A Polymorphic Gene within the Mycobacterium smegmatis esx1 Locus Determines Mycobacterial Self-Identity and Conjugal Compatibility.

Mycobacteria mediate horizontal gene transfer (HGT) by a process called distributive conjugal transfer (DCT) that is mechanistically distinct from oriT-mediated plasmid transfer. The transfer of multiple, independent donor chromosome segments generates transconjugants with genomes that are mosaic blends of their parents. Previously, we had characterized contact-dependent conjugation between two independent isolates of Mycobacterium smegmatis. Here, we expand our analyses to include five independent isolates of M. smegmatis and establish that DCT is both active and prevalent among natural isolates of M. smegmatis. Two of these five strains were recipients but exhibited distinct conjugal compatibilities with donor strains, suggesting an ability to distinguish between potential donor partners. We determined that a single gene, Msmeg0070, was responsible for conferring mating compatibility using a combination of comparative DNA sequence analysis, bacterial genome-wide association studies (GWAS), and targeted mutagenesis. Msmeg0070 maps within the esx1 secretion locus, and we establish that it confers mycobacterial self-identity with parallels to kin recognition. Similar to other kin model systems, orthologs of Msmeg0070 are highly polymorphic. The identification of a kin recognition system in M. smegmatis reinforces the concept that communication between cells is an important checkpoint prior to DCT commitment and implies that there are likely to be other, unanticipated forms of social behaviors in mycobacteria. IMPORTANCE Conjugation, unlike other forms of HGT, requires direct interaction between two viable bacteria, which must be capable of distinguishing between mating types to allow successful DNA transfer from donor to recipient. We show that the conjugal compatibility of Mycobacterium smegmatis isolates is determined by a single, polymorphic gene located within the conserved esx1 secretion locus. This gene confers self-identity; the expression of identical Msmeg0070 proteins in both donor-recipient partners prevents DNA transfer. The presence of this polymorphic locus in many environmental mycobacteria suggests that kin identification is important in promoting beneficial gene flow between nonkin mycobacteria. Cell-cell communication, mediated by kin recognition and ESX secretion, is a key checkpoint in mycobacterial conjugation and likely plays a more global role in mycobacterial biology.

A Mycobacterial Systems Resource for the Research Community.

Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulceransIMPORTANCE Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using Mycobacterium smegmatis as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.

Early-onset type 2 diabetes: higher burden of atherogenic apolipoprotein particles during statin treatment.

AIMS: To determine the burden of atherogenic apolipoprotein particles in early-onset type 2 diabetes (T2D) compared to those with later-onset disease during statin treatment. METHODS: Early and later-onset T2D was defined as current age below and above 40 years respectively. Conventional lipid profile, LDL, non-HDL cholesterol, apolipoprotein B and A1 were determined in those without cardiovascular disease treated with simvastatin to achieve LDL cholesterol <2 mmol/l. RESULTS: Fifty subjects were recruited (early-onset n=24 and later-onset n=26). The mean age was 34.5 and 59.6 years and mean age of diagnosis was 29.1 and 49.1 years for early and later-onset T2D respectively. Obesity, dyslipidaemia, microalbuminuria, glycaemic control and diabetes complication burden were similar in both cohorts. Early-onset subjects received non-significantly higher simvastatin dose (37.5 vs. 31.9 mg daily, p=NS). On-treatment LDL cholesterol was similar in both cohorts (early vs. later-onset; 2.12 vs. 1.97 mmol/l, p=NS). Fasting triglyceride, non-HDL, apo B and B/A1 ratio were significantly higher in early-onset cohort. There was no difference in apo A1, HDL and total cholesterol/HDL ratio. Apo B level remained significantly higher among early-onset subjects after adjustment for insulin treatment. Lower current age and age of diagnosis were significant predictors of higher apo B level. CONCLUSION: The burden of atherogenic apolipoprotein particles was greater in early-onset T2D despite adequate statin treatment indicating an adverse phenotype for vascular disease.

Individualised patient care as an adjunct to standard care for promoting adherence to ocular hypotensive therapy: an exploratory randomised controlled trial.

