Validated assays for the quantification of C9orf72 human pathology.

A repeat expansion mutation in the C9orf72 gene is the leading known genetic cause of FTD and ALS. The C9orf72-ALS/FTD field has been plagued by a lack of reliable tools to monitor this genomic locus and its RNA and protein products. We have validated assays that quantify C9orf72 pathobiology at the DNA, RNA and protein levels using knock-out human iPSC lines as controls. Here we show that single-molecule sequencing can accurately measure the repeat expansion and faithfully report on changes to the C9orf72 locus in what has been a traditionally hard to sequence genomic region. This is of particular value to sizing and phasing the repeat expansion and determining changes to the gene locus after gene editing. We developed ddPCR assays to quantify two major C9orf72 transcript variants, which we validated by selective excision of their distinct transcriptional start sites. Using validated knock-out human iPSC lines, we validated 4 commercially available antibodies (of 9 tested) that were specific for C9orf72 protein quantification by Western blot, but none were specific for immunocytochemistry. We tested 15 combinations of antibodies against dipeptide repeat proteins (DPRs) across 66 concentrations using MSD immunoassay, and found two (against poly-GA and poly-GP) that yielded a 1.5-fold or greater signal increase in patient iPSC-motor neurons compared to knock-out control, and validated them in human postmortem and transgenic mouse brain tissue. Our validated DNA, RNA and protein assays are applicable to discovery research as well as clinical trials.

Nitrogen-doped carbon quantum dots conjugated isoreticular metal-organic framework-3 particles based luminescent probe for selective sensing of trinitrotoluene explosive.

Amine group-containing isoreticular metal-organic framework (IRMOF-3) particles are utilized for the first time as a trinitrotoluene (TNT) sensing material. IRMOF-3 particles are synthesized using zinc nitrate as a metal precursor and 2-amino-1,4-benzenedicarboxylic acid as a linker. The nitrogen-doped carbon quantum dots (NCQDs) are synthesized from citric acid and ethylenediamine as carbon and nitrogen precursor, respectively. The NCQDs are conjugated with IRMOF-3 particles as IRMOF-3/NCQDs. The TEM micrograph revealed the average size of IRMOF-3 particles to be 363.66 nm. The photoluminescence emission intensity of IRMOF-3 particles at λem 430 nm is highly increased in the presence of NCQDs (λex 330 nm). Both the as-synthesized IRMOF-3 and IRMOF-3/NCQD particles are explored for TNT detection to compare the effect of NCQDs on the IRMOF-3 particle surface. Lower limit of detection (7.5 × 10-8 M) and higher Stern-Volmer constant (4.46 × 106 M-1) are achieved by IRMOF-3/NCQD particles. The association constant also increased from 5.3 × 104 to 2.78 × 106 M-1 after the conjugation of IRMOF-3 particles with NCQDs. Moreover, enhanced selectivity for TNT over trinitrophenol is achieved using the IRMOF-3/NCQD particles. Graphical Abstract.

Influence of non-thermal TiCl4/Ar+O2 plasma-assisted TiOx based coatings on the surface of polypropylene (PP) films for the tailoring of surface properties and cytocompatibility.

