MiRNA-Mediated Fibrosis in the Out-of-Target Heart following Partial-Body Irradiation.

Recent reports have shown a link between radiation exposure and non-cancer diseases such as radiation-induced heart disease (RIHD). Radiation exposures are often inhomogeneous, and out-of-target effects have been studied in terms of cancer risk, but very few studies have been carried out for non-cancer diseases. Here, the role of miRNAs in the pathogenesis of RIHD was investigated. C57Bl/6J female mice were whole- (WBI) or partial-body-irradiated (PBI) with 2 Gy of X-rays or sham-irradiated (SI). In PBI exposure, the lower third of the mouse body was irradiated, while the upper two-thirds were shielded. From all groups, hearts were collected 15 days or 6 months post-irradiation. The MiRNome analysis at 15 days post-irradiation showed that miRNAs, belonging to the myomiR family, were highly differentially expressed in WBI and PBI mouse hearts compared with SI hearts. Raman spectral data collected 15 days and 6 months post-irradiation showed biochemical differences among SI, WBI and PBI mouse hearts. Fibrosis in WBI and PBI mouse hearts, indicated by the increased deposition of collagen and the overexpression of genes involved in myofibroblast activation, was found 6 months post-irradiation. Using an in vitro co-culture system, involving directly irradiated skeletal muscle and unirradiated ventricular cardiac human cells, we propose the role of miR-1/133a as mediators of the abscopal response, suggesting that miRNA-based strategies could be relevant for limiting tissue-dependent reactions in non-directly irradiated tissues.

Vibrational spectroscopy of liquid biopsies for prostate cancer diagnosis.

BACKGROUND: Screening for prostate cancer with prostate specific antigen and digital rectal examination allows early diagnosis of prostate malignancy but has been associated with poor sensitivity and specificity. There is also a considerable risk of over-diagnosis and over-treatment, which highlights the need for better tools for diagnosis of prostate cancer. This study investigates the potential of high throughput Raman and Fourier Transform Infrared (FTIR) spectroscopy of liquid biopsies for rapid and accurate diagnosis of prostate cancer. METHODS: Blood samples (plasma and lymphocytes) were obtained from healthy control subjects and prostate cancer patients. FTIR and Raman spectra were recorded from plasma samples, while Raman spectra were recorded from the lymphocytes. The acquired spectral data was analysed with various multivariate statistical methods, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and classical least squares (CLS) fitting analysis. RESULTS: Discrimination was observed between the infrared and Raman spectra of plasma and lymphocytes from healthy donors and prostate cancer patients using PCA. In addition, plasma and lymphocytes displayed differentiating signatures in patients exhibiting different Gleason scores. A PLS-DA model was able to discriminate these groups with sensitivity and specificity rates ranging from 90% to 99%. CLS fitting analysis identified key analytes that are involved in the development and progression of prostate cancer. CONCLUSIONS: This technology may have potential as an alternative first stage diagnostic triage for prostate cancer. This technology can be easily adaptable to many other bodily fluids and could be useful for translation of liquid biopsy-based diagnostics into the clinic.

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma.

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)-FTIR and HT-Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.

Monitoring Radiotherapeutic Response in Prostate Cancer Patients Using High Throughput FTIR Spectroscopy of Liquid Biopsies.

Radiation therapy (RT) is used to treat approximately 50% of all cancer patients. However, RT causes a wide range of adverse late effects that can affect a patient's quality of life. There are currently no predictive assays in clinical use to identify patients at risk of normal tissue radiation toxicity. This study aimed to investigate the potential of Fourier transform infrared (FTIR) spectroscopy for monitoring radiotherapeutic response. Blood plasma was acquired from 53 prostate cancer patients at five different time points: prior to treatment, after hormone treatment, at the end of radiotherapy, two months post radiotherapy and eight months post radiotherapy. FTIR spectra were recorded from plasma samples at all time points and the data was analysed using MATLAB software. Discrimination was observed between spectra recorded at baseline versus follow up time points, as well as between spectra from patients showing minimal and severe acute and late toxicity using principal component analysis. A partial least squares discriminant analysis model achieved sensitivity and specificity rates ranging from 80% to 99%. This technology may have potential to monitor radiotherapeutic response in prostate cancer patients using non-invasive blood plasma samples and could lead to individualised patient radiotherapy.