PURPOSE: To evaluate the impact of individualised patient care, as an adjunct to standard care, on adherence to ocular hypotensive therapy. METHODS: A two-arm, single-masked exploratory randomised controlled trial recruited patients newly prescribed ocular hypotensive therapy. The intervention involved an individual assessment of health-care needs and beliefs and a 1-year follow-up period according to need. The primary outcome was refill adherence, measured by collating prescription and dispensing data for 12 months. Secondary outcomes included self-reported adherence, glaucoma knowledge, beliefs about illness and medicines, quality of care, intraocular pressure (IOP) fluctuation, and changes in clinical management assessed at 12 months. The strength of the intervention was measured following withdrawal by reviewing clinical outcomes for a further 12 months. RESULTS: In all, 127 patients were recruited (91% response rate). Intervention-arm patients collected significantly more prescriptions than control-arm patients. Self-report adherence was significantly better in the intervention-arm for patients who forgot drops and those who intentionally missed drops. The intervention group demonstrated significantly more glaucoma knowledge, expressed a significantly stronger belief in the necessity of eye drops and believed that they had more personal control over managing their condition. Control-arm patients had more IOP fluctuation and changes in clinical management. However, this finding only reached significance at 24 months. CONCLUSION: Modelling patient care according to health-care needs and beliefs about illness and medicines can have a significant impact on improving adherence to therapy for this patient group, with the potential benefit of improving clinical outcomes.

Preliminary survey of educational support for patients prescribed ocular hypotensive therapy.

PURPOSE: to establish the impact of educational support on patients' knowledge of glaucoma and adherence, in preparation for an intervention study. METHODS: structured observation encapsulated the educational support provided during clinical consultations and patient interviews captured the depth of glaucoma knowledge, problems associated with glaucoma therapy, and adherence issues. RESULTS: one hundred and thirty-eight patients completed the study. Education was didactic in nature, limited for many patients and inconsistent across clinics. Patients showed generally poor knowledge of glaucoma with a median score of 6 (range 0-16). A significant association was found between educational support and knowledge for newly prescribed patients (Kendall's tau=0.30, P=0.003), but no association was found for follow-up patients (Kendall's tau=0.11, P=0.174). Only five (6%) patients admitted to a doctor that they did not adhere to their drop regimen, yet 75 (94%) reported at interview that they missed drops. CONCLUSIONS: although important, knowledge alone may not sufficiently improve adherence: a patient-centred approach based on ongoing support according to need may provide a more effective solution for this patient group.

A survey of laboratory practice in the clinical authorization and reporting of results.

BACKGROUND: The aim of this survey was to determine the practice of authorization and reporting of results in clinical biochemistry laboratories in the UK. METHOD: Questionnaires were distributed to the heads of clinical biochemistry departments through the National Audit Committee of the Association of Clinical Biochemists. The standards surveyed were based on guidelines for the reporting of results produced by the Royal College of Pathologists and the relevant Clinical Pathology Accreditation standards. RESULTS: Completed questionnaires were received from 137 laboratories. Workload ranged from 15,000 to 750,000 requests per annum (median 276,883). Most laboratories (98%) release results electronically to at least some wards in real-time. Areas where difficulties were identified included identifying requests that had posed specific questions and access to clinical information at the authorization stage, recording clinical advice given, and ensuring comments remained attached to printed reports or were on the same screen on computer reports. All but six laboratories had consultant advice available, including out of hours, but only 17% had an arrangement for clinical authorization to occur out of hours. CONCLUSION: Only 45 laboratories (33%) were able to achieve 100% compliance with the standards that currently exist, but many others showed evidence of good practice. The practical obstacles still to be overcome include limitations in the capabilities of laboratory computer systems, the lack of accessible electronic clinical records, the difficulties of covering work out of hours and insufficient appropriately trained staff.

Quarts out of pint pots -- expanding the pint. Clinical budgeting in practice.

Clinical budgeting is the process whereby clinical users are charged for the resources they use. A system for recharging users for the costs of tests was introduced at the Northern General Hospital, Sheffield, in 1995, and has been in operation since. The system has allowed pathology to maintain budgetary balance, has automatically compensated for workload increases, has allowed the introduction of new tests, and has encouraged clinical users to include pathology costs in their bids for funding for clinical developments. The system works according to rules agreed between pathology and its users at the outset, but once set up takes a minimal amount of work to operate and maintain.

Phylogenetic conservation of the makorin-2 gene, encoding a multiple zinc-finger protein, antisense to the RAF1 proto-oncogene.

Natural endogenous antisense RNAs have been reported in multiple loci, with evidence in some cases supporting a regulatory role for the antisense transcript. Here, we describe a novel gene, makorin RING zinc finger-2 (MKRN2), that overlaps and is antisense to the gene RAF1 in mammals. Phylogenetic analysis of the 3' untranslated region of RAF1 orthologues suggests that this relationship may have existed for up to 450 million years. We have also identified MKRN2 orthologues in two species of fish. This places the gene duplication event that created this locus from an ancestral MKRN1 gene early in vertebrate evolution, over 450 million years ago. Northern blot analyses show that human MKRN2 and RAF1 are co-expressed in tissues and cell lines, raising the possibility of mRNA duplex formation. The encoded makorin-2 protein is likely a ribonucleoprotein of unknown function, but its conservation suggests an important cellular role. The data presented here describe a conserved vertebrate MKRN2 gene that is closely associated with the RAF1 locus.

