Global transcription of CRISPR loci in the human oral cavity

© 2015 Lum et al.; licensee BioMed Central. Background: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are active in acquired resistance against bacteriophage and plasmids in a number of environments. In the human mouth, CRISPR loci evolve to counteract oral phage, but the expression of these CRISPR loci has not previously been investigated. We sequenced cDNA from CRISPR loci found in numerous different oral bacteria and compared with oral phage communities to determine whether the transcription of CRISPR loci is specifically targeted towards highly abundant phage present in the oral environment. Results: We found that of the 529,027 CRISPR spacer groups studied, 88 % could be identified in transcripts, indicating that the vast majority of CRISPR loci in the oral cavity were transcribed. There were no strong associations between CRISPR spacer repertoires and oral health status or nucleic acid type. We also compared CRISPR repertoires with oral bacteriophage communities, and found that there was no significant association between CRISPR transcripts and oral phage, regardless of the CRISPR type being evaluated. We characterized highly expressed CRISPR spacers and found that they were no more likely than other spacers to match oral phage. By reassembling the CRISPR-bearing reads into longer CRISPR loci, we found that the majority of the loci did not have spacers matching viruses found in the oral cavities of the subjects studied. For some CRISPR types, loci containing spacers matching oral phage were significantly more likely to have multiple spacers rather than a single spacer matching oral phage. Conclusions: These data suggest that the transcription of oral CRISPR loci is relatively ubiquitous and that highly expressed CRISPR spacers do not necessarily target the most abundant oral phage

Dynamin-related protein 1 is required for normal mitochondrial bioenergetic and synaptic function in CA1 hippocampal neurons.

Disrupting particular mitochondrial fission and fusion proteins leads to the death of specific neuronal populations; however, the normal functions of mitochondrial fission in neurons are poorly understood, especially in vivo, which limits the understanding of mitochondrial changes in disease. Altered activity of the central mitochondrial fission protein dynamin-related protein 1 (Drp1) may contribute to the pathophysiology of several neurologic diseases. To study Drp1 in a neuronal population affected by Alzheimer's disease (AD), stroke, and seizure disorders, we postnatally deleted Drp1 from CA1 and other forebrain neurons in mice (CamKII-Cre, Drp1lox/lox (Drp1cKO)). Although most CA1 neurons survived for more than 1 year, their synaptic transmission was impaired, and Drp1cKO mice had impaired memory. In Drp1cKO cell bodies, we observed marked mitochondrial swelling but no change in the number of mitochondria in individual synaptic terminals. Using ATP FRET sensors, we found that cultured neurons lacking Drp1 (Drp1KO) could not maintain normal levels of mitochondrial-derived ATP when energy consumption was increased by neural activity. These deficits occurred specifically at the nerve terminal, but not the cell body, and were sufficient to impair synaptic vesicle cycling. Although Drp1KO increased the distance between axonal mitochondria, mitochondrial-derived ATP still decreased similarly in Drp1KO boutons with and without mitochondria. This indicates that mitochondrial-derived ATP is rapidly dispersed in Drp1KO axons, and that the deficits in axonal bioenergetics and function are not caused by regional energy gradients. Instead, loss of Drp1 compromises the intrinsic bioenergetic function of axonal mitochondria, thus revealing a mechanism by which disrupting mitochondrial dynamics can cause dysfunction of axons

A cluster of cases of severe acute respiratory syndrome in Hong Kong

BACKGROUND: Information on the clinical features of the severe acute respiratory syndrome (SARS) will be of value to physicians caring for patients suspected of having this disorder. METHODS: We abstracted data on the clinical presentation and course of disease in 10 epidemiologically linked Chinese patients (5 men and 5 women 38 to 72 years old) in whom SARS was diagnosed between February 22, 2003, and March 22, 2003, at our hospitals in Hong Kong, China. RESULTS: Exposure between the source patient and subsequent patients ranged from minimal to that between patient and health care provider. The incubation period ranged from 2 to 11 days. All patients presented with fever (temperature, >38°C for over 24 hours), and most presented with rigor, dry cough, dyspnea, malaise, headache, and hypoxemia. Physical examination of the chest revealed crackles and percussion dullness. Lymphopenia was observed in nine patients, and most patients had mildly elevated aminotransferase levels but normal serum creatinine levels. Serial chest radiographs showed progressive air-space disease. Two patients died of progressive respiratory failure; histologic analysis of their lungs showed diffuse alveolar damage. There was no evidence of infection by Mycoplasma pneumoniae, Chlamydia pneumoniae, or Legionella pneumophila. All patients received corticosteroid and ribavirin therapy a mean (±SD) of 9.6±5.42 days after the onset of symptoms, and eight were treated earlier with a combination of beta-lactams and macrolide for 4±1.9 days, with no clinical or radiologic efficacy. CONCLUSIONS: SARS appears to be infectious in origin. Fever followed by rapidly progressive respiratory compromise is the key complex of signs and symptoms from which the syndrome derives its name. The microbiologic origins of SARS remain unclear.published_or_final_versio

Patterns of HIV prevalence among injecting drug users in the cross-border area of Lang Son Province, Vietnam, and Ning Ming County, Guangxi Province, China

