Identification of Dermatophilus congolensis from lower leg dermatitis of cattle in Kerala, India.

This study was conducted to identify the aetiological agents associated with a particular type of lower leg dermatitis, locally called pododermatitis, among dairy cattle in Kerala. Skin scabs and scrapings were collected aseptically from 82 naturally occurring cases of lower leg dermatitis in cattle and were subjected to direct microscopical examination and bacterial and fungal culture. Microscopical examination of the skin scrapings with 10% potassium hydroxide revealed fungal spores in hair shafts from only two samples and did not reveal the presence of mites or other parasites. Fungal culture yielded dermatophytes from only five samples; these were identified as Trichophyton mentagrophytes in two cases, T verrucosum in one case, Epidermophyton floccosum in one case and Microsporum nanum in one case. Microscopical examination of Giemsa- and Gram-stained smears of the scab material from the lesions from 72 cases revealed characteristic Gram-positive septate branching filaments with multiple rows of spherical to ovoid cocci, with a typical 'tram-track' appearance suggestive of Dermatophilus congolensis. Culture of the scab materials on sheep blood agar in the presence of 10% carbon dioxide yielded typical beta haemolytic colonies of D. congolensis from 75 samples. The isolates were further confirmed by the macroscopic and microscopic morphology of the colonies, and biochemical test results. This study confirmed the presence of dermatophilosis caused by D. congolensis in cattle in Kerala.

Interferometric spectroscopy of scattered light can quantify the statistics of subdiffractional refractive-index fluctuations.

Despite major importance in physics, biology, and other sciences, the optical sensing of nanoscale structures in the far zone remains an open problem due to the fundamental diffraction limit of resolution. We establish that the expected value of spectral variance (Σ[over ˜](2)) of a far-field, diffraction-limited microscope image can quantify the refractive-index fluctuations of a label-free, weakly scattering sample at subdiffraction length scales. We report the general expression of Σ[over ˜] for an arbitrary refractive-index distribution. For an exponential refractive-index spatial correlation, we obtain a closed-form solution of Σ[over ˜] that is in excellent agreement with three-dimensional finite-difference time-domain solutions of Maxwell's equations. Sensing complex inhomogeneous media at the nanoscale can benefit fields from material science to medical diagnostics.

Targeted alteration of real and imaginary refractive index of biological cells by histological staining.

Various staining techniques are commonly used in biomedical research to investigate cellular morphology. By inducing absorption of light, staining dyes change the intracellular refractive index due to the Kramers-Kronig relationship. We present a method for creating 2D maps of real and imaginary refractive indices of stained biological cells using their thickness and absorptance. We validate our technique on dyed polystyrene microspheres and quantify the alteration in refractive index of stained biological cells. We reveal that specific staining of individual organelles can increase their scattering cross-section by orders of magnitudes, implying a major impact in the field of biophotonics.

Machine Learning in Differentiating Gliomas from Primary CNS Lymphomas: A Systematic Review, Reporting Quality, and Risk of Bias Assessment.

BACKGROUND: Differentiating gliomas and primary CNS lymphoma represents a diagnostic challenge with important therapeutic ramifications. Biopsy is the preferred method of diagnosis, while MR imaging in conjunction with machine learning has shown promising results in differentiating these tumors. PURPOSE: Our aim was to evaluate the quality of reporting and risk of bias, assess data bases with which the machine learning classification algorithms were developed, the algorithms themselves, and their performance. DATA SOURCES: Ovid EMBASE, Ovid MEDLINE, Cochrane Central Register of Controlled Trials, and the Web of Science Core Collection were searched according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. STUDY SELECTION: From 11,727 studies, 23 peer-reviewed studies used machine learning to differentiate primary CNS lymphoma from gliomas in 2276 patients. DATA ANALYSIS: Characteristics of data sets and machine learning algorithms were extracted. A meta-analysis on a subset of studies was performed. Reporting quality and risk of bias were assessed using the Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis (TRIPOD) and Prediction Model Study Risk Of Bias Assessment Tool. DATA SYNTHESIS: The highest area under the receiver operating characteristic curve (0.961) and accuracy (91.2%) in external validation were achieved by logistic regression and support vector machines models using conventional radiomic features. Meta-analysis of machine learning classifiers using these features yielded a mean area under the receiver operating characteristic curve of 0.944 (95% CI, 0.898-0.99). The median TRIPOD score was 51.7%. The risk of bias was high for 16 studies. LIMITATIONS: Exclusion of abstracts decreased the sensitivity in evaluating all published studies. Meta-analysis had high heterogeneity. CONCLUSIONS: Machine learning-based methods of differentiating primary CNS lymphoma from gliomas have shown great potential, but most studies lack large, balanced data sets and external validation. Assessment of the studies identified multiple deficiencies in reporting quality and risk of bias. These factors reduce the generalizability and reproducibility of the findings.

