The use of real-time digital video in the assessment of post-operative outcomes of breast reconstruction.

INTRODUCTION: Outcome measures of breast reconstruction have used panel assessment of photographs. This provides limited information to the assessor as these images are static.. The aim of this study was to assess whether the use of digital video was a valid assessment tool and to compare its use against photography. METHODS: 35 patients post-reconstruction underwent photography, digital video capture and completed Breast Cancer Treatment Outcomes Scale (BCTOS) questionnaires. The photographs/video clips were randomised and shown to a 21 member panel. Opinions on aesthetic aspects of the reconstruction were assessed using the BCTOS and Harris scale. Panel inter-rater agreement and patient-panel correlation was assessed using Kendall's Coefficient of Concordance and Spearman's rank correlation tests respectively. RESULTS: There was a "moderate" degree of inter-rater agreement amongst panel members in all categories. Greater agreement occurred using video footage to assess overall cosmesis (0.548 vs 0.507) and shape (0.505 vs 0.486). Video showed a greater degree of correlation with patient self-assessment scores in comparison to photography (0.311 vs 0.281). CONCLUSION: Video footage coupled with panel assessment is a valid method of assessing post-operative outcomes of breast reconstruction and appears superior to still photographs in terms of inter-rater agreement and correlation with patient self-assessment.

Short, natural, and extended photoperiod response in BC2F4 lines of bread wheat with different photoperiod-1 (Ppd-1) alleles.

Flowering is a critical period in the life cycle of flowering plant species, resulting in an irreversible commitment of significant resources. Wheat is photoperiod sensitive, flowering only when daylength surpasses a critical length; however, photoperiod insensitivity (PI) has been selected by plant breeders for >40 years to enhance yield in certain environments. Control of flowering time has been greatly facilitated by the development of molecular markers for the Photoperiod-1 (Ppd-1) homeoloci, on the group 2 chromosomes. In the current study, an allelic series of BC2F4 lines in the winter wheat cultivars 'Robigus' and 'Alchemy' was developed to elucidate the influence on flowering of eight gene variants from the B- and D-genomes of bread wheat and the A-genome of durum wheat. Allele effects were tested in short, natural, and extended photoperiods in the field and controlled environments. Across genetic background and treatment, the D-genome PI allele, Ppd-D1a, had a more potent effect on reducing flowering time than Ppd-B1a. However, there was significant donor allele effect for both Ppd-D1a and Ppd-B1a, suggesting the presence of linked modifier genes and/or additional sources of latent sensitivity. Development of Ppd-A1a BC2F4 lines derived from synthetic hexaploid wheat provided an opportunity to compare directly the flowering time effect of the A-genome allele from durum with the B- and D-genome variants from bread wheat for the first time. Analyses indicated that the reducing effect of Ppd-A1a is comparable with that of Ppd-D1a, confirming it as a useful alternative source of PI.

Bilateral deep inferior epigastric perforator flaps based on unilateral perforators.

This report highlights the case of an unusual bilateral breast reconstruction using bilateral Deep Inferior Epigastric Perforator flaps based solely upon unilateral perforators. The usual lower paramedian perforating vessels were absent on one side and subsequently the main abdominal flap was split obliquely (rather than midline) to base both individual flaps on perforators arising from the same side. The operation was a success with no flap complications post-operatively.

Epitope-specific anti-nuclear antibodies are expressed in a mouse model of primary biliary cirrhosis and are cytokine-dependent.

Although the hallmark of primary biliary cirrhosis (PBC) is the presence of anti-mitochondrial antibodies (AMA), a significant number of patients have anti-nuclear antibodies (ANA) directed primarily against two nuclear proteins, gp210 and sp100. In PBC, there are considerable data on the specificity of these anti-nuclear antibodies as well as suggestive evidence that antibodies to gp210 predict a poor outcome. However, a further understanding of the significance of these autoantibodies has been hampered by limitations in accessing human subjects in a preclinical or early asymptomatic stage. To overcome this limitation, we have taken advantage of transgenic mice with abrogated transforming growth factor-ß signalling in T cells (dnTGF-ßRII) that develop histological features of PBC as well as the same AMA specificity. We studied these mice for serum ANA, including specific autoantibodies against gp210 and sp100. We further examined sera from dnTGF-ßRII mice with concurrent deletions of the genes encoding interleukin (IL)-12p35, IL-12p40, IL-23p19, IL-17, IL-6, interferon (IFN)-γ or tumour necrosis factor (TNF)-α. Sera from all the dnTGF-ßRII mouse lines contained antibodies against gp210 and sp100. Of significance, mice with germline deletions of the genes encoding IL-12p40, IL-23p19, IL-17, IL-6 and TNF-α had significantly lower titres of anti-gp210 antibodies. These results provide a platform to dissect the mechanisms of gp210 and sp100 autoantibody production in dnTGF-ßRII mice as well as to study the possible role of ANA in the pathophysiology of PBC.

