16S rRNA based metagenomic analysis unveils unique oral microbial signatures in oral squamous cell carcinoma cases from Coastal Karnataka, India.

Oral Squamous cell carcinoma (OSCC) is the 14th most frequent cancer with 300,000 new cases and 100,000 deaths reported annually. Even with advanced therapy, the treatment outcomes are poor at advanced stages of the disease. The diagnosis of early OSCC is of paramount clinical value given the high mortality rate associated with the late stages of the disease. Recently, the role of microbiome in the disease manifestation, including oral cancer, has garnered considerable attention. But, to establish the role of bacteria in oral cancer, it is important to determine the differences in the colonization pattern in non-tumour and tumour tissues. In this study, 16S rRNA based metagenomic analyses of 13 tumorous and contralateral anatomically matched normal tissue biopsies, obtained from patients with advanced stage of OSCC were evaluated to understand the correlation between OSCC and oral microbiome. In this study we identified Fusobacterium, Prevotella, Capnocytophaga, Leptotrichia, Peptostreptococcus, Parvimonas and Bacteroidetes as the most significantly enriched taxa in OSCC lesions compared to the non-cancerous tissues. Further, PICRUSt2 analysis unveiled enhanced expression of metabolic pathways associated with L-lysine fermentation, pyruvate fermentation, and isoleucine biosynthesis in those microbes associated with OSCC tissues. These findings provide valuable insights into the distinctive microbial signatures associated with OSCC, offering potential biomarkers and metabolic pathways underlying OSCC pathogenesis. While our focus has primarily centred on microbial signatures, it is essential to recognize the pivotal role of host factors such as immune responses, genetic predisposition, and the oral microenvironment in shaping OSCC development and microbiome composition.

Cháksu: A glaucoma specific fundus image database.

We introduce Cháksu-a retinal fundus image database for the evaluation of computer-assisted glaucoma prescreening techniques. The database contains 1345 color fundus images acquired using three brands of commercially available fundus cameras. Each image is provided with the outlines for the optic disc (OD) and optic cup (OC) using smooth closed contours and a decision of normal versus glaucomatous by five expert ophthalmologists. In addition, segmentation ground-truths of the OD and OC are provided by fusing the expert annotations using the mean, median, majority, and Simultaneous Truth and Performance Level Estimation (STAPLE) algorithm. The performance indices show that the ground-truth agreement with the experts is the best with STAPLE algorithm, followed by majority, median, and mean. The vertical, horizontal, and area cup-to-disc ratios are provided based on the expert annotations. Image-wise glaucoma decisions are also provided based on majority voting among the experts. Cháksu is the largest Indian-ethnicity-specific fundus image database with expert annotations and would aid in the development of artificial intelligence based glaucoma diagnostics.

Efficacy, safety and tolerability of bosentan as an adjuvant to sildenafil and sildenafil alone in persistant pulmonary hypertension of newborn (PPHN).

Background: Pulmonary Arterial Hypertension (PAH) carries a poor prognosis in both adult and pediatric patients. It is a life-threatening condition in newborns. Current recommendations advocate the use of targeted monotherapy as a first-line approach for the treatment of Persistent Pulmonary Hypertension of the Newborn (PPHN). In case of an inadequate clinical response to treatment, an addition of a second or third agent is considered. PAH is usually managed with a phosphodiesterase 5 inhibitor or an endothelin receptor blocker. There are limited pediatric studies that address questions like which class of therapy should be initiated first or if a combination should be initiated together. With this background, the present study was initiated to compare the efficacy, safety, and tolerability of bosentan as an adjuvant to sildenafil and sildenafil alone in PPHN. Results: A total of 40 patients were enrolled in the study. Out of them, 26 were males (65%) and 14 were females (35%). PPHN was most commonly seen in the 29 (72.5%) of participants with a history of first order birth. Mean duration of symptoms was 14.05 ± 2.06 days. The participants were randomized to two groups. Group A consisted of total 25 participants that received both bosentan and sildenafil and group B had 15 participants that received sildenafil alone. Both groups were comparable in terms of birth weight and present weight, consanguinity, and mode of delivery. Efficacy was determined by the reduction in mean baseline Pulmonary Artery Systolic Pressure (PASP). PASP in group A was 75.56 ± 10.62 mm Hg and in group B was 64.86 ± 12.25 mm Hg which was not statistically significant (P > 0.05). PASP on the third and seventh day in group A were 43.72 ± 8.63 and 24.47 ± 3.52 mm Hg compared to 42.28 ± 9.43 and 27.276 ± 8.38 respectively in group B which was statistically significant (P < 0.05).There were two deaths each in both groups. Two participants in Group A developed liver function abnormalities. None of the participants in Group B had adverse effects. Conclusion: Most common clinical manifestations were nonspecific. Cardiovocal syndrome was common in PPHN. We conclude that oral sildenafil treatment is a safe, simple and effective treatment for persistent pulmonary hypertension in newborn. Combination of bosentan with sildenafil is more effective and safe in reducing pulmonary artery (PA) pressures in high-risk patients with PPHN.