Diverse splicing mechanisms fuse the evolutionarily conserved bicistronic MOCS1A and MOCS1B open reading frames.

Molybdenum is an essential cofactor in many enzymes, but must first be complexed by molybdopterin, whose synthesis requires four enzymatic activities. The first two enzymes of this pathway are encoded by the MOCS1 locus in humans. We describe here a remarkably well-conserved novel mRNA splicing phenomenon that produces both an apparently bicistronic MOCS1AM-OCS1B transcript, as well as a distinct class of monocistronic transcript. The latter are created by a variety of splicing mechanisms (alternative splice donors, alternative splice acceptors, and exon-skipping) to bypass the normal termination nonsense codon of MOCS1A resulting in fusion of the MOCS1A and MOCS1B open reading frames. Therefore, these "no-nonsense" transcripts encode a single bifunctional protein embodying both MOCS1A and MOCS1B activities. This coexpression profile was observed in vertebrates (human, mouse, cow, rabbit, opossum, and chicken) and invertebrates (fruit fly and nematode) spanning at least 700 million years of evolution. Our phylogenetic data also provide evidence that the bicistronic form of MOCS1 mRNA is likely to only produce MOCS1A protein and, combined with Northern analyses, suggests that MOCS1B is translated only as a fusion with MOCS1A. Taken together, the data presented here demonstrate a very highly conserved and physiologically relevant dynamic splicing scheme that profoundly influences the protein-coding potential of the MOCS1 locus.

The ancient source of a distinct gene family encoding proteins featuring RING and C(3)H zinc-finger motifs with abundant expression in developing brain and nervous system.

Intronless genes can arise by germline retrotransposition of a cDNA originating as mRNA from an intron-containing source gene. Previously, we described several members of a family of intronless mammalian genes encoding a novel class of zinc-finger proteins, including one that shows imprinted expression and one that escapes X-inactivation. We report here the identification and characterization of the Makorin ring finger protein 1 gene (MKRN1), a highly transcribed, intron-containing source for this family of genes. Phylogenetic analyses clearly indicate that the MKRN1 gene is the ancestral founder of this gene family. We have identified MKRN1 orthologs from human, mouse, wallaby, chicken, fruitfly, and nematode, underscoring the age and conservation of this gene. The MKRN gene family encodes putative ribonucleoproteins with a distinctive array of zinc-finger motifs, including two to four C(3)H zinc-fingers, an unusual Cys/His arrangement that may represent a novel zinc-finger structure, and a highly conserved RING zinc-finger. To date, we have identified nine MKRN family loci distributed throughout the human genome. The human and mouse MKRN1 loci map to a conserved syntenic group near the T-cell receptor beta cluster (TCRB) in chromosome 7q34-q35 and chromosome 6A, respectively. MKRN1 is widely transcribed in mammals, with high levels in murine embryonic nervous system and adult testis. The ancient origin of MKRN1, high degree of conservation, and expression pattern suggest important developmental and functional roles for this gene and its expressed family members.

Conserved characteristics of heterochromatin-forming DNA at the 15q11-q13 imprinting center.

Nuclear matrix binding assays (NMBAs) define certain DNA sequences as matrix attachment regions (MARs), which often have cis-acting epigenetic regulatory functions. We used NMBAs to analyze the functionally important 15q11-q13 imprinting center (IC). We find that the IC is composed of an unusually high density of MARs, located in close proximity to the germ line elements that are proposed to direct imprint switching in this region. Moreover, we find that the organization of MARs is the same at the homologous mouse locus, despite extensive divergence of DNA sequence. MARs of this size are not usually associated with genes but rather with heterochromatin-forming areas of the genome. In contrast, the 15q11-q13 region contains multiple transcribed genes and is unusual for being subject to genomic imprinting, causing the maternal chromosome to be more transcriptionally silent, methylated, and late replicating than the paternal chromosome. We suggest that the extensive MAR sequences at the IC are organized as heterochromatin during oogenesis, an organization disrupted during spermatogenesis. Consistent with this model, multicolor fluorescence in situ hybridization to halo nuclei demonstrates a strong matrix association of the maternal IC, whereas the paternal IC is more decondensed, extending into the nuclear halo. This model also provides a mechanism for spreading of the imprinting signal, because heterochromatin at the IC on the maternal chromosome may exert a suppressive position effect in cis. We propose that the germ line elements at the 15q11-q13 IC mediate their effects through the candidate heterochromatin-forming DNA identified in this study.

