Reverse pharmacology based clinical protocols for noninvasive integrative management of low grade cervical precancer lesions: Rationale and outcomes.

Prevention is the most efficient and cost-effective method to combat cervical cancer for which High Risk Human Papilloma Virus (HR-HPV) infection is identified as the major causative factor. HPV vaccination is for primary prevention whereas surgical ablation of precancer is for secondary prevention after HPV infection has occurred. Screening of women for early detection of Squamous Intraepithelial Lesions (SILs) with Papanicolou smear (Pap smear) is a desirable pre-requisite. Surgical ablation which invites invasive procedures is not accessible nor affordable to the larger section of the population. We propose here a non-invasive integrative management approach for the early phase of cervical pre-cancer. In tune with the reverse pharmacology approach, 'experience-exploration- experimentation', we have conducted five clinical studies related to Turmeric extracts for chemo-preventive activity and non-surgical feasibility. We were able to achieve arrest or regression in Low-grade SILs in all 41 women participating in these studies. The unique features of this integrative management approach were i) Avoidance of surgery-associated trauma, cost and complications ii) Standard of care for associated genital infections iii) Feasibility when surgery was not accessible iv) Scope for repeating the noninvasive treatment.

Comprehensive assessment of holding urine as a behavioral risk factor for UTI in women and reasons for delayed voiding.

BACKGROUND: Women of reproductive age group have greater predilection to urinary tract infections (UTI). Various risk factors increase the prevalence in women. Emergence of multidrug resistant uropathogens make clinical management of UTI challenging. Here we assess holding of urine as risk factor of UTI in women and reasons for delayed voiding. We also investigate the relationship between frequency of UTIs and overall behavioural features, menstrual hygiene and attitude of women towards their own health issues. METHODS: A questionnaire based cross-sectional study was performed with 816 hostel residents with written consent. Self-reported data was statistically analysed using SPSS software. Urinalysis and urine culture were done for 50 women by random sampling to obtain the information on leading causative agents of UTI in the study population and their antimicrobial resistance profile. RESULTS: The prevalence of UTI among the participants without risk factors was found to be 27.5 (95% CI: 24.4-30.7). Attitude of women towards their own personal health issues and use of public toilets showed a correlation with prevalence of infection. Delay in urination on habitual basis was found to be associated with UTI. Uropathogens isolated by random sampling were resistant to multiple drugs that are generally used to treat UTI. CONCLUSIONS: Holding urine for long time had proven to be an important risk factor and amongst different reasons of holding urine, holding due to poor sanitary condition of public toilets was the most common. Higher frequency of self-reported UTIs is related to holding of urine, behavioural features and attitude of women.

The digitisation of food manufacturing to reduce waste - Case study of a ready meal factory.

Generation of food waste (FW) continues to be a global challenge and high on the political agenda. One of the main reasons for its generation is the absence of detailed data on the amount, timing and reasons for created waste. This paper discusses the design, the application and investigates the Internet of Things (IoT) based FW monitoring system to capture waste data during manufacturing in real-time and make it available to all the stakeholders in a food supply chain (FSC). A case study of ready-meal factory comprises of design and architecture for tracking FW including both hardware and software, its implementation in the factory and the positive data-driven results achieved. The case study demonstrates the benefits of digital FW tracking system including the FW reduction of 60.7%, better real-time visibility of the FW hotspots, reasons for waste generations, reliable data, operational improvements and employee behavioural transformation. Although the system replaced the paper-based manual system of tracking FW in the factory, it still needed human input to confirm the waste and was prone to human errors. Overall, the implementation of an IoT-based FW tracking system resulted in a reduction of FW and created a positive environmental and financial impact.

Differences in the Early Development of Human and Mouse Embryonic Stem Cells.