Automatic Segmentation of Lumen Intima Layer in Longitudinal Mode Ultrasound Images.

We propose an automated method for the segmentation of lumen intima layer of the common carotid artery in longitudinal mode ultrasound images. The method is hybrid, in the sense that a coarse segmentation is first achieved by optimizing a locally defined contrast function of an active oblong considering its five degrees-of-freedom, and subsequently the fine segmentation and delineation of the carotid artery are achieved by post-processing the portion of the ultrasound image spanned by the annulus region of the optimally fitted active oblong. The post-processing includes median filtering and Canny edge detection to retain the lumen intima representative points followed by a smooth curve fitting technique to delineate the lumen intima boundary. The algorithm has been validated on 84 longitudinal mode carotid artery ultrasound images provided by the Signal Processing laboratory, Brno university. The proposed technique results in an average accuracy and Dice similarity index of 98.9% and 95.2%, respectively.

Automatic analysis of normative retinal oximetry images.

Retinal oximetry is an important screening tool for early detection of retinal pathologies due to changes in the vasculature and also serves as a useful indicator of human-body-wide vascular abnormalities. We present an automatic technique for the measurement of oxygen saturation in retinal arterioles and venules using dual-wavelength retinal oximetry images. The technique is based on segmenting an optic-disc-centered ring-shaped region of interest and subsequent analysis of the oxygen saturation levels. We show that the two dominant peaks in the histogram of the oxygen saturation levels correspond to arteriolar and venular oxygen saturations from which the arterio-venous saturation difference (AVSD) can be calculated. For evaluation, we use a normative database of Asian Indian eyes containing 44 dual-wavelength retinal oximetry images. Validations against expert manual annotations of arterioles and venules show that the proposed technique results in an average arteriolar oxygen saturation (SatO2) of 87.48%, venular SatO2 of 57.41%, and AVSD of 30.07% in comparison with the expert ground-truth average arteriolar SatO2 of 89.41%, venular SatO2 of 56.32%, and AVSD of 33.09%, respectively. The results exhibit high consistency across the dataset indicating that the automated technique is an accurate alternative to the manual procedure.

Rim-to-Disc Ratio Outperforms Cup-to-Disc Ratio for Glaucoma Prescreening.

We present a novel and fully automated fundus image processing technique for glaucoma prescreening based on the rim-to-disc ratio (RDR). The technique accurately segments the optic disc and optic cup and then computes the RDR based on which it is possible to differentiate a normal fundus from a glaucomatous one. The technique performs a further categorization into normal, moderate, or severely glaucomatous classes following the disc-damage-likelihood scale (DDLS). To the best of our knowledge, this is the first engineering attempt at using RDR and DDLS to perform glaucoma severity assessment. The segmentation of the optic disc and cup is based on the active disc, whose parameters are optimized to maximize the local contrast. The optimization is performed efficiently by means of a multiscale representation, accelerated gradient-descent, and Green's theorem. Validations are performed on several publicly available databases as well as data provided by manufacturers of some commercially available fundus imaging devices. The segmentation and classification performance is assessed against expert clinician annotations in terms of sensitivity, specificity, accuracy, Jaccard, and Dice similarity indices. The results show that RDR based automated glaucoma assessment is about 8% to 10% more accurate than a cup-to-disc ratio (CDR) based system. An ablation study carried out considering the ground-truth expert outlines alone for classification showed that RDR is superior to CDR by 5.28% in a two-stage classification and about 3.21% in a three-stage severity grading.