Concerted regulation and molecular evolution of the duplicated SNRPB'/B and SNRPN loci.

The human small nuclear ribonucleoprotein SNRPB ' /B gene is alternatively spliced to produce the SmB or SmB' spliceosomal core proteins. An ancestral duplication gave rise to the closely related SNRPN paralog whose protein product, SmN, replaces SmB'/B in brain. However, the precise evolutionary and functional relationship between these loci has not been clear. Genomic, cDNA and protein analyses presented here in chicken, two marsupials (South American opossum and tammar wallaby), and hedgehog, suggest that the vertebrate ancestral locus produced the SmB' isoform. Interestingly, three eutherians exhibit radically distinct splice choice expression profiles, producing either exclusively SmB in mouse, both SmB and SmB' in human, or exclusively SmB' in hedgehog. The human SNRPB ' /B locus is biallelically unmethylated, unlike the imprinted SNRPN locus which is unmethyl-ated only on the expressed paternal allele. Western analysis demonstrates that a compensatory feedback loop dramatically upregulates SmB'/B levels in response to the loss of SmN in Prader-Willi syndrome brain tissue, potentially reducing the phenotypic severity of this syndrome. These findings imply that these two genes encoding small nuclear ribonucleoprotein components are subject to dosage compensation. Therefore, a more global regulatory network may govern the maintenance of stoichiometric levels of spliceosomal components and may constrain their evolution.

Validation of the SpaceLabs 90207 ambulatory blood pressure device for hemodialysis patients.

BACKGROUND: Background Ambulatory blood pressure monitoring (ABPM) has been used increasingly in assessing dialysis patients. However, devices have not been validated formally for this population. Formal device validation is important in order to guarantee adequate performance for populations with special characteristics, such as patients undergoing hemodialysis. OBJECTIVE: To achieve formal validation of the SpaceLabs 90207 ambulatory blood pressure device (SLD) using a modified British Hypertension Society protocol. METHODS: Eighty-five hemodialysis patients were studied, generating 255 readings. Readings were obtained with patients supine (in a dialysis chair) over 5-10 min during hemodialysis. Simultaneous, same-arm readings were obtained through the use of a T connector to a calibrated mercury manometer. The mean differences between readings obtained by observers and those obtained by the device were calculated. Limits of agreement between observers and SLD were determined and plotted according to the method of Bland and Altman. For grading of performance, we determined the number of readings for which the readings obtained by the device were within 5, 10, and 15mmHg of manometer readings. Final gradings were ascribed according to British Hypertension Society criteria. RESULTS: The mean blood pressure (+/-SD) was 141/76+/-31/15mmHg (observers) and 141/77+/-27/15mmHg (SLD), and the mean (+/-SD) difference between observers and device (observer-device) was -0.5+/-7.5mmHg for systolic blood pressure (SBP) and -0.2+/-5.2mmHg for diastolic blood pressure (DBP). The device was less accurate in extreme ranges of SBP. In fact, there was a positive correlation between average [(observer+device)/2] and difference (observer-device) for SBP (r =0. 54, P <0.0001), showing that underestimation in higher ranges, and overestimation in lower ranges of blood pressure occurred for SBP. For SBP, 53% of readings were within 5mmHg of those obtained by the observers, 85% were within 10mmHg, and 97% within 15mmHg. For DBP, 78% were within 5mmHg, 96% within 10mmHg, and 98% within 15mmHg. These observations conferred on the device grade C for SBP and grade B for DBP. The type of vascular access and the presence of non-functioning arteriovenous grafts and fistulas in the ipsilateral arm did not alter these results significantly. CONCLUSIONS: These data validate the use of this device for hemodialysis patients. However, caution should be exercised in the evaluation of upper and lower ranges of SBP.

Chromium homeostasis in patients with type II (NIDDM) diabetes.