Prevalence of Linguatula serrata in domestic ruminants in South India.

Linguatula serrata Frohlich, 1789 is an aberrant endoparasite of occasional zoonotic importance in humans. We report the prevalence of encapsulated nymphal stages of L. serrata in domestic ruminants surveyed in Wayanad, located in Kerala State, South India. Large and small intestine mesentery were examined from 100 goats, cattle and buffaloes each revealing an infection prevalence of 21, 19, and 8%, respectively. The prevalence of L. serrata infection among ruminants is higher than previous reports from India and indicates a strong focus of infection in herbivorous domesticated mammals of South India. The potential importance of these findings to human health is discussed.

Visceral schistosomiasis among domestic ruminants slaughtered in Wayanad, South India.

This short communication reports the prevalence of visceral schistosomiasis by worm counts from the mesentery of domestic ruminants of the hilly district of Wayanad, located in Kerala, one of the states in South India. We found 57.3, 50, and 4.7% of cattle, buffaloes and goats, respectively, had visceral schistosomiasis upon slaughter at a municipal slaughter house in Kalpetta. Our findings show that the prevalence of Schistosoma spindale infection is very high in Wayanad in comparison to previous reports from this and neighboring countries.

Morphometric profile of large intestinal neuronal plexuses in normal perinatal autopsies and Hirschsprung disease.

OBJECTIVES: To derive objective values for the diagnosis of Hirschsprung disease (HSCR) from a comparison of the morphometric profile of large intestinal neuronal plexuses in normal perinatal autopsies and surgical specimens of HSCR. METHODS: A cross-sectional comparative study with 40 subjects each in (i) non-HSCR perinatal group encompassing neonates and stillborn babies beyond 30 weeks of gestation on whom autopsies were conducted and (ii) HSCR group comprising all patients clinicoradiologically diagnosed as HSCR. The morphometric assessment was done on hematoxylin-and-eosin-stained sections. KEY RESULTS: The morphometric profile in terms of average number of ganglia/linear mm of colon, interganglion distance, number of ganglion cells/ganglion, average ganglion cell length, ganglion cell nuclear area, ganglion cell nuclear diameter, nerve trunk thickness, and density has been outlined. On comparison with the neuroanatomically normal zone of HSCR, the cut-offs to identify hypertrophic nerve trunks (nerve trunk thickness of >37.85 µm) and reduced number of ganglia (number of ganglia/linear mm of colon <2.05 and interganglion distance of >229 µm) were derived. CONCLUSIONS & INFERENCES: The determined objective values, after testing on diagnostic rectal biopsies, may serve to formulate a diagnostic algorithm along with immunostaining for diagnosis of HSCR in colorectal specimens.

The Global Relationship between Chromatin Physical Topology, Fractal Structure, and Gene Expression.