The superior bulk properties (corrosion resistance, high strength to weight ratio, relatively low cost and easy processing) of hydrocarbon based polymers such as polypropylene (PP) have contributed significantly to the development of new biomedical applications such as artificial organs and cell scaffolds. However, low cell affinity is one of the main draw backs for PP due to its poor surface properties. In tissue engineering, physico-chemical surface properties such as hydrophilicity, polar functional groups, surface charge and morphology play a crucial role to enrich the cell proliferation and adhesion. In this present investigation TiOx based biocompatible coatings were developed on the surface of PP films via DC excited glow discharge plasma, using TiCl4/Ar+O2 gas mixture as a precursor. Various TiOx-based coatings are deposited on the surface of PP films as a function of discharge power. The changes in hydrophilicity of the TiOx/PP film surfaces were studied using contact angle analysis and surface energy calculations by Fowke's approximation. X-ray photo-electron spectroscopy (XPS) was used to investigate the surface chemical composition of TiOx/PP films. The surface morphology of the obtained TiOx/PP films was investigated by scanning electron and transmission electron microscopy (SEM &TEM). Moreover, the surface topography of the material was analyzed by atomic force microscopy (AFM). The cytocompatibility of the TiOx/PP films was investigated via in vitro analysis (cell viability, adhesion and cytotoxicity) using NIH3T3 (mouse embryonic fibroblast) cells. Furthermore the antibacterial activities of TiOx/PP films were also evaluated against two distinct bacterial models namely Gram positive Staphylococcus aureus (S.aureus) and Gram negative Escherichia coli DH5α. (E.coli) bacteria. XPS results clearly indicate the successful incorporation of TiOx and oxygen containing polar functional groups on the surface of plasma treated PP films. Moreover the surface of modified PP films exhibited nano structured morphology, as confirmed by SEM, TEM and AFM. The physico-chemical changes have improved the hydrophilicity of the PP films. The in-vitro analysis clearly confirms that the TiOx coated PP films performs as good as the standard tissue culture plates and also are unlikely to impact the bacterial cell viability.

Measurement of adult antimicrobial drug use in tertiary care hospital using defined daily dose and days of therapy.

Widespread overuse and inappropriate use of antimicrobial drugs continues to fuel an increase in antimicrobial resistance and leads to consequent treatment complications and increased healthcare costs. In the present study we aimed to describe antimicrobial drug consumption and predictors and to identify potential targets for antimicrobial stewardship. This was a prospective observational study conducted at adult medicine wards of tertiary care teaching hospital over the period of five months. Antimicrobial drug consumption was measured using days of therapy per 1000 patient days and defined daily dose per 1000 patient days. Additionally, predictors of multiple antimicrobial prescribing were also analyzed. Seven hundred thirty patients were screened and 550 enrolled, receiving 1,512 courses of antimicrobial therapy, mainly intravenously (66%). Most frequently prescribed agents were artesunate (13%), ceftriaxone (11%) and metronidazole (10.5%). Overall consumption was 1,533 days of therapy per 1000 patient days and was mainly attributed to antibiotics (98.3%) for empirical therapy (50%). Median days of antimicrobial drugs prescribing were 3 (inter quartile range 2-5). Most commonly consumed antimicrobials were ceftriaxone (31%, 248.8 g) and artesunate (26%, 29 g). Antimicrobials contributed to 72.5% expense of the total incurred. Multivariate analysis reveals that younger patients (≥45 years) (odds ratio: 1.59, 95% CI 1.14-2.21) were more likely and absence of comorbidities (odds ratio: 0.58, 95% CI 0.42-0.79) and shorter hospital stay (≥6 days)(odds ratio: 0.44, 95% CI 0.32-0.60) were associated with less likelihood of prescribing multiple antimicrobial drugs. Estimating antimicrobial drugs use by defined daily dose method will remain open to criticism because the prescribed dosage is not often in agreement with the "usual" daily dose, which depends on location of and susceptibility of pathogenic organisms and metabolic status of the patient.

Clinical Pulmonary Infection Score to diagnose ventilator-associated pneumonia in children.

BACKGROUND: There is a need to validate and suggest easy clinical method for diagnosis of ventilator-associated pneumonia (VAP) in developing countries. OBJECTIVES: To validate the use of simplified Clinical Pulmonary Infection Score (CPIS) for the diagnosis of VAP. DESIGN: Prospective study. SETTING: Pediatric intensive care unit of a tertiary care teaching hospital. SUBJECTS: 30 children receiving mechanical ventilation for more than 48 hours and with simplified CPIS=6. METHODS: All patients underwent flexible bronchoscopy to obtain bronchoalveolar lavage which was analyzed quantitatively. Colony count = 10(4) cfu/mL was considered reference standard for definite VAP. RESULTS: Of the five variables used for simplified CPIS, only patients temperature (P=0.013) and PaO2/FiO2 ratio were significant (P<0.001) to differentiate the presence of definite VAP. Patients with definite VAP (BAL colony count = 10(4) cfu/mL) had CPIS of 8.4 while in no definite VAP group it was 6.4 (P=0.007). CPIS of 8 was found to have sensitivity of 80%, specificity 80%, PPV 86.9%, NPV 70.5% and accuracy 80%. The area under Receiver operating characteristic curve of CPIS against reference standard was 0.81± 0.069 (P=0.001). CONCLUSION: Simplified CPIS is useful in patients on mechanical ventilation to diagnose ventilator-associated pneumonia.