A novel duplex real-time PCR for HPIV-4 detects co-circulation of both viral subtypes among ill children during 2008.

The two subtypes of the human parainfluenzavirus type 4 (HPIV-4) are rarely sought in testing for acute respiratory illness (ARI) and this may be confounding our understanding of its role. This study presents a novel duplex real-time RT-PCR assay targeting the P gene that can detect and differentiate the two subtypes in a single reaction. Subtype-specific synthetic RNA positive controls were prepared and used to determine an analytical sensitivity of 10 copies per reaction with an 8log(10) dynamic range. The assays were validated using 1140 clinical specimens mostly nasopharyngeal aspirates collected from children during 2008. These included specimens previously determined to be positive for all commonly considered respiratory viruses. The novel assay did not cross-reaction with any other virus. Fourteen HPIV-4 positives, ten detected in the absence of any co-detections (four with rhinovirus), were identified in 2008 and their subtype confirmed by conventional RT-PCR and sequencing of P gene fragments. Most detections were in children two years of age or younger. Our assay proved suitably sensitive and specific for inclusion in future studies seeking to better understand the role HPIV-4 and other respiratory viruses in children with ARI.

The Scottish-Finnish-Swedish PARTNER study of taprostene versus placebo treatment in patients with critical limb ischemia.

AIM: Atherosclerotic peripheral arterial disease is a major health problem in the western world, often manifested as intermittent claudication, affecting 10-20% males above 60 years. Ischemic complications can lead to rest pain, ulceration and gangrene. The treatment of choice for critical limb ischemia (CLI) is vascular reconstruction or endovascular interventions. Medical management with vasodilator antiplatelet prostaglandins, could be considered in patients unsuitable for surgery. Long term follow-up on previous prostaglandin studies has been insufficient to evaluate amputation rates. Hence this study evaluated safety and longer term efficacy of taprostene sodium, a prostacyclin (PGI2) analogue in CLI. The aim of this study was to determine whether Taprostene sodium, a PGI2 analogue, was a safe and effective treatment for CLI. METHODS: This paper reports the data from the Scottish-Finnish-Swedish PARTNER Study Group which consisted of a double-blind placebo controlled multi-centre study evaluating Taprostene compared to placebo. The primary endpoints were pain relief and early ulcer healing response at the end of the four week infusion phase and amputation at six months follow-up. The patients were randomly allocated to receive taprostene or placebo in a two to one randomization of active versus placebo. A total of 111 patients with CLI were recruited. Taprostene was given twice a day over two 2 hour periods for four weeks. The early response was evaluated at the end of the four week infusion phase. In patients with rest pain without ulceration, a positive response was complete pain relief without any requirement for analgesic therapy. However in patients with ulceration, a positive response was defined as a decrease in the ulcer size by >30%. Amputation scores were compared at the end of the 6 months follow-up period for all participants. RESULTS: Seventy-four patients received taprostene and 37 placebo. Overall, 61 male patients were enrolled in the study along with 50 females with 11% more women in the taprostene (active) group. For both patients with and without ulcers there was no statistically significant difference noted in the early response between those receiving taprostene and those receiving placebo infusion. The percentage of patients without any amputations was 43% in the taprostene group compared to 38% in the control group at the end of six months; however, these results were not statistically significant. CONCLUSION: Although a reasonable number of patients enrolled in the study it has not been possible to demonstrate any statistically significant benefit of taprostene over placebo. This may be due to more patients with risk factors for peripheral artery disease (PAD) such as hypertension, diabetes mellitus and cigarette smoking in the actively treated group and also due the increased number of women in the active group who are known to generally respond less favourably to antiplatelet agents.

