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.

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.

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.

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.

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.

WITHDRAWN: Association of uncoupling protein 2 and 3 gene polymorphisms with type 2 diabetes in Asian Indians.

Ahead of Print article withdrawn by publisher.

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.

Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators.

PKC-delta is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-delta slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-delta dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-delta using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-delta caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, P<0.05), while mRNA levels remained unchanged. The PKC agonist, phorbol 12-myristate 13-acetate (TPA, 100 nM, 4 h), induced two-fold higher protein and mRNA levels of p21(Waf1), a cyclin-dependent kinase (cdk) inhibitor in PKC-delta transfectants compared with empty vector (EV) transfected cells, whereas the PKC-delta specific inhibitor rottlerin (3 microM) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21(Waf1) expression. Concomitantly, compared to EV control cells, PKC-delta upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-delta increased binding of cdk inhibitor p27(Kip1) to cdk4. These alterations in cyclin-cdks and their inhibitors are predicted to decrease G1 cyclin kinase activity. As an independent confirmation of the direct role PKC-delta plays in cell growth and cell cycle regulation, we knocked down PKC-delta using specific siRNA oligonucleotides. PKC-delta specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-delta protein by more than 80% in Caco-2 cells. Moreover, PKC-delta knockdown enhanced cell proliferation ( approximately 1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression ( approximately 1.7-fold, P<0.05). This was a specific effect, as nontargeted PKC-zeta was not changed by PKC-delta siRNA oligonucleotides. Consistent with accelerated apoptosis in PKC-delta transfectants, compared to EV cells, PKC-delta upregulation increased proapoptotic regulator Bax two-fold at mRNA and protein levels, while antiapoptotic Bcl-2 protein was decreased by 50% at a post-transcriptional level. PKC-delta specific siRNA oligonucleotides inhibited Bax protein expression by more than 50%, indicating that PKC-delta regulates apoptosis through Bax. Taken together, these results elucidate two critical mechanisms regulated by PKC-delta that inhibit cell cycle progression and enhance apoptosis in colon cancer cells. We postulate these antiproliferative pathways mediate an important tumor suppressor function for PKC-delta in colonic carcinogenesis.

Regulation of the hTERT telomerase catalytic subunit by the c-Abl tyrosine kinase.

BACKGROUND: Telomeres consist of repetitive (TTAGGG) DNA sequences that are maintained by the multisubunit telomerase ribonucleoprotein. Telomerase consists of an RNA, which serves as template for the sequence tracts, and a catalytic subunit that functions in reverse transcription of the RNA template. Cloning and characterization of the human catalytic subunit of telomerase (hTERT) has supported a role in cell transformation. How telomerase activity is regulated, however, is largely unknown. RESULTS: We show here that hTERT associates directly with the c-Abl protein tyrosine kinase. We also found that c-Abl phosphorylates hTERT and inhibits hTERT activity. Moreover, our findings demonstrate that exposure of cells to ionizing radiation induces tyrosine phosphorylation of hTERT by a c-Abl-dependent mechanism. The functional significance of the c-Abl-hTERT interaction is supported by the demonstration that cells deficient in c-Abl show telomere lengthening. CONCLUSIONS: The ubiquitously expressed c-Abl tyrosine kinase is activated by DNA double-strand breaks. Our finding of telomere lengthening in c-Abl-deficient cells and the functional interactions between c-Abl and hTERT support a role for c-Abl in the regulation of telomerase function.

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.

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.

Neurotransmitter receptor expression and activity during neuronal differentiation of embryonal carcinoma and stem cells: from basic research towards clinical applications.

