POLIE suppresses telomerase-mediated telomere G-strand extension and helps ensure proper telomere C-strand synthesis in trypanosomes.

Trypanosoma brucei causes human African trypanosomiasis and sequentially expresses distinct VSGs, its major surface antigen, to achieve host immune evasion. VSGs are monoallelically expressed from subtelomeric loci, and telomere proteins regulate VSG monoallelic expression and VSG switching. T. brucei telomerase is essential for telomere maintenance, but no regulators of telomerase have been identified. T. brucei appears to lack OB fold-containing telomere-specific ssDNA binding factors that are critical for coordinating telomere G- and C-strand syntheses in higher eukaryotes. We identify POLIE as a telomere protein essential for telomere integrity. POLIE-depleted cells have more frequent VSG gene conversion-mediated VSG switching and an increased amount of telomeric circles (T-circles), indicating that POLIE suppresses DNA recombination at the telomere/subtelomere. POLIE-depletion elongates telomere 3' overhangs dramatically, indicating that POLIE is essential for coordinating DNA syntheses of the two telomere strands. POLIE depletion increases the level of telomerase-dependent telomere G-strand extension, identifying POLIE as the first T. brucei telomere protein that suppresses telomerase. Furthermore, depletion of POLIE results in an elevated telomeric C-circle level, suggesting that the telomere C-strand experiences replication stress and that POLIE may promote telomere C-strand synthesis. Therefore, T. brucei uses a novel mechanism to coordinate the telomere G- and C-strand DNA syntheses.

Trypanosoma brucei RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions.

RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1's self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution.IMPORTANCETrypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing.

Detection of multidrug resistant (MDR) bacteria in untreated waste water disposals of hospitals in Dhaka City, Bangladesh.

BACKGROUND: Emergence of MDR bacteria is a global problem and a major burden for treatment of various infectious diseases. This study was performed to detect antibiotic resistant bacteria in untreated hospital waste. METHOD: Waste water samples were collected from sewerage disposal points of two renowned hospital of Dhaka city and a total of 59 Escherichia coli and 29 Klebsiella pneumonia isolates were isolated. Antibiotic susceptibility were measured by disc diffusion method. RESULTS: Resistance among E. coli and K. pneumoniae to 10 frequently used antibiotic tested were respectively as follows: cefotaxime (CTX) 48 and 45%; ceftazidime (CAZ) 40 and 38%; ampicillin (AMP) 71 and 100%; streptomycin (S) 50 and 34%; sulfamethoxazole-trimethoprim (SXT) 58 and 28%; ciprofloxacin (CIP) 71 and 48%; kanamycin (K) 38 and 10%; chloramphenicol (C) 28 and 10%; gentamycin (CN) 19 and 16% and imipenem (IPM) 12 and 7%. Results also demonstrate that the sites that were at the disposal point of hospital waste have higher degree of resistance. High degree of resistance was observed when 23 high-resistant E. coli isolates were further tested with 15 additional antibiotics. CONCLUSION: This study revealed a significant rise of MDR bacteria in the hospital waste and underscore necessity of hospital waste treatment.

5-Hydroxytryptophan, a major product of tryptophan degradation, is essential for optimal replication of human parainfluenza virus.

Interferon (IFN) exerts its antiviral effect by inducing a large family of cellular genes, named interferon (IFN)-stimulated genes (ISGs). An intriguing member of this family is indoleamine 2,3-dioxygenase (IDO), which catalyzes the first and rate-limiting step of the main branch of tryptophan (Trp) degradation, the kynurenine pathway. We recently showed that IDO strongly inhibits human parainfluenza virus type 3 (PIV3), a significant respiratory pathogen. Here, we show that 5-hydoxytryptophan (5-HTP), the first product of an alternative branch of Trp degradation and a serotonin precursor, is essential to protect virus growth against IDO in cell culture. We also show that the apparent antiviral effect of IDO on PIV3 is not due to the generation of the kynurenine pathway metabolites, but rather due to the depletion of intracellular Trp by IDO, as a result of which this rare amino acid becomes unavailable for the alternative, proviral 5-HTP pathway.

Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3.

A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-ß. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3. IMPORTANCE: The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3.

Methodology: a modified method for high-quality DNA extraction for molecular analysis in cereal plants.

Cereal crops that have rigid non-cellulose components in the cell wall tissues of leaves and high starch and protein content in grains face limitations in DNA extraction. Advanced molecular genetic techniques such as mapping and marker-assisted selection programs require pure and quick DNA extraction. In this study, we developed methods for isolating high-quality genomic DNA from leaves and seeds of major cereal crops with minor modifications. DNA yields ranged from 300 to 1800 ng for 0.01 g seed or leaf tissue.

