Multishot tomography for high-resolution in situ subtomogram averaging.

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) can resolve protein complexes at near atomic resolution, and when combined with focused ion beam (FIB) milling, macromolecules can be observed within their native context. Unlike single particle acquisition (SPA), cryo-ET can be slow, which may reduce overall project throughput. We here propose a fast, multi-position tomographic acquisition scheme based on beam-tilt corrected beam-shift imaging along the tilt axis, which yields sub-nanometer in situ STA averages.

The role of tocilizumab in the treatment of inflammatory diseases of the eye and orbit: A useful alternative.

PURPOSE: With uveitis being one of the leading causes of blindness worldwide, biological therapies have arisen as an option for the treatment of refractory cases based on good results shown in clinical practice. The goal of this study is to provide a systematic review of current knowledge of the role and possible uses of tocilizumab in the field of ophthalmology. MATERIALS AND METHODS: We performed a search for records reporting the use of tocilizumab for various diseases in MEDLINE (PubMed and OVID). We conducted an analysis of several individual studies and their reported individual patient data (82 eyes of 45 patients) published from 2011 to 2017. CONCLUSIONS: Tocilizumab may prove to be an effective choice for the treatment of a variety of ocular conditions such as refractory uveitis, inflammatory macular edema, vitreo-retinal tumors and thyroid orbitopathy, leading to control of the inflammation in these patients. Further studies need to be conducted to establish its safety and efficacy.

Experience of Self-Harm and Its Treatment in Looked-After Young People: An Interpretative Phenomenological Analysis.

We report the first Interpretative Phenomenological Analysis examination of self-harm and experience of clinical services in young people in the public care system. Qualitative interviews with 24 looked-after young people were completed. Prevalent themes were 1) Changes in care placement, 2) Feelings of anger, 3) Not wanting/feeling able to talk, 4) Developing coping techniques, 5) Clinical services: A relational mixed bag (subthemes: feeling (i) patronized, not listened to, (ii) nothing being done, (iii) comfortable/able to talk). Placement change and anger were highly salient to self-harm in this group and experiences of clinical services depended on individual relationships with clinicians. Implications include increasing compassion in therapeutic relationships, recognizing and managing emotional dysregulation, and increasing support during placement changes.

Kalyani cohort - the first platform in Eastern India for longitudinal studies on health and disease parameters in peri-urban setting.

The Kalyani cohort created in 2010 by the National Institute of Biomedical Genomics, West Bengal, India, is designed to serve as a platform for conducting prospective basic and translational studies on epidemiology and genomics of health and disease-related parameters, particularly of non-communicable diseases (NCDs). The overall goal is to assess behavioural, biological, genetic, social and environmental factors and obtain necessary evidence for effective health improvement. Collected baseline data comprise 15727 individuals, >14 years of age from seven municipal wards in the Kalyani and Gayeshpur regions. Data are being collected on demographics, current health status, medical history and health-related behaviours. Blood samples were also collected from a subset of individuals (n = 5132) and analysed for estimation of known markers of NCDs. DNA has been extracted from blood samples and stored for future use. Important baseline findings include a high prevalence of diabetes, dyslipidemias and hypothyroidism. Prevalence estimates for these disorders obtained from self-reported data are significantly lower, indicating that participants are unaware of their health problems. The identification of 'at risk' individuals will allow formation of sub-cohorts for further investigations of epidemiological and genetic risk factors for NCDs. Access to the resource, including data and blood samples, created by this study will be provided to other researchers.

Uncovering key patterns in self-harm in adolescents: Sequence analysis using the Card Sort Task for Self-harm (CaTS).