Embryonal carcinoma and embryonic stem cells have served as models to understand basic aspects of neuronal differentiation and are promising candidates for regenerative medicine. Besides being well characterized regarding the capability of embryonal carcinoma and embryonic stem cells to be precursors of different tissues, the molecular mechanisms controlling neuronal differentiation are hardly understood. Neuropeptide and neurotransmitter receptors are expressed at early stages of differentiation prior to synaptogenesis, triggering transient changes in calcium concentration and inducing neurone-specific gene expression. In vitro neuronal differentiation of embryonal carcinoma and embryonic stem cells closely resembles early neuronal development in vivo. Murine P19 EC cells are a well-characterized model for in vitro differentiation, which upon treatment with retinoic acid differentiate into neurones. Expression and activity of various receptor proteins is regulated during their differentiation. Stimulation of kinin-B2, endothelin-B, muscarinic acetylcholine, and N-methyl-D-aspartate receptors results in transient increases of intracellular free calcium concentration [Ca(2+)](i) in P19 cells undergoing neuronal differentiation, whereas embryonal cells do not respond or show a smaller change in [Ca(2+)](i) than differentiating cells. Receptor inhibition, as studied with the example of the kinin-B2 receptor, aborts neuronal maturation of P19 cells, demonstrating the crucial importance of B2 receptors during the differentiation process. Future success in obtaining desired neuronal phenotypes from pluripotent cells in vitro may offer new therapeutic perspectives for curing genetic and acquired dysfunctions of the developing and adult nervous system.

Remarkable variation in the informativeness of RFLP markers linked to hemophilia B locus in Indian population groups: implication in the strategy for carrier detection.

Hemophilia B, an X-linked recessive bleeding disorder, is caused by heterogeneous mutations in the factor IX (F9) gene. Hence, carriers of the disease are usually detected by F9 gene linked RFLP analysis. We aimed to test a set of RFLP markers (DdeI, XmnI, MnlI, TaqI & HhaI), used worldwide for carrier detection, to estimate its heterozygosity in different population groups of India, and identify additional single nucleotide polymorphisms (SNPs) if necessary. A total of 8 population groups encompassing different regions of India, consisting of 107 unrelated normal females without any history of hemophilia B in the family and 13 unrelated obligate carriers were recruited in the study. Regions of F9 gene were amplified by PCR from genomic DNA of the donors followed by restriction enzyme digestion and/or sequencing as appropriate. Combined informativeness for the markers varied between 52-86% among normal females belonging to different geographical locations of India. Haplotype analysis revealed that the most prevalent haplotype lacked the restriction sites for all five RFLP markers. Screening regions of F9 gene that harbor 10 SNPs reported in dbSNP yielded only two SNPs, which increased the overall informativeness in each population group and heterozygosity in the obligate carriers for the disease from 38% to 69%. Our data show that heterozygosity of commonly used RFLP markers is remarkably variable across different regions of India. Thus prudent selection of the markers based on specific population groups including usage of additional markers is recommended for efficient carrier detection.

Cancer mortality in relatives of retinoblastoma patients.

The risk of other cancers in relatives of retinoblastoma (RTB) patients was determined by a survey of the mortality experience of siblings, parents, parental siblings, and grandparents of all U.S. or Canadian RTB patients referred to The University of Texas M.D. Anderson Hospital and Tumor Institute between 1944 and 1980. Expected mortality was ascertained by the application of age-, sex-, race-, and calendar year-specific U.S. mortality rates to the observed person-years. Among 607 relatives of 33 unilateral-sporadic RTB probands, no excess in cancer deaths was observed (observed/expected = 18/22). Among 733 relatives of 47 bilateral-familial RTB probands, a slight excess in cancer deaths was observed (41/31). A significant excess in cancer deaths was occurred in relatives under age 55 years (18/9) and in fathers (7/1) of the bilateral RTB probands. To determine whether the cancer excess was related to some unique allele associated with second tumors in RTB survivors, the cancer mortality of 203 relatives of the 14 RTB patients with second tumors was examined, and no excess was observed (11/11). To determine whether the excess might be attributable to an unexpressed RTB gene or precursor, the mortality experience was examined in 6 kindreds in which parents, unaffected by RTB, had more than 1 child with RTB. Among these 72 relatives a significant excess in cancer deaths was observed (8/2). The findings demonstrate a modest overall cancer excess in relatives of hereditary RTB patients and suggest it may be attributable to an unexpressed RTB gene or precursor in a small number of kindreds. Mechanisms for an apparent "precursor" might involve a delayed mutation, genetic mosaicism, or a submicroscopic balanced chromosomal translocation.

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.

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.