BACKGROUND: To assess patterns of injecting drug use and HIV prevalence among injecting drug users (IDUs) in an international border area along a major heroin trans-shipment route. METHODS: Cross-sectional surveys of IDUs in 5 sites in Lang Son Province, Vietnam (n = 348) and 3 sites in Ning Ming County, Guangxi Province, China (n = 308). Respondents were recruited through peer referral ("snowball") methods in both countries, and also from officially recorded lists of IDUs in Vietnam. A risk behavior questionnaire was administered and HIV counseling and testing conducted. RESULTS: Participants in both countries were largely male, in their 20s, and unmarried. A majority of subjects in both countries were members of ethnic minority groups. There were strong geographic gradients for length of drug injecting and for HIV seroprevalence. Both mean years injecting and HIV seroprevalence declined from the Vietnamese site farthest from the border to the Chinese site farthest from the border. 10.6% of participants in China and 24.5% of participants in Vietnam reported crossing the international border in the 6 months prior to interview. Crossing the border by IDUs was associated with (1) distance from the border, (2) being a member of an ethnic minority group, and (3) being HIV seropositive among Chinese participants. CONCLUSION: Reducing the international spread of HIV among IDUs will require programs at the global, regional, national, and "local cross border" levels. At the local cross border level, the programs should be coordinated on both sides of the border and on a sufficient scale that IDUs will be able to readily obtain clean injection equipment on the other side of the border as well as in their country of residence

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length

Multiple ITS Copies Reveal Extensive Hybridization within Rheum (Polygonaceae), a Genus That Has Undergone Rapid Radiation

During adaptive radiation events, characters can arise multiple times due to parallel evolution, but transfer of traits through hybridization provides an alternative explanation for the same character appearing in apparently non-sister lineages. The signature of hybridization can be detected in incongruence between phylogenies derived from different markers, or from the presence of two divergent versions of a nuclear marker such as ITS within one individual.In this study, we cloned and sequenced ITS regions for 30 species of the genus Rheum, and compared them with a cpDNA phylogeny. Seven species contained two divergent copies of ITS that resolved in different clades from one another in each case, indicating hybridization events too recent for concerted evolution to have homogenised the ITS sequences. Hybridization was also indicated in at least two further species via incongruence in their position between ITS and cpDNA phylogenies. None of the ITS sequences present in these nine species matched those detected in any other species, which provides tentative evidence against recent introgression as an explanation. Rheum globulosum, previously indicated by cpDNA to represent an independent origin of decumbent habit, is indicated by ITS to be part of clade of decumbent species, which acquired cpDNA of another clade via hybridization. However decumbent and glasshouse morphology are confirmed to have arisen three and two times, respectively.These findings suggested that hybridization among QTP species of Rheum has been extensive, and that a role of hybridization in diversification of Rheum requires investigation

Proteomic profiling of neuronal mitochondria reveals modulators of synaptic architecture

Abstract Background Neurons are highly polarized cells consisting of three distinct functional domains: the cell body (and associated dendrites), the axon and the synapse. Previously, it was believed that the clinical phenotypes of neurodegenerative diseases were caused by the loss of entire neurons, however it has recently become apparent that these neuronal sub-compartments can degenerate independently, with synapses being particularly vulnerable to a broad range of stimuli. Whilst the properties governing the differential degenerative mechanisms remain unknown, mitochondria consistently appear in the literature, suggesting these somewhat promiscuous organelles may play a role in affecting synaptic stability. Synaptic and non-synaptic mitochondrial subpools are known to have different enzymatic properties (first demonstrated by Lai et al., 1977). However, the molecular basis underpinning these alterations, and their effects on morphology, has not been well documented. Methods The current study has employed electron microscopy, label-free proteomics and in silico analyses to characterize the morphological and biochemical properties of discrete sub-populations of mitochondria. The physiological relevance of these findings was confirmed in-vivo using a molecular genetic approach at the Drosophila neuromuscular junction. Results Here, we demonstrate that mitochondria at the synaptic terminal are indeed morphologically different to non-synaptic mitochondria, in both rodents and human patients. Furthermore, generation of proteomic profiles reveals distinct molecular fingerprints – highlighting that the properties of complex I may represent an important specialisation of synaptic mitochondria. Evidence also suggests that at least 30% of the mitochondrial enzymatic activity differences previously reported can be accounted for by protein abundance. Finally, we demonstrate that the molecular differences between discrete mitochondrial sub-populations are capable of selectively influencing synaptic morphology in-vivo. We offer several novel mitochondrial candidates that have the propensity to significantly alter the synaptic architecture in-vivo. Conclusions Our study demonstrates discrete proteomic profiles exist dependent upon mitochondrial subcellular localization and selective alteration of intrinsic mitochondrial proteins alters synaptic morphology in-vivo

Global gene expression analysis of canine osteosarcoma stem cells reveals a novel role for COX-2 in tumour initiation

Osteosarcoma is the most common primary bone tumour of both children and dogs. It is an aggressive tumour in both species with a rapid clinical course leading ultimately to metastasis. In dogs and children distant metastasis occurs in >80% of individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to contain a sub-population of cancer stem cells (CSCs), which may drive tumour growth, recurrence and metastasis, suggesting that naturally occurring canine osteosarcoma could act as a preclinical model for the human disease. Here we report the successful isolation of CSCs from primary canine osteosarcoma, as well as established cell lines. We show that these cells can form tumourspheres, and demonstrate relative resistance to chemotherapy. We demonstrate similar results for the human osteosarcma cell lines, U2OS and SAOS2. Utilizing the Affymetrix canine microarray, we are able to definitively show that there are significant differences in global gene expression profiles of isolated osteosarcoma stem cells and the daughter adherent cells. We identified 13,221 significant differences (p = 0.05), and significantly, COX-2 was expressed 141-fold more in CSC spheres than daughter adherent cells. To study the role of COX-2 expression in CSCs we utilized the COX-2 inhibitors meloxicam and mavacoxib. We found that COX-2 inhibition had no effect on CSC growth, or resistance to chemotherapy. However inhibition of COX-2 in daughter cells prevented sphere formation, indicating a potential significant role for COX-2 in tumour initiation