Development of a high throughput (HT) Raman spectroscopy method for rapid screening of liquid blood plasma from prostate cancer patients.

Extensive research has been undertaken on the examination of tissue biopsies using vibrational spectroscopic techniques. However, fewer studies have focused on less invasive and commonly acquired blood samples. Recent studies have shown the ability of Raman and Fourier transform infrared (FTIR) spectroscopy to discriminate between non-cancer controls and cancer cases using blood serum or plasma. Even though many studies have proposed Raman spectroscopy as a potential diagnostic tool in various cancers, the Raman spectroscopic technique has not been introduced as a routine clinical technology. This is due to multiple drawbacks with the application of the technique, including sample preparation, the requirement for expensive substrates and long acquisition times. The current study aims to overcome these limitations and focuses on the translation of Raman spectroscopy into a high throughput clinical diagnostic tool for prostate cancer. In this study, the effect of different instrumental and sample preparation parameters were investigated, with the aim of identifying a combination that would reduce the overall acquisition time for spectra from peripheral blood plasma, reduce the complexity of sample preparation and retain the classification accuracy from Raman spectroscopic diagnostics. A high throughput (HT) system was developed and Raman spectroscopic measurements were performed on plasma samples from 10 prostate cancer patients and 10 healthy volunteers. The spectra were pre-processed and classified by principal component analysis - linear discriminant analysis (PCA-LDA) in the R environment. Statistically significant differences were observed between Raman spectra of prostate cancer patients and non-cancer controls. The (HT) classification resulted in a sensitivity and specificity of 96.5% and 95% respectively. Overall, this study has overcome some of the limitations associated with clinical translation of Raman spectroscopy. The HT-Raman spectroscopy method developed in this study can be used for rapid and accurate diagnosis of prostate cancer using liquid plasma samples.

Multidrug-resistant endemic clonal strain of Candida auris in India.

Candida auris is a recently described rare agent of fungemia. It is notable for its antifungal resistance. A total of 15 C. auris isolates, originating from seven cases of fungemia, three cases of diabetic gangrenous foot, and one case of bronchopneumonia from a tertiary care hospital in south India, were investigated. All of the 15 isolates were identified by sequencing and 14 of these along with 12 C. auris isolates previously reported from two hospitals in Delhi, north India, two each from Japan and Korea were genotyped by amplified fragment length polymorphism (AFLP). In vitro antifungal susceptibility testing (AFST) was done by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. Candida auris isolates were misidentified as Candida haemulonii by VITEK. All were resistant to fluconazole [geometric mean minimum inhibitory concentration (MIC) 64 µg/ml] and 11 isolates were resistant to voriconazole (MIC ≥1 µg/ml). Forty-seven percent of the C. auris isolates were resistant to flucytosine (MIC ≥64 µg/ml) and 40% had high MIC (≥1 µg/ml) of caspofungin. Breakthrough fungemia developed in 28.6% of patients and therapeutic failure in 4 (66.7%) patients. Interestingly, the 26 Indian C. auris isolates from north and south India were clonal and phenotypically and genotypically distinct from Korean and Japanese isolates. The present study demonstrates that C. auris is a potential emerging pathogen that can cause a wide spectrum of human mycotic infections. The prevalence of a C. auris endemic clonal strain resistant to azoles and other antifungals in Indian hospitals with high rates of therapeutic failure in cases of fungemia is worrisome.

Pneumococcusuria: From bench to bedside.

The present study highlights six cases of pneumococcusuria during the time period of May 2008 to May 2010. All the patients had a co-existing predisposing factor with the isolation of Streptococcus pneumoniae in urine. Five of the six patients having signs and symptoms of urinary tract infections (UTI) were treated and cured of the same. It becomes essential to consider pneumococcal UTI in the presence of clinical signs and symptoms associated with urinary tract abnormalities like hydronephrosis and renal stones. S. pneumoniae may be regarded as an emerging pathogen in UTI. Precise microbiological diagnosis must correlate with the clinical signs and symptoms for the administration of appropriate antibiotic therapy.

Targeted treatment of invasive fungal infections accelerates healing of foot wounds in patients with Type 2 diabetes.