Reanalyses of the historical series of UK variety trials to quantify the contributions of genetic and environmental factors to trends and variability in yield over time.

Historical datasets have much to offer. We analyse data from winter wheat, spring and winter barley, oil seed rape, sugar beet and forage maize from the UK National List and Recommended List trials over the period 1948-2007. We find that since 1982, for the cereal crops and oil seed rape, at least 88% of the improvement in yield is attributable to genetic improvement, with little evidence that changes in agronomy have improved yields. In contrast, in the same time period, plant breeding and changes in agronomy have contributed almost equally to increased yields of forage maize and sugar beet. For the cereals prior to 1982, contributions from plant breeding were 42, 60 and 86% for winter barley, winter wheat and spring barley, respectively. These results demonstrate the overwhelming importance of plant breeding in increasing crop productivity in the UK. Winter wheat data are analysed in more detail to exemplify the use of historical data series to study and detect disease resistance breakdown, sensitivity of varieties to climatic factors, and also to test methods of genomic selection. We show that breakdown of disease resistance can cause biased estimates of variety and year effects, but that comparison of results between fungicide treated and untreated trials over years may be a means to screen for durable resistance. We find the greatest sensitivities of the winter wheat germplasm to seasonal differences in rainfall and temperature are to summer rainfall and winter temperature. Finally, for genomic selection, correlations between observed and predicted yield ranged from 0.17 to 0.83. The high correlation resulted from markers predicting kinship amongst lines rather than tagging multiple QTL. We believe the full value of these data will come from exploiting links with other experiments and experimental populations. However, not to exploit such valuable historical datasets is wasteful.

Molecular characterization and distinguishing features of a novel human rhinovirus (HRV) C, HRVC-QCE, detected in children with fever, cough and wheeze during 2003.

BACKGROUND: Human rhinoviruses (HRVs) are associated with more acute respiratory tract infections than any other viral group yet we know little about viral diversity, epidemiology or clinical outcome resulting from infection by strains, in particular the recently identified HRVs. OBJECTIVES: To determine whether HRVC-QCE was a distinct HRV-C strain, by determining its genome and prevalence, by cataloguing genomic features for strain discrimination and by observing clinical features in positive patients. STUDY DESIGN: Novel real-time RT-PCRs and retrospective chart reviews were used to investigate a well-defined population of 1247 specimen extracts to observe the prevalence and the clinical features of each HRV-QCE positive case from an in- and out-patient pediatric, hospital-based population during 2003. An objective illness severity score was determined for each HRVC-QCE positive patient. RESULTS: Differences in overall polyprotein and VP1 binding pocket residues and the predicted presence of a cis-acting replication element in 1B defined HRVC-QCE as a novel HRV-C strain. Twelve additional HRVC-QCE detections (1.0% prevalence) occurred among infants and toddlers (1-24 months) suffering mild to moderate illness, including fever and cough, who were often hospitalized. HRVC-QCE was frequently detected in the absence of another virus and was the only virus detected in three (23% of HRVC-QCE positives) children with asthma exacerbation and in two (15%) toddlers with febrile convulsion. CONCLUSIONS: HRVC-QCE is a newly identified, genetically distinct HRV strain detected in hospitalized children with a range of clinical features. HRV strains should be independently considered to ensure we do not overestimate the HRVs in asymptomatic illness.

Sexual dimorphism in autoimmune disease.

We briefly survey the concept of autoimmunity and nominate the range of autoimmune diseases that include multisystemic and organ-specific disorders, and cite prevalences of autoimmune diseases in males and females, in humans and in experimental animals. Most human autoimmune diseases such as systemic lupus erythematosus (SLE), multiple sclerosis (MS) and autoimmune thyroid disease, have an increased incidence and prevalence in females, but a few others such as autoimmune diabetes, the Guillain Barré syndrome (GBS) and psoriasis are increased in males. Animal models of autoimmunity show an equivalent sexual dimorphism. The possible reasons for the differing incidence and prevalence of autoimmune diseases in females and males engage our attention. Environmental exposures may differ for females and males. There are innate differences in the function of the female and male immune systems, and there is some evidence for differences between females and males in the ability of a target organ for autoimmunity to withstand damage. In seeking reasons for these differences, we review the role of sex hormones in immunity and include results of trials of hormone therapy in autoimmune diseases. The association of autoimmunity and pregnancy, a female-specific condition, is discussed, and the claimed effects of lymphoid cell microchimerism on provocation of autoimmunity are reviewed. Genetic predisposition is an important factor in autoimmune disease and we particularly focus on genes on the X and Y chromosomes, the role of X chromosome inactivation, and the interaction of the sex of the patient with other genetic factors. The possible role of epigenetic mechanisms, including environmental influences, is then surveyed. We assert that sex is a vital variable that must be considered in all immunological studies, as it should be at all levels of biological research.

