Cat-to-Human Transmission of Mycobacterium bovis, United Kingdom.

Human infection with Mycobacterium bovis is reported infrequently in the United Kingdom. Most cases involve previous consumption of unpasteurized milk. We report a rare occurrence of 2 incidents of cat-to-human transmission of M. bovis during a cluster of infection in cats.

Bovine tuberculosis in working foxhounds: lessons learned from a complex public health investigation.

In 2017, Public Health England South East Health Protection Team (HPT) were involved in the management of an outbreak of Mycobacterium bovis (the causative agent of bovine tuberculosis) in a pack of working foxhounds. This paper summarises the actions taken by the team in managing the public health aspects of the outbreak, and lessons learned to improve the management of future potential outbreaks. A literature search was conducted to identify relevant publications on M. bovis. Clinical notes from the Public Health England (PHE) health protection database were reviewed and key points extracted. Animal and public health stakeholders involved in the management of the situation provided further evidence through unstructured interviews and personal communications. The PHE South East team initially provided 'inform and advise' letters to human contacts whilst awaiting laboratory confirmation to identify the infectious agent. Once M. bovis had been confirmed in the hounds, an in-depth risk assessment was conducted, and contacts were stratified in to risk pools. Eleven out of 20 exposed persons with the greatest risk of exposure were recommended to attend TB screening and one tested positive, but had no evidence of active TB infection. The number of human contacts working with foxhound packs can be large and varied. HPTs should undertake a comprehensive risk assessment of all potential routes of exposure, involve all other relevant stakeholders from an early stage and undertake regular risk assessments. Current guidance should be revised to account for the unique risks to human health posed by exposure to infected working dogs.

Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein.

Telomerase is an essential cellular ribonucleoprotein (RNP) that solves the end replication problem and maintains chromosome stability by adding telomeric DNA to the termini of linear chromosomes. Genetic mutations that abrogate the normal assembly of telomerase RNP cause human disease. It is therefore of fundamental and medical importance to decipher cellular strategies for telomerase biogenesis, which will require new insights into how specific interactions occur in a precise order along the RNP assembly pathway. Here we use a single-molecule approach to dissect the individual assembly steps of telomerase. Direct observation of complex formation in real time revealed two sequential steps of protein-induced RNA folding, establishing a hierarchical RNP assembly mechanism: interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs the binding of the telomerase reverse transcriptase to form the functional ternary complex. This hierarchical assembly process is facilitated by an evolutionarily conserved structural motif within the RNA. These results identify the RNA folding pathway during telomerase biogenesis and define the mechanism of action for an essential telomerase holoenzyme protein.

Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability.

Telomerase adds simple-sequence repeats to chromosome 3' ends to compensate for the loss of repeats with each round of genome replication. To accomplish this de novo DNA synthesis, telomerase uses a template within its integral RNA component. In addition to providing the template, the telomerase RNA subunit (TER) also harbors nontemplate motifs that contribute to the specialized telomerase catalytic cycle of reiterative repeat synthesis. Most nontemplate TER motifs function through linkage with the template, but in ciliate and vertebrate telomerases, a stem-loop motif binds telomerase reverse transcriptase (TERT) and reconstitutes full activity of the minimal recombinant TERT+TER RNP, even when physically separated from the template. Here, we resolve the functional requirements for this motif of ciliate TER in physiological RNP context using the Tetrahymena thermophila p65-TER-TERT core RNP reconstituted in vitro and the holoenzyme reconstituted in vivo. Contrary to expectation based on assays of the minimal recombinant RNP, we find that none of a panel of individual loop IV nucleotide substitutions impacts the profile of telomerase product synthesis when reconstituted as physiological core RNP or holoenzyme RNP. However, loop IV nucleotide substitutions do variably reduce assembly of TERT with the p65-TER complex in vitro and reduce the accumulation and stability of telomerase RNP in endogenous holoenzyme context. Our results point to a unifying model of a conformational activation role for this TER motif in the telomerase RNP enzyme.