Most of what we know about gene transcription comes from the view of cells as molecular machines: focusing on the role of molecular modifications to the proteins carrying out transcriptional reactions at a loci-by-loci basis. This view ignores a critical reality: biological reactions do not happen in an empty space, but in a highly complex, interrelated, and dense nanoenvironment that profoundly influences chemical interactions. We explored the relationship between the physical nanoenvironment of chromatin and gene transcription in vitro. We analytically show that changes in the fractal dimension, D, of chromatin correspond to simultaneous increases in chromatin accessibility and compaction heterogeneity. Using these predictions, we demonstrate experimentally that nanoscopic changes to chromatin D within thirty minutes correlate with concomitant enhancement and suppression of transcription. Further, we show that the increased heterogeneity of physical structure of chromatin due to increase in fractal dimension correlates with increased heterogeneity of gene networks. These findings indicate that the higher order folding of chromatin topology may act as a molecular-pathway independent code regulating global patterns of gene expression. Since physical organization of chromatin is frequently altered in oncogenesis, this work provides evidence pairing molecular function to physical structure for processes frequently altered during tumorigenesis.

Procedures for risk-stratification of lung cancer using buccal nanocytology.

Lung cancer is the leading cause of cancer deaths in the U.S. with survival dramatically depending on stage at diagnosis. We had earlier reported that nanocytology of buccal cells can accurately risk-stratify smokers for the presence of early and late-stage lung cancer. To translate the technique into clinical practice, standardization of operating procedures is necessary to consistently yield precise and repeatable results. Here, we develop and validate simple, robust, and easily implementable procedures for specimen collection, processing, etc. in addition to a commercially-viable instrument prototype. Results of this work enable translation of the technology from academic lab to physicians' office.

Dirofilariosis in dogs and humans in Kerala.

There has been an increased occurrence of filarial worms in the subcutaneous tissues and subconjunctival space of human beings belonging to different areas of the state of Kerala. The present work was carried out to identify the worms recovered from human cases, to study the presence of microfilariae in dogs and to discuss the possible mode of infection in human beings. Twelve worm specimens from human patients received in the Department of Veterinary Parasitology, College of Veterinary and Animal Sciences, Mannuthy from 2002 to 2004 were identified as Dirofilaria repens based on morphology. Out of the 160 blood smears of dogs examined during the same period, microfilariae were detected in 11 samples (7%). With 7 per cent dogs positive for microfilariae of D. repens in Kerala, it is suggested that human beings are at an enhanced risk of acquiring dirofilaria infection.

The sGC stimulator riociguat inhibits platelet function in washed platelets but not in whole blood.

BACKGROUND AND PURPOSE: Stimulation of soluble guanylyl cyclase (sGC) is a valuable therapeutic strategy for the treatment of several cardiovascular diseases. The sGC stimulator riociguat has been approved for the treatment of two forms of pulmonary hypertension. Platelets contain large amounts of sGC and play a key role in the regulation of haemostasis. Therefore, we investigated the effects of riociguat on platelet function. EXPERIMENTAL APPROACH: The effect of riociguat treatment on human platelet activation and aggregation was investigated. The sGC-specific effects of riociguat were determined by comparing wild-type and platelet-specific sGC-knockout mice. KEY RESULTS: Riociguat induced cGMP synthesis and subsequent PKG activation in human platelets, suggesting that the inhibitory effects are mediated by cGMP signalling. This finding was confirmed when sGC-knockout platelets were not inhibited by riociguat. In washed human platelets, 100 nM riociguat reduced ADP-induced GPIIb/IIIa activation, while a 10-fold higher concentration was required to reduce convulxin-stimulated GPIIb/IIIa activation. Riociguat inhibited ADP-induced platelet shape change and aggregation, while ATP-induced shape change remained unaffected. However, in PRP and whole blood, 50-100 µM riociguat was required to inhibit platelet activation and aggregation. Riociguat in combination with iloprost significantly inhibited platelet aggregation, even in whole blood. CONCLUSIONS AND IMPLICATIONS: Riociguat inhibits platelet activation in whole blood only at concentrations above 50 µM, while the plasma concentrations in riociguat-treated patients are 150 to 500 nM. This finding indicates that riociguat treatment does not affect platelet function in patients. Nevertheless, the possibility that riociguat acts synergistically with iloprost to inhibit platelet activation should be considered.