Do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections?

BACKGROUND: Human rhinoviruses (HRVs) are often concurrently detected with other viruses found in the respiratory tract because of the high total number of HRV infections occurring throughout the year. This feature has previously relegated HRVs to being considered passengers in acute respiratory infections. HRVs remain poorly characterized and are seldom included as a target in diagnostic panels despite their pathogenic potential, infection-associated healthcare expenditure and relatively unmoderated elicitation of an antiviral state. OBJECTIVES: To test the hypothesis that respiratory viruses are proportionately more or less likely to co-occur, particularly the HRVs. STUDY DESIGN: Retrospective PCR-based analyses of 1247 specimens for 17 viruses, including HRV strains, identified 131 specimens containing two or more targets. We investigated the proportions of co-detections and compared the proportion of upper vs. lower respiratory tract presentations in the HRV positive group. Both univariate contingency table and multivariate logistic regression analyses were conducted to identify trends of association among the viruses present in co-detections. RESULTS: Many of the co-detections occurred in patterns. In particular, HRV detection was associated with a reduced probability of detecting human adenoviruses, coronaviruses, bocavirus, metapneumovirus, respiratory syncytial virus, parainfluenza virus, influenza A virus, and the polyomaviruses KIPyV and WUPyV (p < or = 0.05). No single HRV species nor cluster of particular strains predominated. CONCLUSIONS: HRVs were proportionately under-represented among viral co-detections. For some period, HRVs may render the host less likely to be infected by other viruses.

A quantitative genomic imbalance gene expression assay in a hexaploid species: wheat (Triticum aestivum).

Responses to allopolyploidy include unequal expression of duplicated genes, gene silencing, and sometimes genomic rearrangements. In plants, the relationship between allelic expression differences arising from changes in regulatory regions and the resulting phenotype is poorly understood because of the complexity of their genomes and lack of efficient methodology to identify regulatory variation. Identifying functionally important regulatory variation in crops such as hexaploid wheat (Triticum aestivum) is in its infancy. More knowledge is required about the contribution of participatory genomes to its transcriptome. In this paper, we demonstrate the use of allelic imbalance assays to quantify relative expression levels across tissues and growth regimes of homoeologous transcripts of the A, B, and D genomes. Polymorphisms in the type I thionins have been used as an example. We show that expression levels vary markedly and interactively over all factors. For this gene, the B genome is the smallest contributor to the transcriptome and the D genome the largest. As additional sequence information is accumulated across genomes, this assay will allow the simple study of relative expression across multiple homeologous loci.

Induction of autoimmune cholangitis in non-obese diabetic (NOD).1101 mice following a chemical xenobiotic immunization.

Our laboratory has suggested that loss of tolerance to pyruvate dehydrogenase (PDC-E2) leads to an anti-mitochondrial antibody response and autoimmune cholangitis, similar to human primary biliary cirrhosis (PBC). We have suggested that this loss of tolerance can be induced either via chemical xenobiotic immunization or exposure to select bacteria. Our work has also highlighted the importance of genetic susceptibility. Using the non-obese diabetic (NOD) congenic strain 1101 (hereafter referred to as NOD.1101 mice), which has chromosome 3 regions from B6 introgressed onto a NOD background, we exposed animals to 2-octynoic acid (2OA) coupled to bovine serum albumin (BSA). 2OA has been demonstrated previously by a quantitative structural activity relationship to react as well as or better than lipoic acid to anti-mitochondrial antibodies. We demonstrate herein that NOD.1101 mice immunized with 2OA-BSA, but not with BSA alone, develop high titre anti-mitochondrial antibodies and histological features, including portal infiltrates enriched in CD8(+) cells and liver granulomas, similar to human PBC. We believe this model will allow the rigorous dissection of early immunogenetic cause of biliary damage.