BACKGROUND: Self-harm is a significant clinical issue in adolescence. There is little research on the interplay of key factors in the months, weeks, days and hours leading to self-harm. We developed the Card Sort Task for Self-harm (CaTS) to investigate the pattern of thoughts, feelings, events and behaviours leading to self-harm. METHODS: Forty-five young people (aged 13-21 years) with recent repeated self-harm completed the CaTS to describe their first ever/most recent self-harm episode. Lag sequential analysis determined significant transitions in factors leading to self-harm (presented in state transition diagrams). RESULTS: A significant sequential structure to the card sequences produced was observed demonstrating similarities and important differences in antecedents to first and most recent self-harm. Life-events were distal in the self-harm pathway and more heterogeneous. Of significant clinical concern was that the wish to die and hopelessness emerged as important antecedents in the most recent episode. First ever self-harm was associated with feeling better afterward, but this disappeared for the most recent episode. LIMITATIONS: Larger sample sizes are necessary to examine longer chains of sequences and differences in genders, age and type of self-harm. The sample was self-selected with 53% having experience of living in care. CONCLUSIONS: The CaTs offers a systematic approach to understanding the dynamic interplay of factors that lead to self-harm in young people. It offers a method to target key points for intervention in the self-harm pathway. Crucially the factors most proximal to self-harm (negative emotions, impulsivity and access to means) are modifiable with existing clinical interventions.

Mahogunin regulates fusion between amphisomes/MVBs and lysosomes via ubiquitination of TSG101.

Aberrant metabolic forms of the prion protein (PrP), membrane-associated (Ctm)PrP and cytosolic (cyPrP) interact with the cytosolic ubiquitin E3 ligase, Mahogunin Ring Finger-1 (MGRN1) and affect lysosomes. MGRN1 also interacts with and ubiquitinates TSG101, an ESCRT-I protein, involved in endocytosis. We report that MGRN1 modulates macroautophagy. In cultured cells, functional depletion of MGRN1 or overexpression of (Ctm)PrP and cyPrP blocks autophagosome-lysosome fusion, alleviates the autophagic flux and its degradative competence. Concurrently, the degradation of cargo from the endo-lysosomal pathway is also affected. This is significant because catalytic inactivation of MGRN1 alleviates fusion of lysosomes with either autophagosomes (via amphisomes) or late endosomes (either direct or mediated through amphisomes), without drastically perturbing maturation of late endosomes, generation of amphisomes or lysosomal proteolytic activity. The compromised lysosomal fusion events are rescued by overexpression of TSG101 and/or its monoubiquitination in the presence of MGRN1. Thus, for the first time we elucidate that MGRN1 simultaneously modulates both autophagy and heterophagy via ubiquitin-mediated post-translational modification of TSG101.

Comparative anti-proliferative activity of some new 2-(arylazo)phenolate-palladium (II) complexes and cisplatin against some human cancer cell lines.

In this study, we report the synthesis of four 2-(arylazo)phenol-Pd(II) complexes and their anti-proliferative property against the human lung cancer (A549), cervical cancer (HeLa), and ovarian teratocarcinoma (PA-1) cell lines with cisplatin as the gold standard. One of the complexes, [Pd(L(2))2], induced robust apoptosis in all the chosen cells, as revealed by annexin-V-positive/propidium iodide dual staining, increased sub-G1 cell cycle population, and significant morphological changes in the treated cells. The Pd complex inflicted mitochondrial dysfunction leading to mitochondrial membrane potential loss, reactive oxygen species generation and release of cytosolic cytochrome c that activated caspase-9 and caspase-3 proteins which finally caused programmed cell death.

The effect of aerobic exercise in the maintenance treatment of depression.

We investigated the efficacy of aerobic exercise alongside antidepressant medication as an adjuvant maintenance treatment for depression. Fifty patients in remission were randomly assigned to either medication only or medication plus exercise. Assessment of psychopathology was made at 6-weekly intervals (for 24 weeks) using the Hamilton Rating Scale for Depression. The medication-plus-exercise group showed significantly more improvement at 12 and 24 weeks than the medication-only group. This study adds to a growing evidence base that suggests aerobic exercise is worthy of further development in the treatment of depressive disorders.

Common distal elements orchestrate CIITA isoform-specific expression in multiple cell types.

Major histocompatibility class II (MHC-II) expression is critical for immune responses and is controlled by the MHC-II transactivator CIITA. CIITA is primarily regulated at the transcriptional level and is expressed from three main promoters with myeloid, lymphoid and interferon (IFN)-γ-treated non-hematopoietic cells using promoters pI, pIII and pIV, respectively. Recent studies in non-hematopoietic cells suggest that a series of distal regulatory elements may be involved in regulating CIITA transcription. To identify distal elements in B cells, a DNase I hypersensitivity screen was performed, revealing a series of potential novel regulatory elements. These elements were analyzed computationally and biochemically. Several regions displayed active histone modifications and/or enhanced expression of a reporter gene. Four of the elements interacted with pIII in B cells. These same four regions were also found to interact with pI in splenic dendritic cells (spDC). Intriguingly, examination of the above interactions in pI-knockout-derived spDC showed a switch to the next available promoter, pIII. Extensive DNA methylation was found at the pI region in B cells, suggesting that this promoter is not accessible in B cells. Thus, CIITA expression is likely mediated in hematopoietic cells by common elements with promoter accessibility having a part in promoter choice.