Oxidative phosphorylation system during steady-state hypoxia in the dog brain.

The relationship between biochemical and physiological responses and tissue O2 during hypoxia was investigated in vivo in the dog brain by 31P nuclear magnetic resonance (NMR) spectroscopy. Our findings demonstrate how ATP synthesis in the brain can be maintained during hypoxia because of compensatory changes in NADH, ADP, and Pi. Eleven beagle dogs were anesthetized and mechanically ventilated, and a steady-state graded hypoxia was induced by decreasing the fraction of inspired O2 (FIO2) stepwise at 20-min intervals. Biochemical metabolites were measured using 31P-NMR and fluorescence spectroscopy. When sagittal sinus O2 partial pressure (PVO2) had decreased to 15 Torr, NADH increased by 30%, Pi increased by 50%, and phosphocreatine (PCr) decreased by 20%. In contrast, ATP remained constant. There was a 10% increase in ADP in dogs that maintained a steady temperature, but ADP decreased by as much as 30% in dogs in which body temperature decreased with the falling PVO2. PCr/Pi was logarithmically related to the phosphorylation potential during steady-state hypoxia. Compensation for the O2 lack is attributed to increases in ADP, Pi, and NADH as a result of the reciprocal relationship of the Michaelis-Menten equation. If the Michaelis-Menten constants (Km) of ADP, Pi, and O2 are the same as determined in vitro in mitochondria, the minimum brain cytosolic O2 capable of maintaining a steady-state ATP is near its Km (0.1 Torr) at a PVO2 of 7.5 Torr. At this critical O2 level, PCr/Pi is 0.9, intracellular pH is 6.75, phosphorylation potential is 38.5 mM-1, and the calculated maximum velocity of ATP formation by oxidative phosphorylation is 55% of normal.

Relationship between intracellular pH and energy metabolism in dog brain as measured by 31P-NMR.

The relationships between pHi (intracellular pH) and phosphate compounds were evaluated by nuclear magnetic resonance (NMR) in normo-, hypo-, and hypercapnia, obtained by changing fractional inspired concentration of CO2 in dogs anesthetized with 0.75% isoflurane and 66% N2O. Phosphocreatine (PCr) fell by 2.02 mM and Pi (inorganic phosphate) rose by 1.92 mM due to pHi shift from 7.10 to 6.83 during hypercapnia. The stoichiometric coefficient was 1.05 (r2 = 0.78) on log PCr/Cr against pHi, showing minimum change of ADP/ATP and equilibrium of creatine kinase in the pH range of 6.7 to 7.25. [ADP] varied from 21.6 +/- 4.1 microM in control (pHi = 7.10) to 26.8 +/- 6.3 microM in hypercapnia (pHi = 6.83) and 24.0 +/- 6.8 microM in hypocapnia (pHi = 7.17). ATP/ADP X Pi decreased from 66.4 +/- 17.1 mM-1 during normocapnia to 25.8 +/- 6.3 mM-1 in hypercapnia. The ADP values are near the in vitro Km; thus ADP is the main controller. The velocity of oxidative metabolism (V) in relation to its maximum (Vmax) as calculated by a steady-state Michaelis-Menten formulation is approximately 50% in normocapnia. In acidosis (pH 6.7) and alkalosis (pH 7.25), V/Vmax is 10% higher than the normocapnic brain. This increase of V/Vmax is required to maintain cellular homeostasis of energy metabolism in the face of either inhibition at extremes of pH or higher ATPase activity.

Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle.