Chronic complications and mortality in community-based patients with latent autoimmune diabetes in adults: the Fremantle Diabetes Study.

AIMS: To compare (i) the prevalence and incidence of chronic complications and (ii) cardiac and all-cause mortality in community-based patients with latent autoimmune diabetes in adults (LADA) with those in Type 2 diabetic patients without antibodies to glutamic acid decarboxylase (GAD). METHODS: Of the 1294 patients with clinically-defined Type 2 diabetes recruited to the longitudinal, observational Fremantle Diabetes Study between 1993 and 1996, 1255 (97%) had GAD antibodies measured at baseline. Complications were ascertained using standard criteria in patients returning for annual assessments until November 2001. Data on hospital admissions and mortality were available to the end of June 2006. Cox proportional hazards modelling was used to determine independent predictors of first occurrence of complications and cardiac and all-cause mortality. RESULTS: Forty-five (3.6%) subjects had LADA. Compared with the GAD antibody-negative patients, they had a similar prevalence and incidence of coronary heart (P = 0.48 and 0.80, respectively) and cerebrovascular (P = 0.64 and 0.29) disease and cardiac and all-cause mortality (P = 0.62 and 0.81, respectively). There was also a similar prevalence and incidence of retinopathy (P = 0.22 and 0.64, respectively) and neuropathy (P = 0.25 and 0.95), but microalbuminuria was less frequent both at baseline and during follow-up in the LADA subgroup in unadjusted models (P = 0.046) and after adjustment for other risk factors (P = 0.014 and 0.013). CONCLUSIONS: Except for a lower prevalence and incidence of nephropathy, LADA patients have a similar risk of complications and death to patients with clinically-diagnosed Type 2 diabetes without GAD antibodies. Cardiovascular risk factor management in LADA should, therefore, be as intensive as that for GAD antibody-negative patients.

An actin-myosin functional assay for analysis of smooth muscle (anti-microfilament) autoantibodies in human plasma.

The detection of serum autoantibodies to smooth muscle (SMA) on rodent gastric mucosa by indirect immunofluorescence (IIF) has long been an immunodiagnostic marker for autoimmune hepatitis type 1 (AIH-1). The reactive antigenic moieties are cytoskeletal proteins which include polymeric F-actin as judged by the staining of microfilaments of tissue by IIF. However, their specificity for actin in AIH-1 can be and usually is uncertain. Using an in vitro functional assay, we compared the effects of Fab fragments of immunoglobulin (IgG) prepared from SMA-positive plasma from two patients with the effects of Fabs from 10 healthy subjects. Fabs are incorporated into an assay where actin (the putative antigen) activates skeletal muscle heavy meromyosin (HMM) ATPase activity. The data from these functional assays provide new insights into the significance of anti-microfilament assays in the diagnosis, and perhaps also pathogenesis, of AIH-1.

The genetic diversity of UK, US and Australian cultivars of Triticum aestivum measured by DArT markers and considered by genome.

The genetic diversity of UK, US and Australian wheat varieties over the period of modern plant breeding is estimated using diversity array technology markers. Diversity is assessed by both genetic distance between varieties, by AMOVA and as the volumes of multi-dimensional convex hulls estimated from principal co-ordinate analysis. At the whole genome level the three populations are genetically distinct; this is also true of the B genome. However, the US and Australian D genomes are found to occupy the same region of diversity space and the A genomes for these countries are partially overlapping. The use of high-density genotyping with a common marker set allows an unprecedented direct comparison between the diversities of the national populations, between individual genomes and the fluctuation of diversity over time. The highest genetic diversity amongst varieties is reported in the Australian population followed by the US, which in turn is more diverse than the UK. However the average diversity of loci is higher in the US set than in the Australian. Non-random fluctuations in genetic diversity over time are observed.

A randomised, split-face comparison of facial hair removal with the alexandrite laser and intense pulsed light system.