Cell phones and tumor: still in no man's land.

The use of cell phones is increasing worldwide at a phenomenal pace. While cellular communication has dramatically influenced our lifestyle, its impact on human health has not been completely assessed. Widespread concern continues in the community about the deleterious effects of radiofrequency radiations (with which cell phones operate) on human tissues and the subsequent potential for carcinogenesis. A detailed survey of published studies researching this question was done in preparation of this manuscript. Included in the survey were case reports, in vitro studies, population based retrospective studies and other investigations. The database of indexed journals was searched for key words like 'cell phone', 'radiation', 'cancer' and 'radio waves'. Guidelines issued by the World Health Organization, federal and technical authorities, Institute of Electrical and Electronic Engineers and the International Commission for Non-Ionizing Radiation Protection were reviewed. The evaluation of current evidence provided by various studies to suggest the possible carcinogenic potential of radiofrequency radiation is inconclusive. This risk assumes significance in light of the burgeoning number of people who are continually exposed to the high frequency radiation from cell phones and towers that serve as receiving and transmitting stations. The aim of this review is to identify limitations in past studies, present available data for consideration, and identify gaps in the current knowledge base. This will provide impetus and direction for further research and allow informed decisions pertaining to cell phone use to be made.

Bacterial endocarditis treated with intramuscular teicoplanin.

Right-sided endocarditis caused by Staphylococcus aureus in parenteral drug abusers is potentially life-threatening, more so in the presence of pulmonary embolisation, and a course of parental antibiotics is required for at least four weeks. A combination of intravenous cloxacillin and aminoglycosides has proven efficacious for more than 90 percent of the patients. Intravenous vancomycin can also be used in cases of penicillin allergy or methicillin-resistant staphylococci. Intravenous teicoplanin, a glycopeptide with a similar antimicrobial profile to vancomycin, has been used with a somewhat lesser degree of success in these cases and is not recommended as first line therapy. We describe a 37-year-old man, a parenteral drug user, who had right-sided endocarditis, where in the absence of other alternatives, teicoplanin had to be administered intramuscularly and not intravenously.

Isolation and characterization of microsomal omega-6-desaturase gene (fad2-1) from soybean.

A genomic DNA sequence (fad2-1) encoding seed specific microsomal 0-6 desaturase was isolated from soybean (Glycine max. L cv. Pusa-9702). A positive genomic clone of 1852 nucleotides containing a single uninterrupted 3' end exonic region with an ORF of 1140 bp encoding a peptide of 379 amino acids, a complete 3' UTR of 206 bp and 86 bp of 5' UTR interrupted by a single intron of 420 bp was obtained on screening the sub-genomic library of soybean. Southern blots revealed at least two copies of the gene per haploid genome. Analysis of the translated product showed the presence of three histidine boxes, with the general sequence HXXXH and five probable transmembrane segments reported to be involved in substrate specificity.

Histological findings of a choroidal neovascular membrane removed at the time of macular translocation in a patient previously treated with intravitreal bevacizumab treatment (Avastin).

AIM: To report the findings in a patient treated by repeated intravitreal bevacizumab (Avastin) injections, followed by macular relocation and excision of subfoveal choroidal neovascular membrane (CNV). METHODS: Histopathological evaluation of the CNV specimen, including immunohistochemical assessment. RESULTS: During surgical excision, the CNV seemed to be avascular and its underlying bed did not bleed. Histopathological examination revealed that the CNV comprised avascular fibrous subretinal tissue containing fibroblastic retinal pigment epithelial (RPE) cells, fragments of irregular thickened Bruch's membrane and fibrotic choroidal tissue containing some medium-sized vessels but no choriocapillaris. CONCLUSIONS: The development of an RPE tear during the course of Avastin treatment may reflect contraction of the avascular subretinal tissue, whereas the lack of capillaries in both choroidal and subretinal components may be caused by the increased access of Avastin to the choriocapillaris in the presence of the RPE tear.