We performed a systematic analysis of gene expression features in early (10-21 days) development of human vs mouse embryonic cells (hESCs vs mESCs). Many development features were found to be conserved, and a majority of differentially regulated genes have similar expression change in both organisms. The similarity is especially evident, when gene expression profiles are clustered together and properties of clustered groups of genes are compared. First 10 days of mESC development match the features of hESC development within 21 days, in accordance with the differences in population doubling time in human and mouse ESCs. At the same time, several important differences are seen. There is a clear difference in initial expression change of transcription factors and stimulus responsive genes, which may be caused by the difference in experimental procedures. However, we also found that some biological processes develop differently; this can clearly be shown, for example, for neuron and sensory organ development. Some groups of genes show peaks of the expression levels during the development and these peaks cannot be claimed to happen at the same time points in the two organisms, as well as for the same groups of (orthologous) genes. We also detected a larger number of upregulated genes during development of mESCs as compared to hESCs. The differences were quantified by comparing promoters of related genes. Most of gene groups behave similarly and have similar transcription factor (TF) binding sites on their promoters. A few groups of genes have similar promoters, but are expressed differently in two species. Interestingly, there are groups of genes expressed similarly, although they have different promoters, which can be shown by comparing their TF binding sites. Namely, a large group of similarly expressed cell cycle-related genes is found to have discrepant TF binding properties in mouse vs human.

Neuronal developmental gene and miRNA signatures induced by histone deacetylase inhibitors in human embryonic stem cells.

Human embryonic stem cells (hESCs) may be applied to develop human-relevant sensitive in vitro test systems for monitoring developmental toxicants. The aim of this study was to identify potential developmental toxicity mechanisms of the histone deacetylase inhibitors (HDAC) valproic acid (VPA), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) relevant to the in vivo condition using a hESC model in combination with specific differentiation protocols and genome-wide gene expression and microRNA profiling. Analysis of the gene expression data showed that VPA repressed neural tube and dorsal forebrain (OTX2, ISL1, EMX2 and SOX10)-related transcripts. In addition, VPA upregulates axonogenesis and ventral forebrain-associated genes, such as SLIT1, SEMA3A, DLX2/4 and GAD2. HDACi-induced expression of miR-378 and knockdown of miR-378 increases the expression of OTX2 and EMX2, which supports our hypothesis that HDACi targets forebrain markers through miR-378. In conclusion, multilineage differentiation in vitro test system is very sensitive for monitoring molecular activities relevant to in vivo neuronal developmental toxicity. Moreover, miR-378 seems to repress the expression of the OTX2 and EMX2 and therefore could be a regulator of the development of neural tube and dorsal forebrain neurons.

Comparative immunomodulation potential of Tinospora cordifolia (Willd.) Miers ex Hook. F., Tinospora sinensis (Lour.) Merrill and Tinospora cordifolia growing on Azadirachta indica A. Juss.

Guduchi has been widely used in the traditional medicine as an immunomodulator. Description of guduchi in Ayurvedic literature resemble with T. sinensis rather than with commonly available T. cordifolia and hence this may be used as substitutes for T. sinensis. T. cordifolia growing on Azadirachta indica commonly called Neem-guduchi has more immunomodulatory potential. Thus, immunomodulatory activity of three Tinospora spp. was assessed by checking humoral and cell mediated immune responses to the antigenic challenges with sheep RBCs and by neutrophil adhesion tests on albino Wistar rats using Guduchi-Satwa, a well known dosage form. Results revealed that Neem-guduchi possesses higher immunomodulatory potential at the dose of 300 mg/kg, po and validated the traditional claim. Hence, Neem-Guduchi can be employed in immunomodulatory formulation prepared using guduchi.

Fam40b is required for lineage commitment of murine embryonic stem cells.

FAM40B (STRIP2) is a member of the striatin-interacting phosphatase and kinase (STRIPAK) complex that is involved in the regulation of various processes such as cell proliferation and differentiation. Its role for differentiation processes in embryonic stem cells (ESCs) is till now completely unknown. Short hairpin RNA (shRNA)-mediated silencing of Fam40b expression in ESCs and differentiating embryoid bodies (EBs) led to perturbed differentiation to embryonic germ layers and their derivatives including a complete abrogation of cardiomyogenesis. Pluripotency factors such as Nanog, Oct4 and Sox2 as well as epigenetic factors such as histone acetyltransferase type B (HAT1) and DNA (cytosine-5)-methyltransferase 3-ß (Dnmt3b) were highly upregulated in Fam40b knockdown EBs as compared with control and scrambled EBs. To examine the relevance of Fam40b for development in vivo, Fam40b was knocked down in developing zebrafish. Morpholino-mediated knockdown of Fam40b led to severe abnormalities of the cardiovascular system, including an impaired expression of ventricular myosin heavy chain (vmhc) and of cardiac myosin light chain 2 (cmlc2) in the heart. We identified the gene product of Fam40b in ESCs as a perinuclear and nucleolar protein with a molecular weight of 96 kDa. We conclude that the expression of Fam40b is essential for the lineage commitment of murine embryonic stem cells (mESCs) into differentiated somatic cells via mechanisms involving pluripotency and epigenetic networks.

Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP(+).

Assessment of the network of toxicity pathways by Omics technologies and bioinformatic data processing paves the road toward a new toxicology for the twenty-first century. Especially, the upstream network of responses, taking place in toxicant-treated cells before a point of no return is reached, is still little explored. We studied the effects of the model neurotoxicant 1-methyl-4-phenylpyridinium (MPP(+)) by a combined metabolomics (mass spectrometry) and transcriptomics (microarrays and deep sequencing) approach to provide unbiased data on earliest cellular adaptations to stress. Neural precursor cells (LUHMES) were differentiated to homogeneous cultures of fully postmitotic human dopaminergic neurons, and then exposed to the mitochondrial respiratory chain inhibitor MPP(+) (5 µM). At 18-24 h after treatment, intracellular ATP and mitochondrial integrity were still close to control levels, but pronounced transcriptome and metabolome changes were seen. Data on altered glucose flux, depletion of phosphocreatine and oxidative stress (e.g., methionine sulfoxide formation) confirmed the validity of the approach. New findings were related to nuclear paraspeckle depletion, as well as an early activation of branches of the transsulfuration pathway to increase glutathione. Bioinformatic analysis of our data identified the transcription factor ATF-4 as an upstream regulator of early responses. Findings on this signaling pathway and on adaptive increases of glutathione production were confirmed biochemically. Metabolic and transcriptional profiling contributed complementary information on multiple primary and secondary changes that contribute to the cellular response to MPP(+). Thus, combined 'Omics' analysis is a new unbiased approach to unravel earliest metabolic changes, whose balance decides on the final cell fate.

High density monolayer of diisocyanide on gold surface as a platform of supported Rh-catalyst for selective 1,4-hydrogenation of α,ß-unsaturated carbonyl compounds.

A high density monolayer of diisocyanide on gold surface was utilized as a platform of supported Rh catalyst for selective 1,4-hydrogenation of α,ß-unsaturated carbonyl compounds. The catalyst exhibited high turnover numbers in a range of 50 000 to 150 000 per Rh atom and showed steady catalyst performance over six recycle usages.

A tanscriptomics study to elucidate the toxicological mechanism of methylmercury chloride in a human stem cell based in vitro test.

Traditional approaches in evaluating the hazard of drug candidates on the developing offspring are often time-consuming and cost-intensive. Moreover, variations in the toxicological response of different animal species to the tested substance cause severe problems when extrapolating safety dosages for humans. Therefore, more predictive and relevant toxicological systems based on human cell models are required. In the presented study the environmental toxicant methylmercury chloride (MeHgCl), known to cause structural developmental abnormalities in the brain, was used as reference compound to develop a concept contributing to a mechanistic understanding of the toxicity of an investigated substance. Despite the fact, that there are significant data available from animal studies and from poisonings in Japan and Iraq, uncertainties on the mechanism of MeHgCl during human development are still remaining and qualify the substance for further analysis. Transcriptomics analysis in combination with a human cell based in vitro model has been used in order to elucidate the toxicity of MeHgCl at molecular level. Differentiating neural precursor cells that have been exposed continuously to non- and low-cytotoxic concentrations of MeHgCl were investigated. Quantitative change in the mRNA expression profiles of selected genes demonstrated the sensitivity of the cell model and its qualification for a transcriptomics study screening changes in the expression profile of the complete human genome of MeHgCl-treated human neural cells. Potential biomarkers were identified and these candidate marker genes as well as their involvement in a possible toxic mechanism of MeHgCl during the human neurulation process are hereby introduced. The study confirmed the hypothesis that a cellular model based on a human stem cell line can be applied for elucidating unknown mode of actions of developmental toxicants.