Isolation of genomic DNA from defatted oil seed residue of rapeseed (Brassica napus).

A simple protocol for obtaining pure, restrictable and amplifiable megabase genomic DNA from oil-free seed residue of Brassica napus, an important oil seed plant, has been developed. Oil from the dry seeds was completely recovered in an organic solvent and quantified gravimetrically followed by processing of the residual biomass (defatted seed residue) for genomic DNA isolation. The isolated DNA can be cut by a range of restriction enzymes. The method enables simultaneous isolation and recovery of lipids and genomic DNA from the same test sample, thus allowing two independent analyses from a single sample. Multiple micro-scale oil extraction from the commercial seeds gave approximately 39% oil, which is close to the usual oil recovery from standard oil seed. Most of the amplified fragments were scored in the range of 2.5 to 0.5 kb, best suited for scoring as molecular diagnostics.

Mycobacterium tuberculosis infection of a native polycystic kidney following renal transplantation.

Tuberculosis is a recognized complication following renal transplantation. Patients with autosomal-dominant polycystic kidney disease are increasingly being offered renal transplantation as an alternative to chronic hemodialysis. These patients are uniquely susceptible to serious upper urinary tract infections that are associated with significant morbidity and mortality. While involvement with gram-negative organisms is well described, mycobacterial infection of native polycystic kidneys after transplantation has not been addressed. We report a case of a renal transplant recipient who suffered an isolated Mycobacterium tuberculosis infection of a native polycystic kidney. With a 4-drug anti-tuberculosis therapy (ATT) regimen, the patient responded and became afebrile 8 weeks after initiation of drug therapy. ATT was continued for a total of 1 year. Two years after completion of ATT, the patient enjoys a normal life and has stable graft function. M. tuberculosis, though not common, must be recognized as a potential source of infection of native polycystic kidneys in immunocompromised transplant recipients. Similar to the pattern observed with more common pathogens, these infections may be difficult to eradicate with standard antimicrobial drug regimens.

Early renal damage assessed by the SLICC/ACR damage index is predictor of severe outcome in lupus patients in Pakistan.

We investigated patients with systemic lupus erythematosus with the objective of assessing whether early damage accrued in systemic lupus erythematosus as measured by the SLICC/ ACR Damage Index predicts mortality in lupus patients that have been followed prospectively in a single center. Patients with systemic lupus erythematosus from Aga Khan University hospital presenting between 1992 and 2007 were included. This enabled all patients to be potentially followed for at least 10 years. Yearly SLICC/ACR Damage Index scores were determined for each patient. Early damage was defined as a score ≥1, and no damage as a score of 0 at the initial assessment. Kaplan-Meier and Log rank tests were used to compare the survival experience between those with and without damage, with all patients being assessed at 10 years. In this inception cohort 198 patients were identified and were followed for 10 years. Of these, 47 (23.7%) patients had a SLICC/ACR Damage Index score of 0 (no damage) while 151 patients (76.3%) had at least one SLICC/ACR Damage Index item scored (early damage). Mean renal damage score at 1, 5 and 10 years was 0.16, 0.34 and 0.67, respectively. Of lupus patients who exhibited renal damage at their first SLICC/ACR Damage Index assessment, 31% died within 10 years of their illness as compared with only 13% who had no early renal damage (p < 0.003). Mean renal damage score at 1 year after diagnosis was a significant predictor of death within 10 years of diagnosis (p < 0.002).

Systemic lupus erythematosus in Pakistan.

Clinical features of systemic lupus erythematosus (SLE) have been described from different geographical regions in the world, with some clinical differences among different racial groups. Although data on the characteristics of SLE in Pakistan is scarce, it is not uncommon in the South East Asian region. The purpose of this study was, therefore, to delineate the clinical pattern and disease course in Pakistani patients with SLE and to compare it with international data on lupus patients. A total of 196 patients with SLE fulfilling the clinical and laboratory criteria of the American Rheumatism Association admitted to the hospital between 1986 and 2001 were studied by means of a retrospective review of their records. Demographically, it was seen that SLE is a disease predominantly of females in their third decade, which is consistent with worldwide data. The mean age of presentation was 31 years (range 14-76) and the mean duration of follow up was 34 (4-179) months. Generally, there was less cutaneous (46%), arthritic (38%), serositis (22%) and renal involvement (33%) but more neuropsychiatric symptoms (26%) in our population. Eighty-six percent of patients were ANA positive, whereas anti dsDNA was positive in 74% of patients. Infections, renal involvement, seizures and thrombocytopenia were associated with poor prognosis (P < 0.05). This study is the first of its kind in Pakistan. The clinical and laboratory characteristics of SLE patients in our study place our population in the middle of a spectrum between the Caucasians and other Asian populations. It has shown that the clinical characteristics of SLE patients in this country may be different to those of its neighbors.