Gamma-secretase inhibitors target tumor-initiating cells in a mouse model of ERBB2 breast cancer.

Human breast tumors comprise a minor sub-population of tumor-initiating cells (TICs), commonly termed cancer stem cells. TICs are thought to sustain tumor growth and to confer resistance to current anticancer therapies. Hence, targeting TIC may be essential to achieving durable cancer cures. To identify molecular targets in breast TIC, we employed a transgenic mouse model of ERBB2 breast cancer; tumors arising in this model comprise a very high frequency of TIC, which is maintained in tumor cell populations propagated in vitro as non-adherent tumorspheres. The Notch pathway is dysregulated in human breast tumors and overexpression of constitutively active Notch proteins induces mammary tumors in mice. The Notch pathway has also been implicated in stem cell processes including those of mammary epithelial stem cells. Hence, we investigated the potential that the Notch pathway is required for TIC activity. We found that an antagonist of Notch signaling, a gamma (γ)-secretase inhibitor termed MRK-003, inhibited the survival of tumorsphere-derived cells in vitro and eliminated TIC as assessed by cell transplantation into syngeneic mice. Whereas MRK-003 also inhibited the self-renewal and/or proliferation of mammosphere-resident cells, this effect of the inhibitor was reversible thus suggesting that it did not compromise the survival of these cells. MRK-003 administration to tumor-bearing mice eliminated tumor-resident TIC and resulted in rapid and durable tumor regression. MRK-003 inhibited the proliferation of tumor cells, and induced their apoptosis and differentiation. These findings suggest that MRK-003 targets breast TIC and illustrate that eradicating these cells in breast tumors ensures long-term, recurrence-free survival.

Species-specific AFLP markers for identification of Zingiber officinale, Z. montanum and Z. zerumbet (Zingiberaceae).

The Zingiber genus, which includes the herbs known as gingers, commonly used in cooking, is well known for its medicinal properties, as described in the Indian pharmacopoeia. Different members of this genus, although somewhat similar in morphology, differ widely in their pharmacological and therapeutic properties. The most important species of this genus, with maximal therapeutic properties, is Zingiber officinale (garden ginger), which is often adulterated with other less-potent Zingiber sp. There is an existing demand in the herbal drug industry for an authentication system for the Zingiber sp in order to facilitate their commercial use as genuine phytoceuticals. To this end, we used amplified fragment length polymorphism (AFLP) to produce DNA fingerprints for three Zingiber species. Sixteen collections (six of Z. officinale, five of Z. montanum, and five of Z. zerumbet) were used in the study. Seven selective primer pairs were found to be useful for all the accessions. A total of 837 fragments were produced by these primer pairs. Species-specific markers were identified for all three Zingiber species (91 for Z. officinale, 82 for Z. montanum, and 55 for Z. zerumbet). The dendogram analysis generated from AFLP patterns showed that Z. montanum and Z. zerumbet are phylogenetically closer to each other than to Z. officinale. The AFLP fingerprints of the Zingiber species could be used to authenticate Zingiber sp-derived drugs and to resolve adulteration-related problems faced by the commercial users of these herbs.

DNA methylation dysregulates and silences the HLA-DQ locus by altering chromatin architecture.

