Pyrosequencing for discovery and analysis of DNA sequence variations

Since the invention of pyrosequencing, more than 500 articles have been published describing different applications of this technology, most notably for DNA structure variation and microbial detection. Technological advances have been made to enhance the robustness and accuracy of this technique as well as to reduce the cost and increase the throughput. This review intends to cover recent advances in this technology and discuss its application for low and high-throughput DNA variation studies.     

The immediate and 6-mo reproducibility of pressure-flow studies in men with benign prostatic enlargement

OBJECTIVES: Urodynamics is an objective method of diagnosing bladder outlet obstruction (BOO) in men. This study examined the immediate and 6-mo reproducibility of this investigation. METHODS: Urodynamics was performed in men as part of a multinational, multicentre, double-blind, placebo-controlled drug trial. Each patient had two fill/void cycles both at baseline and 6 mo. The BOO index (BOOI) and bladder contractility index (BCI) were calculated for each cycle and data analysed to look for changes in immediate and 6-mo reproducibility between the two fill/void cycles. RESULTS: A total of 114 patients had urodynamics at baseline. In the immediate term, although there was a small but statistical fall in both the BOOI and BCI, with cycle one figures greater than those in cycle two, 81% and 79% of patients remained in the same BOOI and BCI category, respectively. At 6 mo, the differences were not statistically different with 70% of patients remaining unchanged in their BOOI category in cycle one and 71% in cycle two; 65% remained unchanged in their BCI category in cycle one and 74% in cycle two. No patient with a BOOI > 65 changed category in the second investigation, and only 5 of 103 first cycles with a BOOI > or = 50 changed category to equivocal obstruction. CONCLUSIONS: Urodynamics has good reproducibility when looking at the BOOI and BCI, indicating that a second study is not necessary in most patients and one investigation is sufficient for an accurate diagnosis on which treatment options can be based.     

Prospective cohort study of mother‐to‐infant infection and clearance of hepatitis C in rural Egyptian villages

Although persistent transmission of hepatitis C virus (HCV) from infected mothers to their infants is reported in 4–8%, transient HCV perinatal infection also occurs. This prospective cohort study determined perinatal HCV infection‐ and early and late clearance‐rates in 1,863 mother‐infant pairs in rural Egyptian villages. This study found 15.7% and 10.9% of pregnant women had HCV antibodies (anti‐HCV) and HCV‐RNA, respectively. Among 329 infants born of these mothers, 33 (10.0%) tested positive for both anti‐HCV and HCV‐RNA 2 months following birth—29 (12.5%) having HCV‐RNA positive mothers and 4 (with transient infections) having mothers with only anti‐HCV. Fifteen remained HCV‐RNA positive at one and/or 2 years (persistent infections), while 18 cleared both virus and antibody by 1 year (transient infections). Among the 15 persistent cases, 7 cleared their infections by 2 or 3 years. At 2‐ to 6‐ and at 10‐ to 12‐month maternally acquired anti‐HCV was observed in 80% and 5% of infants, respectively. Four perinatally infected and one transiently infected infant were confirmed to be infected by their mothers by the sequence similarity of their viruses. Viremia was 155‐fold greater in mothers of infants with persistent than mothers of infants with transient infections. Maternal‐infant transmission of HCV is more frequent than generally reported. However, both early and late clearance of infection frequently occurs and only 15 (4.6%) and 8 (2.4%) infants born of HCV‐RNA positive mothers had detectable HCV‐RNA at one and 2–3 years of age. Investigating how infants clear infection may provide important information about protective immunity to HCV. J. Med. Virol. 81:1024–1031, 2009. © 2009 Wiley‐Liss, Inc.     

