Barriers to research productivity amongst postgraduate trainees: results from a survey of 333 medical and surgical residents.

PURPOSE: We aimed to determine the perceptions of, barriers to, and predictors of research engagement amongst residents at a national level in Pakistan. METHODS: This cross-sectional study used REDCap for online survey dissemination to residents from 12 institutes accredited by the national accreditation body (College of Physicians and Surgeons Pakistan) for core medical and surgical specialties. Logistic regression was used to estimate associations between likelihood of publications and participant characteristics. RESULTS: The response rate was 79% (333/423), with 171 (51%) medical and 162 (49%) surgical residents. The mean ± standard deviation age was 28.8 ± 2.7 years; 137 (41%) were males and 195 (59%) females. More than half the residents, 202 (61%), had received research training, but 189 (57%) scored <33% on basic research knowledge. While most residents agreed on the positive impact of research on their careers (P = .012) and realized that they should be involved in it (P = .33), they also strongly believed that it was difficult to engage in research during training (P < .01). Only 60 (18%) trainees had published a paper in local and 37 (11%) in international journals, respectively. The most significant barriers to conducting research included time limitation due to clinical work, lack of financial support, and unavailability of data (P < .01). CONCLUSION: Residents have a positive attitude towards research. However, research engagement among residents is low. Improving research mentorship and creating systems that enable protected time and institutional access to data are needed to increase research output of postgraduate trainees. Key messages What is already known on this topic Postgraduate trainees benefit academically from research conducted during residency training. However, in low- and middle-income countries like Pakistan, research output among residents has remained low over the years. The nation has consistently produced very little research. What this study adds The current study helped shed light on the reasons for low research productivity amongst medical and surgical residents in Pakistan. How this study might affect research, practice, or policy The potential predictors for low research involvement highlighted in this study necessitate modification of the existing national residency curriculum to increase research engagement and productivity among residents. Not only can the potential factors be improved, but the study also helps in bringing stakeholders' attention to increasing research opportunities in Pakistan.

EUS-guided celiac plexus neurolysis for pain in pancreatic cancer patients - a meta-analysis and systematic review.

Background: Endoscopic ultrasound guided celiac plexus neurolysis (EUS- CPN) has been reported to be an effective way to help with pain in pancreatic cancer patient. The aim of our updated meta-analysis is to assess the efficacy of pain relief in patients with pancreatic cancer who underwent EUS guided neurolysis. Methods: Pooled proportions were calculated using both Mantel-Haenszel method (fixed effects model) and DerSimonian Laird method (random effects model). The heterogeneity among studies was tested using Cochran's Q test based upon inverse variance weights.  Results: Initial search identified 176 reference articles, of which 34 were selected and reviewed in detail. Sixteen studies that met the inclusion criteria were included in this analysis. The mean age of patients undergoing neurolysis was 56.31 ± 19.72 years. Number of males, N = 563 (57.4%), was higher than the number of females, N = 417 (42.5%). The pooled proportion of patients who showed pain relief with EUS-guided neurolysis was 71% (95% CI = 68-74). Bias calculated using Begg-Mazumdar was not significant (p = 0.8). In a subgroup analysis, when comparing the central and bilateral techniques, the pooled proportion of patients with pain relief was 66% (95% CI = 61-71) and 57% (95% CI = 48-67), respectively. Conclusions: Our results show that EUS guided CPN could provide relief in as much as 70% of patients with central neurolysis technique having some edge over peripheral neurolysis. Further larger scale randomized controlled trials may further help to elaborate the efficacy of central vs peripheral neurolysis.

Management and outcomes of intramedullary spinal cord tumors: A single center experience from a developing country.