The purpose of this study was to assess chromium handling in non-insulin dependent diabetic patients (NIDDM) compared to healthy volunteers. Chromium handling was evaluated using fasting blood and second morning void urine samples from 93 NIDDM patients and 33 healthy volunteers. Significant differences in chromium homeostasis were seen between patients and controls. NIDDM patients had mean levels of plasma chromium around 33% lower and urine values almost 100% higher than those found in health. Healthy volunteers showed a significant negative correlation between fasting levels of plasma chromium and insulin. This was not evident in NIDDM patients. In the early years of onset of NIDDM, plasma chromium values were inversely correlated with plasma glucose. This was lost in patients with diabetes of more than 2 years duration. We suggest large losses of chromium over many years may exacerbate an already compromised chromium status in NIDDM patients and might contribute to the developing insulin resistance seen in patients with type 2 diabetes.

An imprinted, mammalian bicistronic transcript encodes two independent proteins.

Polycistronic transcripts are common in prokaryotes but rare in eukaryotes. Phylogenetic analysis of the SNRPN (SmN) mRNA in five eutherian mammals reveals a second highly conserved coding sequence, termed SNURF (SNRPN upstream reading frame). The vast majority of nucleotide substitutions in SNURF occur in the wobble codon position, providing strong evolutionary evidence for selection for protein-coding function. Because SNURF-SNRPN maps to human chromosome 15q11-q13 and is paternally expressed, each cistron is a candidate for a role in the imprinted Prader-Willi syndrome (PWS) and PWS mouse models. SNURF encodes a highly basic 71-aa protein that is nuclear-localized (as is SmN). Because SNURF is the only protein-coding sequence within the imprinting regulatory region in 15q11-q13, it may have provided the original selection for imprinting in this domain. Whereas some human tissues express a minor SNURF-only transcript, mouse tissues express only the bicistronic Snurf-Snrpn transcript. We show that both SNURF and SNRPN are translated in normal, but not PWS, human, and mouse tissues and cell lines. These findings identify SNURF as a protein that is produced along with SmN from a bicistronic transcript; polycistronic mRNAs therefore are encoded in mammalian genomes where they may form functional operons.

A novel imprinted gene, encoding a RING zinc-finger protein, and overlapping antisense transcript in the Prader-Willi syndrome critical region.

We describe a complex imprinted locus in chromosome 15q11-q13 that encodes two genes, ZNF127 and ZNF127AS. The ZNF127 gene encodes a protein with a RING (C3HC4) zinc-finger and multiple C3H zinc-finger motifs, the former being closely related to a protein from variola major virus, the smallpox etiological agent. These motifs allow prediction of ZNF127 function as a ribonucleoprotein. The intronless ZNF127 gene is expressed ubiquitously, but the entire coding sequence and 5' CpG island overlaps a second gene, ZNF127AS, that is transcribed from the antisense strand with a different transcript size and pattern of expression. Allele-specific analysis shows that ZNF127 is expressed only from the paternal allele. Consistent with this expression pattern, in the brain the ZNF127 5' CpG island is completely unmethylated on the paternal allele but methylated on the maternal allele. Analyses of adult testis, sperm and fetal oocytes demonstrates a gametic methylation imprint with unmethylated paternal germ cells. Recent findings indicate that ZNF127 is part of the coordinately regulated imprinted domain affected in Prader-Willi syndrome patients with imprinting mutations. Therefore, ZNF127 and ZNF127AS are novel imprinted genes that may be associated with some of the clinical features of the polygenic Prader-Willi syndrome.

Imprinting-mutation mechanisms in Prader-Willi syndrome.

Microdeletions of a region termed the "imprinting center" (IC) in chromosome 15q11-q13 have been identified in several families with Prader-Willi syndrome (PWS) or Angelman syndrome who show epigenetic inheritance for this region that is consistent with a mutation in the imprinting process. The IC controls resetting of parental imprints in 15q11-q13 during gametogenesis. We have identified a larger series of cases of familial PWS, including one case with a deletion of only 7.5 kb, that narrows the PWS critical region to <4. 3 kb spanning the SNRPN gene CpG island and exon 1. Identification of a strong DNase I hypersensitive site, specific for the paternal allele, and six evolutionarily conserved (human-mouse) sequences that are potential transcription-factor binding sites is consistent with this region defining the SNRPN gene promoter. These findings suggest that promoter elements at SNRPN play a key role in the initiation of imprint switching during spermatogenesis. We also identified three patients with sporadic PWS who have an imprinting mutation (IM) and no detectable mutation in the IC. An inherited 15q11-q13 mutation or a trans-factor gene mutation are unlikely; thus, the disease in these patients may arise from a developmental or stochastic failure to switch the maternal-to-paternal imprint during parental spermatogenesis. These studies allow a better understanding of a novel mechanism of human disease, since the epigenetic effect of an IM in the parental germ line determines the phenotypic effect in the patient.