INTRODUCTION: Despite the high incidence of polycystic ovary syndrome (PCOS) in women attending for facial hair removal there are few studies looking specifically at this patient group. We carried out a split-face study directly comparing the efficacy of a 3 milliseconds pulse duration alexandrite laser with the Lumina IPL system in 38 women with PCOS. MATERIALS AND METHODS: Each patient underwent six treatments using both systems, with 1, 3 and 6 months follow-up. Hair counts, hair-free intervals and patient satisfaction were recorded for all patients. RESULTS: After six treatments, alexandrite laser treatment resulted in longer median hair-free intervals when compared to IPL (7 weeks vs. 2 weeks; P < 0.001). Decrease in hair counts was significantly larger on the Alexandrite side compared to the IPL side at 1, 3 and 6 months (52%, 43% and 46% vs. 21%, 21% and 27%; P < 0.001). Patient satisfaction scores, using linear analogue scales (LAS), at 1, 3 and 6 months were significantly higher for the alexandrite laser than the IPL (8.7, 7.8 and 7.7 vs. 5.7, 5.1 and 5.1; P < or = 0.002). CONCLUSIONS: The alexandrite laser resulted in significantly longer hair-free intervals, a larger reduction in hair counts and greater patient satisfaction than the IPL and appeared to be more effective in this patient group. It is clear from the results in this study that the GentleLase alexandrite laser is more effective at reducing facial hirsutism in women with PCOS than the Lumina IPL. It is probable that this is due to the specific wavelength, short pulse duration and single pulse delivery of the GentleLase alexandrite laser, resulting in more follicular destruction than the IPL.

GAD autoantibodies and epitope reactivities persist after diagnosis in latent autoimmune diabetes in adults but do not predict disease progression: UKPDS 77.

AIMS/HYPOTHESIS: Latent autoimmune diabetes in adults (LADA) is a slowly progressive form of autoimmune diabetes, with autoantibodies to islet proteins developing in older patients who have no immediate requirement for insulin therapy. Markers of its clinical course are uncharacterised. The aim of this study was to determine whether persistence of, or changes in, GAD65 autoantibodies (GADAs) in the LADA patients who participated in the United Kingdom Prospective Diabetes Study (UKPDS) were associated with disease progression or insulin requirement. METHODS: GADA levels and their relative epitope reactivities to N-terminal, middle and C-terminal regions of human GAD65 were determined in 242 UKPDS patients who were GADA-positive at diagnosis; samples taken after 0.5, 3 and 6 years of follow-up were tested using a radiobinding assay. Comparisons were made of GADA status with clinical details and disease progression assessed by the requirement for intensified glucose-lowering therapy. RESULTS: GADA levels fluctuated between 0.5 and 6 years but persisted in 225 of 242 patients. No association of GADA levels with disease progression or insulin requirement was observed. Antibody reactivity was directed to C-terminal and middle epitopes of GAD65 in >70% patients, and the N-terminal in <9%. There were no changes in epitope reactivity pattern over the 6 year follow-up period, nor any association between epitope reactivity and insulin requirement. CONCLUSIONS/INTERPRETATION: GADAs persist for 6 years after diagnosis of LADA, but levels and reactivity to different GAD65 epitopes are not associated with disease progression.

Assessment of burn depth: a prospective, blinded comparison of laser Doppler imaging and videomicroscopy.

INTRODUCTION AND AIMS: There is a need, both in clinical and research settings, for an affordable, objective method of assessing burn depth. This study compares burn depth assessment by videomicroscopy with laser Doppler imaging (LDI) in patients with dermal burns. The videomicroscope is inexpensive compared to LDI, and can visualise the dermal capillary structure, therefore potentially allowing objective assessment of dermal burn injuries. METHODS: Patients admitted <72 h post-injury were included in the trial. Blinded LDI and videomicroscopy assessments were carried out. The patients were then followed up to one of three end-points: primary healing without surgery; early surgery; delayed healing and subsequent split skin grafting. The incidence of infection was also noted. RESULTS: Twenty-seven burn wounds were examined. In superficial partial thickness injuries, the videomicroscope reliably demonstrated an intact or nearly intact dermal vascular structure, progressing through to large amounts of capillary destruction and haemoglobin deposition in deep partial thickness injuries and complete destruction in full thickness injuries. The videomicroscope findings correlated strongly with both those of the LDI (p<0.001) and with clinical outcome (p<0.001). DISCUSSION: The videomicroscope is capable of accurately and objectively assessing burn depth. The results correlated well with both the clinical outcome and the laser Doppler findings. In addition, videomicroscopy is significantly cheaper than LDI and avoids several of the disadvantages of LDI.