Radiocontrast nephropathy: is it dose related or not?

OBJECTIVE: To assess the safety of high dose non-ionic contrast media during a single radiological procedure in patients with pre-existing renal impairment. METHODS: One hundred eighteen patients, with serum Creatinine greater than 1.3 mg/dl who were undergoing coronary angiography or percutaneous transluminal coronary angiography (PTCA) were included in the study. All patients received the nonionic dye ULTRAVIST (lopromide). Serum creatinine were measured before, 48 hours and 1 week after the administration of contrast agent. An acute contrast induced reduction in renal function was defined as an increase in Serum Creatinine concentration of >=0.5 mg/dl, 48 hours after the administration of contrast agent. All patients with end stage renal disease or patients undergoing coronary bypass surgery within a week after coronary angiography or had any concomitant factors that could cause acute renal failure e.g., sepsis, hypotention, etc., were excluded. Patients receiving a dose of upto 100 ml of contrast agent (low dose group) were separated from those who received greater than 100 ml of contrast agent (high dose group). Patients in both groups had similar characteristics in terms of sex, age, weight and underlying disease. Student's t-test was used for statistical analysis. RESULTS: The mean age of our patients was 62.3+/-8.83 (range 40-84 years). There were 93 (78.8%) males and 25 (21.2%) females. The mean pre-contrast creatinine in the low contrast group was 1.97+/-0.92 and high dose group was 2.16+/-1.90 (p=0.48). The post-contrast Creatinine at 48 hours was 2.11+/-1.11 and 2.06+/-1.39 in the groups receiving low and high dose contrast agents respectively (p=0.830), while at 7 days post-contrast it was 2.17+/-1.28 and 1.95+/-1.43 respectively in the two groups (p=0.391). The contrast-induced reduction in renal function (rise in serum Cr >=0.5 mg/dl above base line) occurred in 14% (n=8) of patients in low dose and in 11% (n=7) in high dose contrast group (p=0.830, insignificant). CONCLUSION: The results of our study confirm that high dose non-ionic contrast is not associated with increased risk of contrast-mediated nephrotoxicity in patients with pre-existing renal insufficiency undergoing cardiac angiography (p=0.830, insignificant).

Maximum tolerable dose of cyclophosphamide and azathioprine in Pakistani patients with primary renal disease.

OBJECTIVE: The immunosuppressive regimens, at present, mainly rely on western guidelines that were derived from studies conducted in western populations. No such study exists for South Asian population, which is home to almost two billion people different in both genetics and environment from west. Locally derived thresholds for side effects markedly different from western figures may warrant re-adjustment of current local immunosuppressive regimens that are at present based largely on western guidelines. In order to define optimum dose for Cyclophosphamide (CYC) and Azathioprine (AZA) based immunosuppressive therapy, we conducted this study to find out maximum tolerable doses of azathioprine (AZA) and cyclophosphamide (CYC) beyond which neutropenia and thrombocytepenia are most likely to occur in patients with primary renal pathology. METHOD: Patients with systemic vasculitis and idiopathic glomerulonephritis who were on CYC and AZA were identified through review of medical records at a tertiary care hospital in Pakistan (The Aga Khan University Hospital, Karachi). Patients were categorized under three principal diagnosis i.e. systemic lupus erythematosus (SLE), primary (idiopathic) glomerulonephritis (GN) and Wegener's granulomatosis (WG). The Receiver Operating Curve (ROC) was used to calculate the maximum tolerable dose for both CYC and AZA. RESULTS: We identified 94 patients aged 6-82 years (median 44.5 years) with primary renal disease (Wegener's granulomatosis n=13, Systemic lupus erythematosis n=62 and idiopathic glomerulonephritis n=19) who received CYC or AZA. Of these 94 patients, 36.2% (n=34) received CYC and 63.8% (n=60) received AZA. The mean dose of CYC was 1.54 +/- 0.50 mg/kg of body weight (range: 0.77-2.93). The mean dose of AZA was 1.64 +/- 0.59 mg/kg of body weight (range: 0.47-2.97). The maximum tolerable doses calculated for CYC and AZA were 1.25 mg/kg and 1.30 mg/kg of body weight respectively. The maximum tolerable dose for CYC and AZA among males could not be calculated, because of insufficient number of patients who developed neutropenia and thrombocytopenia. The maximum tolerable doses for CYC and AZA among females were 1.34 mg/kg and 1.03 mg/kg of body weight respectively. Also we found out that AZA was relatively more likely to cause neutropenia and thrombocytopenia (p=0.07). CONCLUSION: We thereby recommend that CYC should be initiated at a dose no more than 1 mg/kg of body weight and AZA at an initial dose of 0.75-1.0 mg/kg of body weight. The dose may be adjusted later on the basis of clinical response and laboratory reports.