Biochemical and pharmacological characterization of three toxic phospholipase A2s from Daboia russelii snake venom.

Three isoenzymes of phospholipase A2 (PLA2), VRV-PL-IIIc, VRV-PL-VII, and VRV-PL-IX were isolated from Daboia russelii snake venom. The venom, upon gel filtration on Sephadex G-75 column, resolved into six peaks (DRG75 I-VI). The VRV-PL-IIIc was purified by subjecting DRG75II to homogeneity by rechromatography in the presence of 8M urea on Sephadex G-75 column. The other two isoenzymes VRV-PL-VII and VRV-PL-IX were purified by subjecting DRG75III to ion exchange chromatography on CM-Sephadex C-25 column. Mol wt. for the three PLA2s, VRV-PL-IIIc, VRV-PL-VII, and VRV-PL-IX are 13.003kDa, 13.100kDa and 12.531kDa respectively. The VRV-PL-IIIc is not lethal to mice up to 14mg/kg body weight but it affects blood sinusoids and causes necrosis of the hepatocytes in liver. It causes hemorrhage in kidney and shrinkage of renal corpuscles and renal tubules. The LD50s for VRV-PL-VII and VRV-PL-IX are 7 and 7.5mg/kg body weight respectively. They induced neurotoxic symptoms similar to VRV-PL-V. All the three PLA2s are anticoagulant and induced varying degree of edema in the foot pads of mice. VRV-PL-V and VRV-PL-VII are shown to act as pre and post synaptic toxins, while VRV-PL-IX acts as presynaptic toxin. This is evident from experiments conducted on cultured hippocampal neurons by patch clamp electrophysiology.

Profile of EBV associated infectious mononucleosis.

During a 5 year period, 33 children (22 males) were diagnosed to have infectious mononucleosis (M:F::2:1; age 9 mo-15 y). The common clinical features observed were fever (100%), lymphadenopathy (84%) hepatosplenomegaly (81%), tonsillar enlargement (45%), neck swelling (30%), upper respiratory symptoms (21%), epitrochlear node enlargement (20%), vomiting and diarrhea (1%). Ten children had complications; upper airway obstruction and hemophagocytic lymphohistocytosis occurred in four each and septic shock in two. EBV associated infectious mononucleosis in hospitalized children was found to affect mainly preschool children and had a favorable prognosis.

Isolation and characterization of "Reprotoxin", a novel protein complex from Daboia russelii snake venom.

In snake venoms, non-covalent protein-protein interaction leads to protein complexes with synergistic and, at times, distinct pharmacological activities. Here we describe a new protein complex containing phospholipaseA(2) (PLA(2)), protease, and a trypsin inhibitor. It is isolated from the venom of Daboia russelii by gel permeation chromatography, on a Sephadex G-75 column. This 44.6 kDa complex exhibits only phospholipase A(2) activity. In the presence of 8M urea it is well resolved into protease (29.1 kDa), PLA(2) (13 kDa), and trypsin inhibitor (6.5 kDa) peaks. The complex showed an LD(50) of 5.06 mg/kg body weight in mice. It inhibited the frequency of spontaneous release of neurotransmitter in hippocampal neurons. It also caused peritoneal bleeding, and edema in the mouse foot pads. Interestingly, the complex caused degeneration of both the germ cells and the mouse Leydig cells of mouse testis. A significant reduction in both the diameter of the seminiferous tubules and height of the seminiferous epithelia were observed following intraperitoneal injection of the sub-lethal dose (3 mg/kg body weight). This effect of the toxin is supported by the increase in the activities of acid and alkaline phosphatases and the nitric oxide content in the testes, and a decrease in the ATPase activity. Because of its potent organ atrophic effects on the reproductive organs, the toxin is named "Reprotoxin". This is the first report demonstrating toxicity to the reproductive system by a toxin isolated from snake venom.