Type II lepra reaction: an unusual presentation.

Ulcers with maculo-papular rash are an unusual presenting feature of leprosy. They occur as result of neuropathy, type-2 lepra reaction or Lucio's phenomenon. The hall mark of type-2 reaction is erythema nodosum. Very rarely it manifests as ulcerative skin lesions. We describe one such unusual case of a young male who presented with multiple ulcers and maculo-papular rash over the legs, chest and abdomen. In addition to this, he had fever, heart murmur, pulmonary infiltrates, neuropathy, and deranged liver function. A clinical differential diagnosis of infective endocarditis and systemic nectrozing vasculitis was made. Skin biopsy showed dense inflammation with lepra bacilli consistent with type-2 lepra reaction.

Ageing and circadian variation in cardiovascular events.

INTRODUCTION: Circadian variation in cardiovascular events is well recognised in vascular events. This study aims to observe any significant difference in circadian variability in geriatric patients when compared to their younger counterparts. METHODS: This prospective study was conducted at medical emergency at the Government Medical College and Hospital, Chandigarh, India. All the patients attending medical emergency with symptoms suggestive of coronary artery disease were included. The time of occurrence of first symptom and subsequent symptoms was noted. Electrocardiography, cardiac enzymes and echocardiography were performed to establish the diagnosis of acute coronary syndrome. The 24-hour day was divided into 12 equal parts of two hours each. For the final analysis, the two-hour periods were grouped into six equal periods of four hours each and four quarters of six hours each. RESULTS: We studied 559 patients, out of whom 459 were 65 years old or younger, and 100 patients were older than 65 years old. 459 patients had 498 episodes in all. 100 patients above the age of 65 years had 104 episodes. Both the groups had peaking of acute myocardial infarction in the early morning hours with patients older than 65 years of age having a slightly early peak. However, there was considerable variability for peaking of unstable angina and non-Q myocardial infarction. In patients older than 65 years, early morning peaking of events was noted whereas in 65 years or younger, the peaking of events was noted in the evening hours. CONCLUSION: Circadian variability exists in occurrence of acute coronary events and is variable in geriatric patients when compared to their younger counterparts.

Accurate blood pressure recording: is it difficult?

BACKGROUND: Blood pressure (BP) measurement is a routine procedure but errors are frequently committed during BP recording. AIMS AND SETTINGS: The aim of the study was to look at the prevalent practices in the institute regarding BP recording. The study was conducted in the Medicine Department at Government Medical College, Chandigarh, a teaching institute for MBBS students. METHODS: A prospective, observational study was performed amongst the 80 doctors in a tertiary care hospital. All of them were observed by a single observer during the act of BP recording. The observer was well versed with the guidelines issued by British Hypertension Society (BHS) and the deviations from the standard set of guidelines issued by BHS were noted. The errors were defined as deviations from these guidelines. STATISTICAL METHODS: The results were recorded as percentage of doctors committing these errors. RESULTS: In our study, 90% used mercury type sphygmomanometer. Zero error of the apparatus, hand dominance was not noted by any one. Every one used the standard BP cuff for recording BP. 70% of them did not let the patient rest before recording BP. 80% did not remove the clothing from the arm. None of them recorded BP in both arms. In out patient setting, 80% recorded blood pressure in sitting position and 14% in supine position. In all the patients where BP was recorded in sitting position BP apparatus was below the level of heart and 20% did not have their arm supported. 60% did not use palpatory method for noticing systolic BP and 70% did not raise pressure 30-40 mm Hg above the systolic level before checking the BP by auscultation. 80% lowered the BP at a rate of more than 2 mm/s and 60% rounded off the BP to nearest 5-10 mm Hg. 70% recorded BP only once and 90% of the rest re inflated the cuff without completely deflating and allowing rest before a second reading was obtained. CONCLUSION: The practice of recording BP in our hospital varies from the standard guidelines issued by the BHS.

A prospective study of the long-term efficacy of local methyl prednisolone acetate injection in the management of mild carpal tunnel syndrome.