The major histocompatibility complex class II (MHC-II) locus encodes a cluster of highly polymorphic genes HLA-DR, -DQ and -DP that are co-expressed in mature B lymphocytes. Two cell lines were established over 30 years ago from a patient diagnosed with acute lymphocytic leukemia. Laz221 represented the leukemic cells of the patient; whereas Laz388 represented the normal B cells of the patient. Although Laz388 expressed both HLA-DR and HLA-DQ surface and gene products, Laz221 expressed only HLA-DR genes. The discordant expression of HLA-DR and HLA-DQ genes was due to epigenetic silencing of the HLA-DQ region CCCTC transcription factor (CTCF)-binding insulators that separate the MHC-II sub-regions by DNA methylation. These epigenetic modifications resulted in the loss of binding of the insulator protein CTCF to the HLA-DQ flanking insulator regions and the MHC-II-specific transcription factors to the HLA-DQ promoter regions. These events led to the inability of the HLA-DQ promoter regions to interact with flanking insulators that control HLA-DQ expression. Inhibition of DNA methylation by treatment with 5'-deoxyazacytidine reversed each of these changes and restored expression of the HLA-DQ locus. These results highlight the consequence of disrupting an insulator within the MHC-II region and may be a normal developmental mechanism or one used by tumor cells to escape immune surveillance.

Transcription regulation of caspase-1 by R393 of HIPPI and its molecular partner HIP-1.

Earlier we have shown that exogenous expression of HIPPI, a molecular partner of Huntingtin interacting protein HIP-1, induces apoptosis and increases expression of caspases-1, -8 and -10 in HeLa and Neuro2A cells. The C-terminal pseudo death effector domain of HIPPI (pDED-HIPPI) specifically interacts with the putative promoter sequences of these genes. In the present manuscript, we predict from structural modeling of pDED-HIPPI that R393 of HIPPI is important for such interaction. R393E mutation in pDED-HIPPI decreases the interaction with the putative promoter of caspase-1 in cells. Expression of caspase-1 is decreased in cells expressing mutant pDED-HIPPI in comparison to that observed in cells expressing wild type pDED-HIPPI. Using HIP-1 knocked down cells as well as over expressing HIP-1 with mutation at its nuclear localization signal and other deletion mutations, we demonstrate that translocation of HIPPI to the nucleus is mediated by HIP-1 for the increased expression of caspase-1. HIPPI-HIP-1 heterodimer is detected in cytoplasm as well as in the nucleus and is associated with transcription complex in cells. Taking together, we are able to show the importance of R393 of HIPPI and the role of HIPPI-HIP-1 heterodimer in the transcription regulation of caspase-1.

WITHDRAWN: Association of uncoupling protein 2 and 3 gene polymorphisms with type 2 diabetes in Asian Indians.

Ahead of Print article withdrawn by publisher.

Down-regulation of the Notch pathway mediated by a gamma-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia.

BACKGROUND AND PURPOSE: gamma-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy. EXPERIMENTAL APPROACH: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Abeta(40) and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. KEY RESULTS: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis. CONCLUSIONS AND IMPLICATIONS: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

A frequency stabilization technique for diode lasers based on frequency-shifted beams from an acousto-optic modulator.

We present a simple method for diode laser frequency stabilization that makes use of a Doppler-broadened vapor cell absorption signals of two frequency-shifted laser beams. Using second-order-diffracted, double-passed beams from an acousto-optic modulator, we achieve a frequency separation roughly equal to the Doppler half width. The differential transmission signals of the two beams provide an error signal with a very large linear feature, allowing frequency stabilization over a range of greater than 1 GHz by means of standard proportional-integral-derivative servo feedback to the piezoelectric control of the grating in our external cavity diode laser. We have applied this technique to two different diode laser systems, one used to lock to the 410 nm E1 transition in indium and another for locking to the M1/E2 transition in thallium at 1283 nm. In both cases the technique reduces frequency fluctuation to roughly 1 MHz over time scales from 10(-3) to 10(2) s.

Laryngocoele: our experince.

Laryngocoele is an extremely rare condition. Three cases of Laryngocoeles are presented. Two cases had infected laryngocoeles. The diagnosis was done clinically and confirmed radiologically. No predisposing factor was found except in one case who was a singer by profession and initially presented with stridor which was an unusual presentation. Surgical excision were done in two cases (case 1 and 3)and both the case were symptom free.

Portability of tag SNPs across isolated population groups: an example from India.

