which can only modify free histones, Dot1 activity is specific to nucleosomal substrates. This was also confirmed using native chromatin purified from yeast cells. We also demonstrate that, like its mammalian homolog PRMT1, Rmt1 specifically dimethylates an arginine residue at position 3 of histone H4 N-terminal tail. In surprising contrast, methylation by Dot1 occurs in the globular domain of nucleosomal histone H3. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis suggests that H3 lysine 79 is trimethylated by Dot1. The intrinsic nucleosomal histone H3 methyltransferase activity of Dot1 is certainly a key aspect of its function in gene silencing at telomeres, most likely by directly modulating chromatin structure and Sir protein localization. In agreement with a role in regulating localization of histone deacetylase complexes like SIR, an increase of bulk histone acetylation is detected in dot1- cells.