A telomerase holoenzyme protein enhances telomerase RNA assembly with telomerase reverse transcriptase.

Telomerase maintains the simple sequence repeats at chromosome ends, protecting cells from genomic rearrangement, proliferative senescence and death. The telomerase reverse transcriptase (TERT) and telomerase RNA (TER) alone can assemble into active enzyme in a heterologous cell extract, but the physiological process of telomerase biogenesis is more complex. The endogenous accumulation of Tetrahymena thermophila TERT and TER requires an additional telomerase holoenzyme protein, p65. Here, we reconstitute this cellular pathway for telomerase ribonucleoprotein biogenesis in vitro. We demonstrate that tandem RNA interaction domains in p65 recognize the sequence of the TER 3' stem. Notably, the p65-TER complex recruits TERT much more efficiently than does TER alone. Using bacterially expressed p65 and TERT polypeptides, we show that p65 enhances TERT-TER interaction by a mechanism involving a conserved bulge in the protein-bridging TER molecule. These findings reveal a pathway for telomerase holoenzyme biogenesis that preassembles TER for TERT recruitment.

The predictive value of early neurological examination in neonatal hypoxic-ischaemic encephalopathy and neurodevelopmental outcome at 24 months.

AIM: The clinical and electrographic signs of hypoxic-ischaemic encephalopathy (HIE) evolve over the first days of life. We examined the evolution of neurological signs over the first 3 days of life, and determined whether serial administration of the Amiel-Tison Neurological Assessment at Term (ATNAT) would predict neurodevelopmental outcome at 24 months. METHOD: Term (>37 wks' gestation) neonates born with suspected HIE between May 2003 and May 2005 in a Cork maternity unit were recruited prospectively. Modified Sarnat grading was assigned. The ATNAT was administered on days 1, 2, and 3 of life and a discharge neurological examination. Time to oral feeding and demographic variables were recorded. Developmental status was assessed using the revised Griffiths Mental Development Scales at 6, 12, and 24 months. RESULTS: Fifty-seven infants were recruited, with 51 (31 males, 20 females) included for follow-up. Neurological examination evolved and normalized over the first 3 days of life in many cases. At 24 months, 21 children had an adverse outcome, including six deaths. Examination at all time points correlated significantly with neurological outcome at 24 months. The best correlations were found to be (1) neurological examination at discharge (r=0.65, p<0.001), (2) Sarnat grading (r=0.64, p<0.001), and (3) ATNAT on day 3 (r=0.46, p<0.001). The best predictive value was seen with neurological examination at discharge (positive and negative predictive values of 86% and 72% respectively). INTERPRETATION: Persistence of abnormal neurological signs correlates significantly with adverse outcome. The later a neonatal neurological examination was performed, the better its predictive ability.

A novel RNA binding domain in tetrahymena telomerase p65 initiates hierarchical assembly of telomerase holoenzyme.

Telomerase reverse transcriptase (TERT) and telomerase RNA (TER) assemble as part of a holoenzyme that synthesizes telomeric repeats at chromosome ends. Genetic approaches have identified proteins that are required for in vivo association of TERT and TER, including the Tetrahymena telomerase holoenzyme protein p65. Here, we use quantitative assays to define the mechanisms underlying p65 function in holoenzyme biogenesis. We demonstrate that four modules of p65 contribute affinity for TER, including a C-terminal domain that recognizes the conserved dinucleotide bulge of central stem IV. This C-terminal domain is necessary and sufficient for p65's function in enhancing the recruitment of TERT to TER. Finally, we show that p65 and TERT assemble on TER with hierarchical rather than cooperative binding. These findings elucidate an extensive network of p65-TER recognition specificity and define a novel p65 RNA binding domain that initiates telomerase holoenyzme biogenesis.