Immunostimulatory principles from Chlorella pyrenoidosa--part 1: isolation and biological assessment in vitro.

Our proprietary preparation obtained by extraction of Chlorella pyrenoidosa cells, ONC-107 (Respondin), was recently found to selectively boost antibody response to the influenza vaccine in a human clinical trial. Respondin is a potent stimulator of mouse B cell proliferation and an activator of macrophages. Bioactivity-guided resolution concluded that Respondin is composed of a mixture of immunostimulatory principles of different chemical nature. A combination of size exclusion, anion exchange and hydrophobic interaction chromatography revealed that the bulk of the immunostimulatory activity resides in polysaccharide/protein complexes with molecular masses larger than 100 kDa that are composed primarily of galactose, rhamnose and arabinose.

A rapid and sensitive extractive spectrophotometric determination of copper(II) in pharmaceutical and environmental samples using benzildithiosemicarbazone.

Benzildithiosemicarbazone (BDTSC) is proposed as a sensitive and selective analytical reagent for the extractive spectrophotometric determination of copper(II). BDTSC reacts with copper(II) in the pH range 1.0-7.0 to form a yellowish complex. Beer's law is obeyed in the concentration range 0.5-0.4 microg cm(-3). The yellowish Cu(II)-BDTSC complex in chloroform shows a maximum absorbance at 380 nm, with molar absorptivity and Sandell's sensitivity values of 1.63 x 10(4) dm3 mol(-1) cm(-1) and 0.00389 microg cm(-2), respectively. A repetition of the method is checked by finding the relative standard deviation (RSD) (n = 10), which is 0.6%. The composition of the Cu(II)-BDTSC complex is established as 1:1 by slope analysis, molar ratio and Asmus' methods. An excellent linearity with a correlation coefficient value of 0.98 is obtained for the Cu(II)-BDTSC complex. The instability constant of the complex calculated from Edmond and Birnbaum's method is 7.70 x 10(-4) and that of Asmus' method is 7.66 x 10(-4), at room temperature. The method is successfully employed for the determination copper(II) in pharmaceutical and environmental samples. The reliability of the method is assured by analyzing the standard alloys (BCS 5g, 10g, 19e, 78, 32a, 207 and 179) and by inter-comparison of experimental values, using an atomic absorption spectrometer.

A rapid and sensitive extractive spectrophotometric determination of palladium(II) in synthetic mixtures and hydrogenation catalysts using pyridoxal-4-phenyl-3-thiosemicarbazone.

A rapid and sensitive extractive spectrophotometric method has been developed for the determination of palladium(II) in synthetic mixtures and hydrogenation catalysts using pyridoxal-4-phenyl-3-thiosemicarbazone (PPT) as an analytical reagent. The reagent forms a red-color complex with the metal at pH 3.0, which is extracted into benzene. The absorbance is measured at 460 nm. The method adheres to Beer's law up to a concentration range of 0.4-6.4 microg cm(-3). The molar absorptivity and Sandell's sensitivity are 2.20 x 10(4) dm3 mol(-1) cm(-1) and 4.85 x 10(-3) microg cm(-2), respectively. The correlation coefficient of the Pd(II)-PPT complex is 0.99, which indicates an excellent linearity between two variables. The detection limit of this method is 0.05 microg cm(-3). The instability constant of the Pd(II)-PPT complex calculated from Edmond and Birnbaum's method is 2.90 x 10(-5) and that of Asmus' method is 2.80 x 10(-5) at room temperature. The concurrent repetition of the method is checked and the relative standard deviation (RSD) (n = 5) was derived as 1.84 percent. The present method was applied to the determination of palladium(II) in synthetic mixtures and hydrogenation catalysts. The results were compared by employing an atomic-absorption spectrometer.

Comparative studies on the tolerance to photoinduced cutaneous inflammatory reactions by psoralen and rose bengal.