Isolated population groups are useful in conducting association studies of complex diseases to avoid various pitfalls, including those arising from population stratification. Since DNA resequencing is expensive, it is recommended that genotyping be carried out at tagSNP (tSNP) loci. For this, tSNPs identified in one isolated population need to be used in another. Unless tSNPs are highly portable across populations this strategy may result in loss of information in association studies. We examined the issue of tSNP portability by sampling individuals from 10 isolated ethnic groups from India. We generated DNA resequencing data pertaining to 3 genomic regions and identified tSNPs in each population. We defined an index of tSNP portability and showed that portability is low across isolated Indian ethnic groups. The extent of portability did not significantly correlate with genetic similarity among the populations studied here. We also analyzed our data with sequence data from individuals of African and European descent. Our results indicated that it may be necessary to carry out resequencing in a small number of individuals to discover SNPs and identify tSNPs in the specific isolated population in which a disease association study is to be conducted.

Interactions of HIPPI, a molecular partner of Huntingtin interacting protein HIP1, with the specific motif present at the putative promoter sequence of the caspase-1, caspase-8 and caspase-10 genes.

To investigate the mechanism of increased expression of caspase-1 caused by exogenous Hippi, observed earlier in HeLa and Neuro2A cells, in this work we identified a specific motif AAAGACATG (- 101 to - 93) at the caspase-1 gene upstream sequence where HIPPI could bind. Various mutations in this specific sequence compromised the interaction, showing the specificity of the interactions. In the luciferase reporter assay, when the reporter gene was driven by caspase-1 gene upstream sequences (- 151 to - 92) with the mutation G to T at position - 98, luciferase activity was decreased significantly in green fluorescent protein-Hippi-expressing HeLa cells in comparison to that obtained with the wild-type caspase-1 gene 60 bp upstream sequence, indicating the biological significance of such binding. It was observed that the C-terminal 'pseudo' death effector domain of HIPPI interacted with the 60 bp (- 151 to - 92) upstream sequence of the caspase-1 gene containing the motif. We further observed that expression of caspase-8 and caspase-10 was increased in green fluorescent protein-Hippi-expressing HeLa cells. In addition, HIPPI interacted in vitro with putative promoter sequences of these genes, containing a similar motif. In summary, we identified a novel function of HIPPI; it binds to specific upstream sequences of the caspase-1, caspase-8 and caspase-10 genes and alters the expression of the genes. This result showed the motif-specific interaction of HIPPI with DNA, and indicates that it could act as transcription regulator.

P19 embryonal carcinoma cells as in vitro model for studying purinergic receptor expression and modulation of N-methyl-D-aspartate-glutamate and acetylcholine receptors during neuronal differentiation.

The in vitro differentiation of P19 murine embryonal carcinoma cells to neurons resembles developmental stages which are encountered during neuronal development. Three days following induction to neuronal differentiation by retinoic acid, most cells of the P19 population lost expression of the stage specific embryonic antigen (SSEA-1) and expressed the neural progenitor cell specific antigen nestin. Beginning from day 4 of differentiation nestin expression was down-regulated, and expression of neuron-specific enolase as marker of differentiated neurons increased. The molecular mechanisms underlying neuronal differentiation are poorly understood. We have characterized the participation of purinergic ionotropic (P2X) and metabotropic (P2Y) receptors at mRNA transcription and protein levels as well as ATP-induced Ca2+ transients during neuronal differentiation of P19 cells. Gene and protein expression of P2X2, P2X6, P2Y2, and P2Y6 receptors increased during the course of differentiation, whereas P2X3, P2X4, P2Y1 and P2Y4 receptor expression was high in embryonic P19 cells and then decreased following induction of P19 cells to differentiation. P2X1 receptor protein expression was only detected on days 2 and 4 of differentiation. Although P2X5 and P2X7 mRNA transcription was present, no protein expression for this receptor subunit could be detected throughout the differentiation process. In undifferentiated cells, mainly ionotropic P2X receptors contributed to the ATP-induced Ca2+-response. In neuronal-differentiated P19 cells, the ATP-induced Ca2+-response was increased and the metabotropic component predominated. Purinergic receptor function is implicated to participate in neuronal maturation, as cholinergic and glutamate-N-methyl-D-aspartate (NMDA) induced calcium responses were affected when cells were differentiated in the presence of purinergic receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin or reactive blue-2. Our data suggest that inhibition of P2Y1 and possibly P2X2 receptors led to a loss of NMDA receptor activity whereas blockade of possibly P2X2 and P2Y2 purinergic receptors during neuronal differentiation of P19 mouse led to inhibition of cholinergic receptor responses.