The inhibition of glioma growth in vitro and in vivo by a chitosan/ellagic acid composite biomaterial

This study has developed a chitosan-based delivery system to locally administer ellagic acid for brain cancer treatment. We fabricated chitosan/ellagic acid composite films with various concentrations of ellagic acid. In vitro release study was performed by using a UV spectrophotometer, and enzymatic degradation rate was determined by analyzing the increased free amino groups. Viability of brain cancer cells (human U87 glioblastomas and rat C6 glioma cells) was measured via direct and indirect cell culture on the films by MTS assay. Caspase-3 activation, Western blot for p53, and anti-angiogenesis assays were also examined. In the in vivo study, GFP-tagged rat C6 glioma cells were implanted subcutaneously at the right flank region of nude mice and treatments were initiated by implanting the films subcutaneously. Tumor growth was evaluated by measuring tumor volume using a caliper, an ultrasound machine, and an optical imaging system. The chitosan/ellagic acid composite films were enzymatically degradable and exhibited a sustained slow release of ellagic acid. These materials could inhibit the cancer cell growth in an ellagic acid concentration-dependent manner by inducing apoptosis of cancer cells as well as suppressing angiogenesis. These materials also significantly suppressed tumor tissue growth in vivo.     

Bridging to transplantation in acute liver failure in a 7-month-old infant

BACKGROUND: Acute liver failure (ALF) in children is a rare but often fatal event. At present, liver transplantation is the only successful therapy in most cases. In the face of deteriorating hepatic encephalopathy in these children, some bridging therapy using artificial detoxification can be necessary to enable successful transplantation. In adults, albumin dialysis using the molecular absorbent recycling system (MARS) has been described as effective for bridging to liver transplantation. CASE REPORT: A previously healthy 7-month-old infant was admitted with ALF due to autoimmune hepatitis. King's College criteria for children with ALF indicated the need for transplantation (bilirubin 13.7 mg/dl, leukocytes 18,980/mm3, INR 5.83, age<2 years). Despite moderate hyperammonemia (75 microm/l) along with the development of pneumonia, the child deteriorated hemodynamically and neurologically, showing grade III encephalopathy proven by EEG. Albumin dialysis using MARS was used to bridge 36 hours to successful living-donor split-liver transplantation, and resulted in improvements in EEG, plasma levels of amino acids and hemodynamics. Twenty-four months after transplantation the child shows normal liver function and normal neuropsychological development. The explanted liver showed 80 % tissue destruction from autoimmune hepatitis. CONCLUSION: Albumin dialysis as described can be used successfully in infants < 1 year old for bridging to liver transplantation in cases of acute hepatic failure with deteriorating encephalopathy.     

Serum Bactericidal Antibody Response 1 Year after Meningococcal Polysaccharide Vaccination of Patients with Common Variable Immunodeficiency

Some patients with common variable immunodeficiency (CVID) can generate an antibody response following vaccination with Neisseria meningitidis polysaccharide, but the duration of this protection is unknown. In this study, serum bactericidal antibody (SBA) responses to serogroup C N. meningitidis of 23 patients with CVID and 23 sex- and age-matched controls were measured 1 year after vaccination with the plain A/C meningococcal polysaccharide vaccine. The fold rise in serum bactericidal antibody geometric mean titers of the control group from prevaccination to 1 year postvaccination was significantly higher than that of the patient group (5.41- versus 2.96-fold, P = 0.009). Of 23 CVID patients, 8 had a poor response to vaccine (<4-fold rise) 3 weeks after vaccination, and low titers remained when measured 1 year later. Of the 15 CVID patients who had a normal response to vaccine (≥4-fold rise) 3 weeks after vaccination, 6 cases failed to maintain protective SBA titers, whereas the remaining 9 had protective titers 1 year after vaccination. Only one of the 23 controls, who developed protective SBA titers after 3 weeks, lost the protective titers after 1 year. Among the patients, the presence of bronchiectasis and/or splenomegaly at enrollment was associated with poor SBA response to vaccine at 3 weeks and/or failure to maintain protective levels at 1 year. The results of this study demonstrate that a number of CVID patients can produce protective antibody titers that can persist for 1 year after vaccination, which lends strong support to the inclusion of polysaccharide vaccine in the immunization program for CVID patients.Common variable immunodeficiency (CVID) is the commonest symptomatic primary immunodeficiency disease and is a heterogeneous group of disorders, characterized by severe reduction of serum levels of IgG and IgA, with normal or low numbers of B cells in the absence of any recognized genetic abnormality (2, 11, 16, 30). Patients with CVID usually experience recurrent bacterial infections (1, 14) and carry an increased risk of autoimmunity (12, 28) and malignancies (4, 24). Various defects of B cells, T cells, and dendritic cells have been reported for CVID (26, 29, 34-36); however, the exact pathophysiology of the disease is still unclear (3, 15).Deployment of polysaccharide and protein vaccines in CVID is a subject of active debate. Although it is intuitive that CVID patients should have poor antibody responses to vaccine, it is apparent that some patients can produce normal antibody titers (5, 18, 21, 32, 33). We have reported that a protective antibody response was achieved 3 weeks following vaccination with polysaccharide meningococcal vaccine of a group of CVID patients (32, 33). In the current study, we measured serum bactericidal antibody (SBA) titers (7) of the same cohort of patients 1 year after the initial vaccination.     