The photochemotherapeutic value of topical 8-methoxypsoralen (8-MOP) plus UVA irradiation has been well recognized. The phototoxicity associated with psoralen plus UVA (PUVA) therapy is hallmarked by an increase in vascular permeability (iVP), the accumulation of polymorphonuclear leukocytes (aPMN) and erythema formation in situ. Rose bengal (RB) plus UVA-VIS light (320-700 nm) produces a similar acute inflammatory response, but without immediate or delayed erythema and perceptible edema. This study describes some of the parameters involved in inflammatory reactions evoked by PUVA and the results are compared with RB-induced phototoxic reactions. The rates of iVP and aPMN with a 3 h pulse were quantified using 125I-albumin and 51Cr-labelled PMNs respectively. The erythemal response was graded visually. 8-MOP cream was applied topically, while RB was injected intradermally in rabbit skin before UVA-VIS (9.4 J cm-2) irradiation. The data show that there is no significant difference in the rates of iVP, aPMN and erythema formation between normal skin sites and mast cell-depleted skin sites when challenged with 8-MOP plus light. These results suggest that in situ mast cells do not play a significant role in 8-MOP-photoinduced acute cutaneous inflammatory reactions, in contrast with RB-photoinduced reactions. The iVP and aPMN responses are minimal or absent in sites subjected to repeated exposure to 8-MOP plus light for three or more consecutive days, suggesting the establishment of a desensitized/unresponsive state. Moreover, 8-MOP-photo-desensitized sites do not produce iVP and aPMN of the same magnitude as the normal (naive) skin sites when challenged with RB plus light. Similarly, RB-photo-desensitized sites do not produce iVP and aPMN of the same magnitude as the native skin sites when challenged with 8-MOP plus light. The desensitization and cross-desensitization of skin sites to 8-MOP- or RB-photoinduced reactions suggest that there is either direct attack on the target cell(s), thereby removing the ability to express adhesion molecules, such as endothelial leukocyte adhesion molecule 1 (ELAM-1) or intercellular adhesion molecule 1 (ICAM-1), involved in the accumulation of inflammatory cells, or downregulation of the secretion/release of putative agent(s), such as interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), responsible for the initiation and progression of cutaneous inflammations.

Modulation of 8-methoxypsoralen-photoinduced cutaneous inflammatory reactions by various chemotherapeutic agents in vivo.

Exposure of albino rabbits to UVA-VIS (320-700 nm) radiation after the topical application of 8-methoxypsoralen (8-MOP) cream is associated with acute cutaneous inflammatory reactions in situ. In the present studies the effects of various agents on 8-MOP plus light induced cutaneous inflammatory response viz. increase in vascular permeability (iVP), accumulation of polymorphonuclear leukocytes (aPMN) and erythema formation were investigated. The inflammatory reactions were induced by a single exposure of 8-MOP-sensitized sites to UVA-VIS (9.4J/cm2) light. Indomethacin, p-bromophenacyl bromide (BPAB), MK886 (trade name of Merck Sharpe & Dome), ibuprofen (IB), nordihydroguaiaretic acid (NDGA) or quinacrine were applied topically in cream base at various times prior to 8-MOP application. The iVP and aPMN were quantitated 24 h postirradiation using 125I-HSA and 51Cr-labeled PMN respectively, while erythema was graded visually. The rate of iVP, aPMN and erythema was inhibited almost completely by indomethacin (7.5-10%) when applied twice, 18 h and 3 h prior to 8-MOP. At lower concentrations of indomethacin (< or = 5%) iVP was inhibited whereas aPMN was augmented. The BPAB (0.25%) inhibited more than 90% of 8-MOP-photoinduced iVP and aPMN while there was partial reduction in erythema. The MK886 (0.1%) cream inhibited about 50% of iVP and aPMN but erythema persisted. The agents that are somewhat nonspecific such as IB, quinacrine and NDGA inhibited 8-MOP-photoinduced inflammation only marginally at the concentrations tested. The fact that iVP, aPMN and erythema can be dissociated suggests that there are independent variables in 8-MOP-photoinduced reactions, which involve multifactorial mechanisms probably controlled by different cell-signalling pathways and mediators.