BACKGROUND: Intraoperative neurophysiology, high magnification microscopes, and ultrasonic aspirators are considered essential aid for the safe resection of intramedullary spinal cord tumors (IMSCTs). Most centers in developing countries such as Pakistan still lack these facilities. The purpose of this study was to review the management of IMSCTs at our hospital and to determine factors associated with the outcomes of surgery. METHODS: This was a retrospective review of medical records of adult patients undergoing surgery for IMSCT over 12 years. The institutional ethical review committee approved this study. Data were collected regarding demographics, clinical and radiological features, and surgical details. Modified McCormick Scale was used to grade patients' neurological status at admission, discharge, and follow-up. Statistical analysis was performed using the Statistical Package for Social Sciences version 22. RESULTS: Forty three cases were reviewed. Mean age was 33.8 ± 15.1 years whereas median follow-up was 5 months (range: 0.25-96 months). Most patients had ependymoma (n = 16; 73%). Cervical region was the most commonly involved (n = 15; 34.9%). Gross total resection (GTR) was achieved in 30 cases (69.8%). The preoperative McCormick grade was significantly associated with follow-up McCormick grade (P value = 0.002). Eight patients (18.6%) underwent intraoperative electrophysiological monitoring, out of which GTR was achieved in all cases, and none had disease progression or recurrence. Ten patients received postoperative radiotherapy. Thirty five patients (81.4%) had progression free survival at last follow-up. CONCLUSIONS: We achieved a GTR rate of 68.9% for IMSCTs with limited resources. In few cases, where intraoperative electrophysiology was used, the rate of GTR was 100%. Preoperative neurological status was associated with better postoperative McCormick score.

Novel H6PDH mutations in two girls with premature adrenarche: 'apparent' and 'true' CRD can be differentiated by urinary steroid profiling.

CONTEXT: Inactivating mutations in the enzyme hexose-6-phosphate dehydrogenase (H6PDH, encoded by H6PD) cause apparent cortisone reductase deficiency (ACRD). H6PDH generates cofactor NADPH for 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1, encoded by HSD11B1) oxo-reductase activity, converting cortisone to cortisol. Inactivating mutations in HSD11B1 cause true cortisone reductase deficiency (CRD). Both ACRD and CRD present with hypothalamic-pituitary-adrenal (HPA) axis activation and adrenal hyperandrogenism. OBJECTIVE: To describe the clinical, biochemical and molecular characteristics of two additional female children with ACRD and to illustrate the diagnostic value of urinary steroid profiling in identifying and differentiating a total of six ACRD and four CRD cases. DESIGN: Clinical, biochemical and genetic assessment of two female patients presenting during childhood. In addition, results of urinary steroid profiling in a total of ten ACRD/CRD patients were compared to identify distinguishing characteristics. RESULTS: Case 1 was compound heterozygous for R109AfsX3 and a novel P146L missense mutation in H6PD. Case 2 was compound heterozygous for novel nonsense mutations Q325X and Y446X in H6PD. Mutant expression studies confirmed loss of H6PDH activity in both cases. Urinary steroid metabolite profiling by gas chromatography/mass spectrometry suggested ACRD in both cases. In addition, we were able to establish a steroid metabolite signature differentiating ACRD and CRD, providing a basis for genetic diagnosis and future individualised management. CONCLUSIONS: Steroid profile analysis of a 24-h urine collection provides a diagnostic method for discriminating between ACRD and CRD. This will provide a useful tool in stratifying unresolved adrenal hyperandrogenism in children with premature adrenarche and adult females with polycystic ovary syndrome (PCOS).

Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells.

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or -2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography-MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

The p85 subunit of phosphoinositide 3-kinase is associated with beta-catenin in the cadherin-based adhesion complex.

Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell-cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via beta-catenin. We also show that the interaction of p85 and beta-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.

Interaction of the type Ialpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4, 5-bisphosphate in the regulation of PLD2 activity.

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane trafficking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P(2): phosphatidylinositol 4-phosphate 5-kinase alpha (Type Ialpha PIPkinase). We find that both PLD1 and PLD2 interact with the Type Ialpha PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Ialpha PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P(2) is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid.

Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-biphosphate-specific PH domain.

The signalling pathway leading, for example, to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. Two PLD genes (PLD1 and PLD2) with similar domain structures have been doned and progress has been made in identifying the protein regulators of PLD1 activation, for example Arf and Rho family members. The activities of both PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and our sequence analysis suggested the presence of a pleckstrin homology (PH) domain in PLD1, although its absence has also been daimed. Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Furthermore, PLD1 bound specifically and with high affinity to lipid surfaces containing PI(4,5)P2 independently of the substrate phosphatidylcholine, suggesting a key role for the PH domain in PLD function. Importantly, a glutathione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayers containing PI(4,5)P2. Point mutations within the PLD1 PH domain inhibited enzyme activity, whereas deletion of the domain both inhibited enzyme activity and disrupted normal PLD1 localisation. Thus, the functional PH domain regulates PLD by mediating its interaction with polyphosphoinositide-containing membranes; this might also induce a conformational change, thereby regulating catalytic activity.

Characterization of the regulation of phospholipase D activity in the detergent-insoluble fraction of HL60 cells by protein kinase C and small G-proteins.

Phospholipase D (PLD) activity has been shown to be GTP-dependent both in vivo and in vitro. One protein that confers GTP sensitivity to PLD activity in vitro is the low-molecular-mass G-protein ADP-ribosylation factor (Arf). However, members of the Rho family and protein kinase C (PKC) have also been reported to activate PLD in various cell systems. We have characterized the stimulation of PLD in HL60 cell membranes by these proteins. The results demonstrate that a considerable proportion of HL60 PLD activity is located in a detergent-insoluble fraction of the cell membrane that is unlikely to be a caveolae-like domain, but is probably cytoskeletal. This PLD activity required the presence of Arf1, a Rho-family member and PKC for efficient catalysis of the lipid substrate, suggesting that the activity represents PLD1. We show that recombinant human PLD1b is regulated in a similar manner to HL60-membrane PLD, and that PKCalpha and PKCdelta are equally effective PLD activators. Therefore maximum PLD activity requires Arf, a Rho-family member and PKC, emphasizing the high degree of regulation of this enzyme.

3T3-L1 adipocytes express two isoforms of phospholipase D in distinct subcellular compartments.

Phospholipase D has been implicated as an important enzyme in a range of cellular responses, including regulated secretion and the formation of secretory vesicles, cell proliferation and control of cell morphology. As insulin treatment of adipocytes has been shown to stimulate a phosphatidylcholine-specific phospholipase D and also modulates membrane trafficking, we wished to determine which isoform(s) of phospholipase D were present within adipocytes, to identify their subcellular distribution, and examine how this distribution may change in response to insulin. Using RT-PCR, 3T3-L1 adipocytes were found to express two isoforms of phospholipase D, specifically PLD1b and PLD2a. Using isoform-specific antibodies, PLD1 and PLD2 were found to be present predominantly in intracellular membranes, unlike the situation reported in other cells. Detailed analysis of the intracellular localisation of PLD1 and PLD2 revealed that these isoforms are differentially localised within adipocytes, implying functionally distinct roles for PLD activity in distinct subcellular compartments.

Phospholipase D1 localises to secretory granules and lysosomes and is plasma-membrane translocated on cellular stimulation.