Spectrum of complications and mortality of bacterial meningitis: an experience from a developing country.

OBJECTIVE: The aim of this study was to obtain data on predisposing factors, causative organisms and their associated mortality and complications related to acute bacterial meningitis. METHODS: The chart review of all patients in whom acute bacterial meningitis was diagnosed at The Aga Khan University Hospital from January 1995 through December 2001. RESULTS: One hundred ninety-four patients were included in study. There were 146 males and 48 females. The mean age of our study population was 41 +/- 12.3 years. One hundred and ninety (97.9%) patients had community-acquired meningitis; only 4 (2.0%) patients developed meningitis nosocomially. The two most common predisposing factors were diabetes mellitus (13.9%) and otitis media (7.7%) among all 194 patients. A significant proportion of patients with complications had diabetes mellitus (24.6%, p < 0.001). CSF and blood cultures were positive in 53 (27.3%) and 42 (21.6%) patients respectively; there was no statistical difference found. The most common organisms isolated were Streptococcus pneumoniae in 35 (36.8%) patients followed by Neisseria meningitides in 30 (31.5%) patients. Approximately 68% of positive cultures yielded S. pneumoniae and N. meningitides (p < 0.0001). The overall mortality rate was 22.1%. The mortality rate for Streptococcus pneumoniae was 17.1%. The highest mortality was observed in patients with Pseudomonal meningitis where all four patients expired followed by mortality rate of 85.7% in Escherichia coli afflicted patients (p < 0.001). Complications occurred in 73 (37.6%) patients with persistent complications in 31 (42.4%) patients. Complications resolved in 34 (46.5%) patients. The most common complications were seizures (12.8%) and cranial nerve palsies (11.3%). Seizures were more likely to occur in older patients (p < 0.05) whereas hydrocephalus was more common in younger patients (p < 0.05). CONCLUSION: Bacterial Meningitis remains a serious disease associated with substantial morbidity and mortality. Most cases are community acquired with S. Pneumoniae being the most common pathogen. Old age, diabetes mellitus, a positive culture, seizures as a complication and late stage in the disease are the important predictors of a poor outcome.

Cutaneous manifestations of systemic lupus erythematosus in Pakistani patients.

OBJECTIVE: Systemic Lupus Erythematosus (SLE) is an autoimmune process in which cutaneous lesions occur in majority of patients. This study from Karachi, Pakistan was conducted to determine the pattern and prevalence of such lesions in SLE in Pakistani patients. METHODS: One hundred ninety eight patients with SLE fulfilling the clinical and laboratory criteria of the American Rheumatology Association were examined between 1986 and 2001 for the presence of cutaneous manifestations. RESULTS: Skin changes noted were: noncicatricial diffuse alopecia (22%), malar rash (31%), mucosal lesions (20%), discoid eruptions (15%), photosensitivity (33%), vascular lesions (20%), pruritus (17%), and pigmentary changes (22%). Peripheral gangrene, chronic ulcers, Raynauds phenomenon, urticaria, chilblains, thrombophlebitis, palmar erythema, and erythema multiform were rare. Anti ANA and anti dsDNA were positive in 93% and 83% patients respectively. CONCLUSION: A different clinical pattern was noted in our patients than reported previously.

Sustained remission in a case of lupus nephritis with cyclosporin therapy.

Systemic lupus erythematosus (SLE) in severe form still presents a major therapeutic challenge. Aggressive treatment of severe renal lesions has improved the prognosis of renal disease over the last decade. However, this benefit is quite frequently offset by the side effects and toxicity of the treatment. Moreover, the disease may appear to be poorly responsive to treatment with steroids and cytotoxic drugs. We report a case of lupus nephritis that relapsed despite having adequate steroid and cytotoxic therapy, but later was successfully treated with cyclosporin. Fifteen months after discontinuing the treatment with cyclosporin, the patient continued to remain in remission.