Photoinduced cutaneous inflammatory response by psoralens.

Our studies describe the inflammatory response in rabbit skin induced by topical application of 8-methoxypsoralen (8-MOP) and UVA-visible irradiation (320-700 nm). Increase in vascular permeability (iVP) and accumulation of polymorphonuclear leucocytes (aPMN) at the test sites were quantitated using 125I-albumin and 51Cr-labelled PMNs respectively. Erythema was graded visually. 8-MOP cream was applied topically and irradiated. The erythemal response, aPMN and iVP at the test sites were quantitated at 6, 24, 48 and 72 h post-irradiation. The iVP and aPMN were maximal at 24 h; the erythemal response was the same at 24-48 h. The responses were dependent on 8-MOP concentration and irradiation dose. Topical application of 200 micrograms 8-MOP cream followed by irradiation for 2 h (9.4 J cm-2) produced 3-7 times iVP, 2-4 times aPMN and intense erythema at the test sites after 24 h. Neither aPMN nor iVP was detected before 6 h and erythemal response was not observable up to 16 h after irradiation. The aPMN and iVP gradually subsided in 72 h, although the erythemal response was still present. The repeated exposure of 8-MOP-treated sites for three consecutive days 24 h apart did not produce appreciable iVP or aPMN at 72 h or 24 h after the last exposure; however, erythema persisted. The 8-MOP-treated sites previously exposed for three consecutive days on reapplication of 8-MOP cream plus irradiation showed significantly less response compared with non-pretreated sites. Our results suggest that the erythemal response is not directly related to either iVP or aPMN.

Desensitization of rabbit skin by repeated exposure to UV-visible light of sites injected with Rose Bengal.

We have shown in a previous paper that irradiation of rabbit skin sites injected with Rose Bengal (RB) produces immediate increase in vascular permeability and accumulation of PMNs. Studies on the development of temporary tolerance and the biological parameters related to the development of such tolerant state by repeated exposure to light of RB-injected sites are reported here. The increase in VP and PMN migration induced by RB (10 nmol) are of an immediate nature, i.e., occur within the first 3 h of irradiation, and the reaction subsides gradually after 24 h. When such moderate insult is repeated, the skin becomes tolerant to subsequent exposure to light in the presence of RB. This tolerant state is temporary, i.e., the desensitized sites are fully recovered in 72 h. The loss of responsiveness of RB-injected sites previously exposed to light was not due to diffusion of the injected dye from the sites since reinjected sites also showed reduced response and the sites injected three days before but not irradiated showed normal response. The sites that were made tolerant to RB-induced phototoxic reactions, when injected with compound 48/80, an agent known to degranulate mast cells, did not show an increase in VP. This suggests that either the mast cells were depleted from the sites or the mast cells in the sites were rendered refractory by previous exposure to light. It was also found that the sites made tolerant to RB plus light were unresponsive to exogenously injected histamine. The sites tolerant to RB plus light when injected with zymosan-activated serum (ZAS) did not stimulate the migration of PMNs. This loss of chemotactic response to ZAS may have relevance to photodamage of vascular endothelium. These observations are discussed in relation to the development of the tolerant state by repeated exposures to subthreshold doses of light in solar urticaria.

Comparison of filtration leukapheresis and centrifugation leukapheresis in treatment of lymphosarcoma cell leukemia.

A patient with lymphosarcoma cell leukemia resistant to chemotherapy was treated with filtration leukapheresis and centrifugation leukapheresis. Filtration leukapheresis removed only 10(11) leukemia cells per 8.5 liters of blood processed, while centrifugation leukapheresis removed approximately six times as many leukemia cells from the same amount of blood. Lymph nodes and spleen diminished in size and the WBC count decreased after nine treatments. However, the patient remained markedly thrombocytopenic, and his bone marrow remained infiltrated with lymphosarcoma cells on repeat biopsy. This study shows that centrifugation leukapheresis is superior to filtration leukapheresis in removing significant numbers of circulating lymphosarcoma cells, though the clinical benefit of leukapheresis in this situation remains to be determined.