Phospholipase D (PLD) activity has been implicated in the regulation of membrane trafficking [1,2], superoxide generation and cytoskeletal remodelling [3,4]. Several PLD genes have now been identified and it is probable that different isoforms regulate distinct functions. Defining the subcellular localisation of each isoform would facilitate understanding of their roles. Previous PLD localisation studies have been based largely on enzyme activity measurements, which cannot distinguish between isoforms [2,5]. We have cloned the cDNAs encoding human PLD1a and PLD1b from an HL60 cell cDNA library and expressed them as catalytically active fusion proteins with green fluorescent protein (GFP) in COS-1 cells and RBL-2H3 cells, a mast cell model which degranulates upon cross-linking of the high-affinity immunoglobulin E (IgE) receptor. In unstimulated cells, GFP-PLD1b colocalised with secretory granule and lysosomal markers; it was not found at the plasma membrane or nucleus and did not colocalise with markers for the Golgi. Stimulation or RBL-2H3 cells through IgE receptor cross-linking caused plasma membrane recruitment of GFP-PLD1b. Inhibition of IgE-receptor-stimulated, PLD-catalysed phosphatidate formation suppressed secretion of granule and lysosomal contents, but did not affect translocation of GFP-PLD1b. These experiments suggest that PLD1 plays a role in regulated exocytosis rather than endoplasmic reticulum (ER) to Golgi membrane transport.

Phospholipase D.

Phospholipase D catalyses the hydrolysis of phosphatidylcholine to generate phosphatidate. The regulation of PLD activity is complex involving a number of small GTP binding proteins, but in particular Arf and Rho, phosphatidylinositol 4,5-bisphosphate and protein kinase C. The cDNA for PLD1 has recently been cloned and shows homology to the yeast and plant genes but only within four domains. Domains I and IV each contain a putative catalytic triad. PLD activity has been detected in plasma membranes, Golgi membranes and in nuclear membranes; it is unclear if different isoenzymes are responsible for this variation, or if the PLDs are differently regulated. The product of PLD activity, PA, appears to be a messenger molecule regulating the actin cytoskeleton and maybe playing a role in the control of membrane traffic and secretion.

The expression of Escherichia coli diaminopimelate decarboxylase in mouse 3T3 cells.

We have subcloned the coding sequence for the Escherichia coli lysA gene coding for diaminopimelic acid decarboxylase (DAP decarboxylase) into a eukaryotic expression vector based on the SV40 early promoter. The activities of a series of constructs with different lengths of non-coding DNA at the 5' and 3' ends of the coding region have been compared by measuring the synthesis of lysine from diaminopimelic acid (DAP) in mouse 3T3 cells. A short non-coding sequence at the 3' end reduced the expression of enzyme activity. Stable lines of 3T3 cells have been produced by co-transfection of the chimeric gene with a plasmid coding for G-418 resistance. Cells were grown in medium containing G-418 and resistant clones were screened for an ability to synthesise lysine from DAP. [3H]Lysine produced from [3H]DAP was incorporated into cell proteins. An enzyme extract from a cell line which had incorporated two copies of the gene synthesised 0.082 nmol of lysine/min per mg protein. In the intact cell the rate of lysine synthesis is limited by the uptake of DAP which is taken up at only 5% of the rate of lysine. lysA has a potential as a reporter gene in studies of gene expression in mammalian cells.

Threonine synthesis from homoserine as a selectable marker in mammalian cells.

The plasmid pSVthrBC expresses the Escherichia coli thrB (homoserine kinase) and thrC (threonine synthase) genes in mouse cells and enables them to synthesize threonine from homoserine. After transfection with pSVthrBC and culture in medium containing homoserine, only cells that have incorporated pSVthrBC survive. Homoserine at concentrations greater than 1 mM is toxic to mammalian cells. Mouse cells selected from medium containing 5 mM homoserine had incorporated 20-100 copies of the plasmid per cell and had homoserine kinase activities of 0.001-0.012 nmol/min per mg of protein per copy. Cells selected from medium containing 10 mM homoserine had incorporated one or two copies of the plasmid per cell and had homoserine kinase activities of 0.06-0.39 nmol/min per mg of protein per copy. By using high concentrations of homoserine, it is possible to use pSVthrBC to select and isolate cell lines that have one or two copies of the plasmid incorporated into an active region of chromatin. CHO and HeLa cells have also been successfully transfected with pSVthrBC. COS-7 cells are naturally resistant to homoserine as they are able to metabolize homoserine.