Endurance muscle performance is highly dependent on ATP production from mitochondrial oxidative phosphorylation. To study the role of the mitochondrial oxidative enzymes in muscle fatigue, we analyzed the relationship between the concentrations of substrates associated with ATP synthesis and the muscle performance of electrically stimulated rabbit muscle under CO2-induced acidosis. Two different conditions of pacing-induced muscle performance were produced in the gastrocnemius and soleus muscle groups in anesthetized rabbits by stimulating the sciatic nerve submaximally at two frequencies. Phosphorus nuclear magnetic resonance was used to measure ATP, phosphocreatine, and Pi and to provide data for a calculation of intracellular pH and free ADP. To induce acidosis, the animal was ventilated with 20% CO2. The administration of CO2 effectively reduced the intracellular pH from 6.9 to 6.7 and reduced the isometric tension-time integral (TTI) to below half the value measured in normocapnia at the low pacing frequency. A twofold increase in the pacing frequency resulted in a doubling of the TTI in normocapnia and a tripling of TTI in hypercapnia. The increases in TTI corresponded with increases in free ADP and Pi concentrations. Under the various conditions, all free ADP values were near the in vitro Michaelis-Menten constant (Km) of ADP. The Michaelis-Menten relationship of the oxidative phosphorylative enzymes was applied to the change in substrate concentrations with respect to TTI. From this relationship we observed that the in vivo Km of free ADP was 26 microM, which is close to the in nitro Km, and that Km and maximal reaction velocity did not change under hypercapnia and increased pacing frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental brain ischaemia: assessment of injury by magnetic resonance spectroscopy and histology.

Phosphorus magnetic resonance spectroscopy (31P-MRS) was made to measure changes in brain high energy phosphate compounds, adenosine triphosphate (ATP) and phosphocreatine (PCr), inorganic phosphorus (Pi) and intracellular pH (pHi) during a prolonged period of incomplete brain ischaemia produced, in anaesthetized dogs, by bilateral carotid occlusion together with haemorrhagic hypotension for intervals of up to 300 min. Mean arterial blood pressure (MABP) was lowered in a stepwise fashion, until signs of metabolic decompensation (as estimated by MRS) occurred. At that point MABP was varied against further evidence of metabolic decompensation in an attempt to maintain a more constant degree of insult. At the end of the ischaemic period MABP was restored and the animals observed during a 3 h recovery period. At the end of the recovery period the brains were perfusion-fixed for histological examination. A semi-quantitative method of histological evaluation was used to determine the degree of histological damage. This permitted assignment of an 'ischaemic score' to the tissue sampled from each animal. Comparisons were then made between the magnitude of this 'ischaemic score' and the changes in metabolic and physiological variables (ATP, PCr, pHi and MABP) as well as an estimator of phosphorylation potential (PCr/Pi), which were all measured during the ischaemic insult. Histological examination showed a wide variety of neuronal alterations, including dark and pale type injury, which correlated directly with the metabolic derangements brought about by ischaemia. The degree of damage determined from this histological assessment correlated best with the duration and degree of change in PCr/Pi, supporting the use of this ratio as a critical index of cellular energy state. In particular there was a strong linear relationship between the degree of leucocyte recruitment and changes in PCr/Pi. To summarize, metabolic changes, determined by MRS, correlate with the degree of histological damage, and in turn, the classical descriptions of acute ischaemic neuronal injury appear to be validated by MRS determinations of metabolic changes during ischaemia.

Age dependence of steady state mitochondrial oxidative metabolism in the in vivo hypoxic dog brain.

Mitochondrial bioenergetics were investigated in newborn, neonatal and adult dog brains during normoxia and hypoxia. The ratio of the rate of ATP synthesis to the maximum synthesis rate (V/Vmax), phosphorylation potential, [ADP] and PCr/Pi, were used to evaluate age related mitochondrial hypoxic tolerance. These indicators were calculated from the phosphorus compounds measured by in vivo 31P MRS quantitatively using ATP as an internal reference. Indicators and substrates of mitochondrial function, V/Vmax, ADP, and Pi reached a peak value during the neonatal (3-21 days) period of development, suggesting that the oxidative metabolism of the neonate is more vulnerable to stress when compared to newborns and adults. Distinction among newborns and neonates became apparent during hypoxia. Newborns (0-2 days old) showed substantial tolerance by maintaining V/Vmax until exposure to severe hypoxia. Older neonates (3-21 days old) showed increases in V/Vmax, [Pi] and [ADP] under less than severe conditions of hypoxia. Adults exhibited low V/Vmax values even during exposure to severe hypoxia, further indicating that mitochondrial oxidative processes are more stable in adults than in newborns and neonates. This study provides evidence that newborns and adults are more capable of maintaining mitochondrial function under conditions of minimal to moderate hypoxia than 3-21 day old neonates.