OBJECTIVE: Local glucocorticoid injections are used to treat carpal tunnel syndrome (CTS). However, this treatment is associated with frequent relapses. An important limitation of studies with higher relapse rates is that no attempt has been made to identify patients with mild or severe disease. We evaluated the efficacy of local glucocorticoid injection in patients with mild CTS. METHOD: Mild CTS was defined as intermittent symptoms without absence of sensations, muscle atrophy or weakness of the thenar muscles. Forty-eight patients with idiopathic mild CTS were evaluated before and 3 and 12 months after a single local injection of 40 mg methyl prednisolone acetate. Outcome was assessed by overall satisfaction on a 100 mm visual analogue scale, the Boston self-administered questionnaire for symptom severity and functional scores and improvement in the electrophysiological parameters. RESULTS: At 3 months, 93.7% of the patients reported marked improvement in their symptoms, with significant improvement in the mean values of the nerve conduction parameters distal motor latency at the wrist (DML) (P = 0.00001), distal sensory latency at mid-palm (DSL MP) (P = 0.014) and distal sensory latency at the wrist (DSL W) (P = 0.0003), and symptom severity (P = 4.96 x 10(-8)) and the functional scores (P = 3.56 x 10(-5)). Significant improvement was still present for DML (P = 1.39 x 10(-5)) at 12 months. Almost 50% of the patients achieved normalization in the electrophysiological study. At a median follow-up of 16 months, 79% patients continued to have improvement in their symptoms. Eight patients (16.6%) relapsed following the initial response. CONCLUSIONS: Local glucocorticoid injection results in long-term improvement in nerve conduction parameters, symptom severity and functional scores in patients with mild CTS.

A partial sequence of lipoxygenase gene from genomic DNA of aromatic rice (Oryza sativa L.).

Aromatic (Bas-370, PB-1) and non-aromatic (Pusa-677, Pusa-834) rice were selected for the characterization and for distribution of lipoxygenase (Lox) genes. Polymorphism was observed when genomic DNA of rice varieties was hybridized with a heterologous lipoxygenase probe. A distinct polymorphic fragment (approximately 1.2 kb) was found in Bas-370. Sub-genomic library of Bas-370 was constructed and screened with LoxA probe. The smallest putative clone (pBas-14) of approximately 1.2 kb was sequenced. Complete nucleotide and deduced amino acid sequence showed the clone was 1134 bp long and comprised of 378 amino acid residues. PCR amplification of genomic DNA from four rice varieties with a soybean Lox primer also showed a polymorphic fragment of size approximately 600 bp (amplicon) in aromatic varieties that was sequenced directly. Nucleotide sequence alignment between pBas-14 and amplicon concluded that the amplicon was a part of the insert pBas-14.

Unusual haematological alterations in rheumatoid arthritis.

Three cases of rheumatoid arthritis (RA), presenting with refractory anaemia, thrombocytopenia and peripheral lymphocytosis respectively, were observed. In all the cases haematological manifestations were unrelated to disease activity or drug toxicity. These patients were detected to have pure red cell aplasia (PRCA) (normocytic normochromic anaemia, reticulocytopenia and absence of erythroid precursors in the bone marrow), immune thrombocytopenia (IT) (absence of splenomegaly and presence of increased megakaryocytes in the bone marrow) and multiple myeloma (MM) (lytic lesions on skull, paraproteinaemia and bone marrow plasmacytosis) respectively. PRCA and IT responded to glucocorticoids. Association with these three haematological alterations has rarely been reported. Our report highlights the need to regularly monitor blood counts in patients with RA.

Autoimmune hemolytic anemia associated with benign ovarian cyst: a case report and review of literature.

A 28-years-old female presented with severe autoimmune hemolytic anemia (AIHA), warm antibody mediated, and right side tubo-ovarian (TO) mass. There was sub-optimal response to immunosuppressive therapy (IST). Eight months later she underwent ovarian cystectomy which resulted in complete remission of AIHA without the need for IST. Pathological examination of the TO mass revealed benign ovarian cyst. Our case highlights the need to search for ovarian cyst/mass in females presenting with AIHA. Since immunosuppressive therapy is usually ineffectual in AIHA associated with ovarian cyst, it may be appropriate to contemplate surgery before embarking on IST in such cases.