Characterisation of a neural teratogenicity assay based on human ESCs differentiation following exposure to valproic acid.

The development of in vitro testing strategies for chemical and drug screening is a priority need in order to protect human health, to increase safety, to reduce the number of animals required for conventional testing methods and finally to meet the deadlines of current legislations. The aim of this work was to design an alternative testing method based on human embryonic stem cells for the detection of prenatal neural toxicity. For this purpose we have created a model based on the generation of neural rosettes, reproducing in vitro the gastrulation events recapitulating the formation of the neural tube in vivo. To validate the model we have exposed this complex cell system to increasing concentrations of valproic acid, a known teratogenic agent, to analyse the morphological and molecular changes induced by the toxicant. Specific assays were applied to discriminate between cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with a microarray Affimetrix platform and validated by quantitative real time RT-PCR for the expression of genes involved in early neural development, neural tube formation and neural cells migration, key biological processes in which the effect of valproic acid is most relevant. The results demonstrated that neural rosette cells respond to valproic acid exposure with molecular and morphological changes similar to those observed in vivo, indicating that this method represents a promising alternative test for the detection of human prenatal neural toxicity.

A liver-derived rat epithelial cell line from biliary origin acquires hepatic functions upon sequential exposure to hepatogenic growth factors and cytokines.

Withdrawal of promising drug candidates is often due to the detection of liver toxicity. In particular the parenchymal liver cells or hepatocytes are targeted since they are the major sites of drug transport and of metabolite formation and thus also the place where not only detoxification, but also activation of new chemical (NCE) and biological (NBE) entities may occur. Therefore, primary hepatocyte- based cultures are currently the preferred in vitro model to screen for liver toxicity. However, within a few days, they undergo dedifferentiation with loss of liver-specific functionality, including xenobiotic biotransformation capacity, making them only suitable for short-term applications. A plausible alternative to primary hepatocyte cultures that can be maintained for longer periods of time could be the use of liver-derived epithelial cell lines and their optimized derivatives. Therefore, in the present study, we evaluated the stability and the hepatic differentiation potential of a neonatal liver-derived rat epithelial cell line from biliary origin (rLEC). Undifferentiated rLEC stably express the hepatic progenitor markers CEBPA, FOXA2, GJA1, ONECUT1, KRT18 and KRT19 for at least 15 consecutive passages after cryopreservation. Upon sequential exposure to hepatogenic growth factors and cytokines, rLEC generate functional hepatic progeny, expressing mature hepatic markers including Alb, Ahr, Car, C/ebpα, Cx32, Foxa2, Hnf1α, Hnf1ß and Onecut1. Furthermore, an active polarization is observed for the hepatic drug transporters Oatp4 and Ntcp. rLEC-derived hepatic cells also acquire the ability to store glycogen, express genes encoding for key hepatic enzymes as shown by Affymetrix microarray data, and display stable CYP1A1/2- and CYP2B1/2-dependent activities for several weeks at levels comparable to those observed in cultured primary rat hepatocytes. The acquisition of such a stable and active biotransformation capacity is key for the applicability of liver-based in vitro models for long-term toxicity testing.

Dissofilm: a novel approach for delivery of phenobarbital; design and characterization.

An attempt to develop and evaluate mouth-dissolving film of phenobarbital for quick effect in treatment of epilepsy occurring in pediatric population has been made in the present study. Suitable film formers and plasticizers are selected based on optimization studies. Effect of superdisintegrants in formulation of mouth dissolving films at different concentrations has been investigated. Films were prepared by solvent casting method. The prepared films were evaluated for physicochemical parameters, in vitro disintegration and dissolution time, in vitro release rate study, stability study, and in vivo animal safety study. The best formulation was found to be F3 showing 96.57% drug release in 14 min, following first-order kinetics. X-Ray diffraction studies show change in crystalline nature of drug in formulation. In vivo studies in hamster reports effective and safe use of formulation in animals.

Cytosine arabinoside induces ectoderm and inhibits mesoderm expression in human embryonic stem cells during multilineage differentiation.

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here, we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs. EXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points, day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C. KEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2, TUBB III, PAX6, TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2, PITX2, GATA5, MYL4, TNNT2, COL1A1 and COL1A2. In addition, no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated. CONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments.