Orai1-induced store-operated Ca(2+) entry enhances phospholipase activity and modulates canonical transient receptor potential channel 6 function in murine platelets.

BACKGROUND: Orai1, the major store-operated Ca(2+) entry (SOCE) channel in platelets, is not only critical for enhancing diverse signaling pathways, but may also regulate receptor-operated Ca(2+) entry (ROCE). Dynamic coupling of the Orai1 signalosome to canonical transient receptor potential channels (TRPCs) has been suggested as an essential step in the activation of SOCE and ROCE. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms remains controversial. OBJECTIVE: We aimed to elucidate the role of Orai1 in diacylglycerol (DAG)-mediated ROCE. METHODS: Trpc6(-/-) , Orai1(-/-) and Orai1(-/-) /Trpc6(-/-) mice were generated, and their platelets were analyzed. RESULTS: Thapsigargin (TG)-induced SOCE was further reduced in Orai1(-/-) /Trpc6(-/-) platelets as compared with Orai1(-/-) platelets, thus revealing that TG-induced signaling pathways can activate TRPC6. Thapsigargin-induced SOCE leads to enhanced phospholipase C and D activity in wild-type platelets. The activity of both enzymes was significantly reduced in Orai1(-/-) platelets upon TG stimulation, whereas receptor-induced phospholipase activity was not affected. Furthermore, TG-induced and glycoprotein VI-mediated thromboxane A2 release was strongly dependent on Orai1-mediated SOCE. CONCLUSION: The regulation of TRPC6 activity can occur independently of the physical interaction with Orai1. TRPC6 operates in crosstalk with Orai1 through Orai1-induced DAG production via phospholipase activation. Orai1-induced DAG production and thromboxane release amplify the second phase of Ca(2+) signaling in platelets.

Phosphorylation of CalDAG-GEFI by protein kinase A prevents Rap1b activation.

BACKGROUND: Signaling via protein kinase A (PKA) and protein kinase G (PKG) is critical for maintaining platelets in the resting state. Both kinases down-regulate the activity of the small GTPase Rap1b, a critical signaling switch for integrin activation and platelet aggregation. However, the mechanism of Rap1b regulation by PKA and PKG is largely unknown. OBJECTIVE: To identify the PKA phosphorylation sites in calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), the main GEF for Rap1b in platelets, and the effect of CalDAG-GEFI phosphorylation in Rap1b activation. METHODS: The phosphorylation sites in CalDAG-GEFI were identified by radio-active phosphate incorporation assay and mass spectrometry. Phospho-antibody was developed to detect CalDAG-GEFI phosphorylation in Western blots. Rap1b activation was detected by Rap1-GTP pull-down assay. RESULTS: S587 was identified as the major PKA phosphorylation site in CalDAG-GEFI, while S116/117 was weakly phosphorylated. Phosphorylation of S587 correlated with the inhibitory effect of PKA on Rap1b activation in platelets. In HEK293 cells, expression of a phospho-mimetic mutant of CalDAG-GEFI (S587D) abolished agonist-induced Rap1b activation. Mutation of S587 to alanine partially reversed the inhibitory effect of PKA signaling on Rap1b activation, while mutation of S116, S117 and S587 to alanine completely abolished the inhibitory effect of PKA on Rap1b activation. CONCLUSION: Our study strongly suggests that phosphorylation of CalDAG-GEFI is a critical mechanism by which PKA controls Rap1b-dependent platelet aggregation.