Pancreaticoduodenectomy in a tertiary referral center in Saudi Arabia: A retrospective case series

ContextPerioperative outcome of pancreaticoduodenectomy is related to work load volume and to whether the procedure is carried out in a tertiary specialized hepato-pancreatico-biliary (HPB) unit.ObjectiveTo evaluate the perioperative outcome associated with pancreaticoduodenectomy in a newly established HPB unit.PatientsAnalysis of 32 patients who underwent pancreaticoduodenectomy (PD) for benign and malignant indications.DesignRetrospective collection of data on preoperative, intraoperative and postoperative care of all patients undergoing PD.ResultsThirty-two patients (16 male and 16 female) with a mean age of 59.5 ± 12.7 years were analyzed. The overall morbidity rate was high at 53%. The most common complication was wound infection (n = 11; 34.4%). Pancreatic and biliary leaks were seen in 5 (15.6%) and 2 (6.2%) cases, respectively, while delayed gastric emptying was recorded in 7 (21.9%). The female sex was not associated with increased morbidity. Presence of co-morbid illness, pylorus-preserving PD, intra-operative blood loss ?1 L, and perioperative blood transfusion were not associated with significantly increased morbidity. The overall hospital mortality was 3.1% and the cumulative overall (OS) and disease free survival (DFS) at 1 year were 80% and 82.3%, respectively. The cumulative overall survival for pancreatic cancer vs ampullary tumor at 1 year were 52% vs 80%, respectively.ConclusionPD is associated with a low risk of operative death when performed by specialized HPB surgeons even in a tertiary referral hospital. However, the postoperative morbidity rate remains high, mostly due to wound infection. Further improvement by reducing postoperative infection may help curtail the high postoperative morbidity.     

A large scale analytical study on efficacy of different photo(chemo)therapeutic modalities in the treatment of psoriasis,vitiligo and mycosis fungoides

Psoriasis, vitiligo, and mycosis fungoides (MF) are among the most frequently treated dermatological diseases by photo(chemo)therapy. The objectives are to determine which photo (chemo) therapeutic modality could achieve the best response in the treatment of psoriasis, vitiligo, and MF. The design used in this study is retrospective analytical study. The study included 745 patients' records; 293 with psoriasis, 309 with vitiligo, and 143 with early MF, treated in the Phototherapy Unit, Dermatology Department, Kasr El‐Aini Hospital, Cairo University by either psoralen and ultraviolet A (PUVA), narrow band ultraviolet B (NB‐UVB), psoralen and narrow band UVB (P‐NBUVB), broad band UVB (BB‐UVB), or broad band UVA (ΒΒ‐UVA). Data were retrieved from the computer database of the unit and statistically analyzed. In psoriasis, oral and topical PUVA and NB‐UVB were found to be equally effective, whereas oral PUVA had significantly better results than both UVA and BB‐UVB at the end of therapy. In generalized vitiligo, PUVA and P‐NBUVB had significantly better results than NB‐UVB alone. In early MF, there was no statistically significant difference between the response to oral PUVA and NB‐UVB. PUVA and NB‐UVB are good choices in patients with psoriasis and early stage MF, whereas PUVA appears the best choice in the treatment of vitiligo.