AIM: To test the hypothesis that fluconazole plus standard care is superior to the standard care for diabetic foot wounds infected with deep-seated fungal infections. METHODS: We carried out a randomized, controlled, open-label, parallel-arm study in 75 patients with both fungal and bacterial infections in deep tissues of diabetic foot wounds. Thirty-seven patients (control group) were given standard care (surgical debridement + culture-specific antibiotics + offloading + glycaemic control) and 38 patients (treatment group) were given fluconazole 150 mg daily plus standard care. Wound surface area was measured every 2 weeks until the endpoints (complete epithelialization or skin grafting) were met. RESULTS: By week 4, the mean wound surface area reduced to 27.3 from 111.5 cm(2) in the treatment group, as opposed to 67.1 from 87.3 cm(2) in the control group. Subsequently, the mean wound surface areas were remarkably smaller in the treatment group compared with the control group, and statistically significant differences (P ≤ 0.05) in mean wound surface area were observed between the treatment group and the control group at week 6. However, no statistically significant (P ≤ 0.47) difference in complete healing was observed between the treatment group and the control group, 20 vs. 24. The mean wound healing time for the treatment group was 7.3 weeks, whereas for the control group it was 11.3 weeks (P ≤ 0.022). Similarly, the probability of wound healing in the treatment group was 50 vs. 20% in the control group at week 10. CONCLUSIONS: Fluconazole plus standard care was superior to standard care alone in accelerating wound reduction among patients with diabetes with deep-seated fungal infections in diabetic foot wounds. Those in the treatment group who did heal, healed more quickly (P ≤ 0.022), but overall healing was not different.

Chronic subdural empyema and cranial vault osteomyelitis due to Salmonella paratyphi A.

Intracranial infections, especially subdural empyema, due to salmonella are rare. Subdural empyema caused by Salmonella paratyphi A has been documented only once earlier in the literature. Hence, we report a case of subdural empyema and osteomyelitis of cranial vault due to S. paratyphi A. A 42- year-old male presented with headache and purulent discharge from right parietal burr hole wound site. Patient gave a history of head injury two years ago. He underwent burr hole evacuation of chronic subdural haematoma, excision of outer membrane and right parietal craniectomy. The cultures grew S. paratyphi A. Recovery was uneventful following surgical intervention and antibiotic therapy.

Inheritance of RAPD markers in the guppy fish, Poecilia reticulata.

Random Amplified Polymorphic DNA (RAPD) fingerprinting offers a rapid and efficient method for generating a new series of DNA markers in fishes. Three oligonucleotide primers (two 10-mers and one 9-mer) and their paired combinations were found to generate different but reproducible RAPD fingerprints in the guppy. Of these, a 10-mer primer (designated S3D2) was used to detect DNA polymorphisms in two guppy varieties, Green Snakeskin (GSS) and 3/4Black (3/4B). High Genetic Similarity (SI) was found among individuals of the GSS and 3/4B varieties indicating low intra-variety genetic variability. The average SI values for the Green Snakeskin and 3/4Black varieties were 0.78 +/- 0.104 and 0.81 +/- 0.083, respectively. The average SI value between individuals of the GSS and 3/4B varieties was 0.66 +/- 0.066, indicating higher genetic variability between the two varieties. To study the inheritance of RAPD markers, single-pair crosses were set up between males of the GSS variety and females of the 3/4B variety. The S3D2 primer was used to generate RAPD fingerprints of the parents and their F1 offsprings. A total of 14 RAPD markers were scored from these crosses. Of these markers, eight (60.0%) of them were polymorphic. The RAPD markers were shown by the F1 to exhibit dominant Mendelian inheritance and could thus be used for subsequent genetic linkage mapping of the guppy.

RAPD analysis: an efficient method of DNA fingerprinting in fishes.

Random Amplified Polymorphic DNA (RAPD) generated using arbitrary primers of 9, 10, 16 and 20 nucleotide lengths by Polymerase Chain Reaction (PCR) was investigated in 12 species of fishes. We found that the amplification products were best resolved by Urea-SDS-PAGE and detected by silver staining. The amplification products ranged from 25 to 75 depending on the primer and template combination. The random primers generated unique fingerprints for each species of fish in terms of number and position of RAPDs. Our results showed that the fish species can be distinguished from each other by RAPDs. The complexity of the RAPDs in the fingerprints may be manipulated to suit the requirement of the study. The use of RAPD in taxonomy, fishery management and fish culture is discussed.