Chemopreventive potential and safety profile of a Curcuma longa extract in women with cervical low-grade squamous intraepithelial neoplasia.

OBJECTIVE: To determine whether Curcuma longa Linn extract, NBFR-03, can arrest low-grade squamous intraepithelial neoplasia (LSIL) a 12 week intervention study was performed. METHODS: Of a total of 1473 women undergoing Pap smear screening, 88 cases had LSIL. Only those with persistent LSIL subsequent to antimicrobial therapy, and willing to follow the protocol (N=21), were included for clinical examination, Pap smears, colposcopy, clinical biochemistry, urinalysis and assessment of serum IL-6, being conducted before and after treatment. Standardised NBFR-03 (0.2gm) capsules were administered, twice daily, for 12 weeks. RESULTS: None progressed to higher grade lesion as assessed by Pap smears and colposcopy. Sixteen cases regressed to atypia, ASCUS or inflammatory pattern; 3 persisted as LSIL, 1 discontinued early because of itching, and 1 did not start. None developed any significant abnormality clinically or biochemically. Micrometry showed a significant reduction in nuclear diameter and nucleocytoplasmic ratio after treatment (p<0.02, and <0.05 respectively). Serum IL-6 levels showed a significant decline (mean 248∓ 156 (SEM) vs 27.7∓ 10.5 (SEM) pg/ ml; p<0.05). CONCLUSION: Use of NBFR-03 for 12 weeks was associated with an arrest or regression of LSIL in Pap smears and colposcopy, with reduction in the circulating IL-6 levels.

Serum IL-6 and micrometry of pap smears in women with cervical low-grade intraepithelial lesions.

OBJECTIVE: To assess serum IL-6 in women with or without low-grade squamous intraepithelial lesions (LSILs) in Pap smears and correlate with the nucleo/cytoplasmic (N/C) ratio. METHODS: Manual micrometry was carried out on Pap smears for N/C ratios: Group A, negative findings (N=15); Group B, inflammation without abnormality (N=14); Group C, LSIL with inflammation (N=13). Serum IL-6 was measured in Groups B and C after treatment of nonviral genital infections. Women with pelvic inflammatory or systemic diseases were excluded. RESULTS: The N/C ratio was significantly higher in Group C vs Group B, both before and after treatment of nonviral infections and also vs group A (p<0.001, Students t test). After treatment for non-viral infections serum IL-6 levels were >50 pg/ml in 5/13 cases of Group C and significantly higher than levels in Group B (p<0.05), correlating positively with the N/C ratio in the 13 cases of LSIL (Pearson's coefficient r=0.659, p<0.05). CONCLUSIONS: High peripheral circulating level of IL-6, despite prior treatment of nonviral infections, was observed in more than one third of women with persistent LSIL in Pap smears, and may serve as an additional biomarker for early cervical neoplasia. Long term follow up is indicated.

Invasive Papillary Breast Carcinoma.

We present the case of a 55-year-old postmenopausal female who presented with complaints of a gradually increasing painless subareolar mass in the left breast of 4 months' duration. Left-sided modified radical mastectomy was performed and the specimen was histopathologically diagnosed as invasive papillary carcinoma. Immunohistochemistry confirmed this diagnosis. All 8 excised axillary lymph nodes were negative for malignant cells. Postoperative chemotherapy was given and for the past 6 months, the patient has maintained a regular follow-up on an outpatient basis. She does not have any evidence of either local or distant recurrence of tumour metastases.

Metaplastic carcinoma with predominant chondrosarcoma of the right breast.

A 45-year-old female presented with complaint of a lump in the right breast for the last 6 months. Excisional biopsy had been performed outside our institution, which was suggestive of a pure mammary chondrosarcoma. Modified radical mastectomy of the right breast was performed and histopathology was suggestive of a pure mammary chondrosarcoma of the right breast. One axillary lymph node was positive for metastasis, the rest of the lymph nodes were free of metastasis. Immunohistochemistry was suggestive of a metaplastic carcinoma with a predominant chondrosarcoma in the right breast. Postoperative radiotherapy was given to the patient with a further plan of chemotherapy.