The ability of early serial developmental assessment to predict outcome at 5years following neonatal hypoxic-ischaemic encephalopathy.

BACKGROUND: Neurodevelopmental difficulties in children following hypoxic-ischaemic encephalopathy (HIE) may not emerge until school age. AIMS: To evaluate the value and stability of early serial developmental assessments in predicting long-term outcome. STUDY DESIGN: Prospective study of infants with neonatal HIE and early continuous EEG at birth. SUBJECTS: Term infants with HIE were recruited at birth. Development was measured at 6, 12 and 24months using the Revised Griffiths' Scales (GMDS-R). OUTCOME MEASURES: Intellectual abilities at age five were measured using the Wechsler Preschool & Primary Scale of Intelligence (WPPSI-IIIUK) and the 'numbers' subtest from the Children's Memory Scale. Overall five-year outcome was also reported. RESULTS: IQ outcome was available in forty-seven surviving children (28 male, 19 female: mean (SD) age 64.0(5.7) months. Mean processing speed (p=0.01) and short-term verbal memory (p=0.005) were below the norm. Global development (GDQ) at 6, 12 and 24months correlated (p<0.01) with five-year global, verbal and performance IQ with improved correlation over time. Normal GDQ throughout early childhood predicted normal IQ at 5years (24month AUROC value=0.941, p=0.001). An abnormal early GDQ score at any stage in the first 24months had excellent negative predictive values, superior to those for neonatal Sarnat and EEG grading. CONCLUSIONS: Normal early development predicts normal 5year IQ with prediction increasing over time. Repeated measurement is warranted due to instability of findings across the first two years. Follow-up for children with abnormal early development is warranted given high sensitivity for school-age global abnormal outcome.

Early EEG Grade and Outcome at 5 Years After Mild Neonatal Hypoxic Ischemic Encephalopathy.

OBJECTIVE: More than half of all infants with neonatal hypoxic ischemic encephalopathy (HIE) are graded as mild and do not meet current criteria for therapeutic hypothermia. These infants are often not enrolled in follow-up, and hence our knowledge of their long-term outcome is sparse. We wished to compare 5-year outcomes in a group of infants with mild, moderate, and severe HIE, graded with both early EEG and clinical assessment, none of whom were treated with therapeutic hypothermia. METHODS: Term infants with HIE and a healthy comparison group were recruited at birth. Both groups had early continuous EEG recordings. Cognitive and motor outcome was assessed at 5 years. RESULTS: Outcome was available in 53 infants with HIE and 30 infants in the comparison group at 5 years. Infants with mild HIE at birth (n = 22) had significantly lower full-scale IQ, verbal IQ, and performance IQ than comparison infants (n = 30) at 5 years (P = .001, .001, and 0.004, respectively). No difference in cognitive measures was seen between infants with mild and moderate grades HIE. Intact survival at 5 years varied across EEG grade HIE at 6 hours after birth; 75% in mild, 46% in moderate, 43% in major abnormalities, and 0% with inactive EEGs, compared with 97% in the comparison group. CONCLUSIONS: Survivors of mild HIE, graded clinically or by early EEG, have higher rates of disability than their peers and have cognitive outcomes similar to that of children with moderate encephalopathy in an uncooled HIE cohort.

An ecological and comparative perspective on the control of bovine tuberculosis in Great Britain and the Republic of Ireland.

Disease ecology involves a systematic approach to understanding the interactions and evolution of host-pathogen systems at the population level, and is essential for developing a comprehensive understanding of the reasons for disease persistence and the most likely means of control. This systems or ecological approach is being increasingly recognised as a progressive method in disease control and is exploited in diverse fields ranging from obesity management in humans to the prevention of infectious disease in animal populations. In this review we discuss bovine tuberculosis (bTB) in Great Britain (GB) within a disease ecology context, and suggest how a comparative ecological perspective helps to reconcile apparent conflicts with the evidence on the effectiveness of badger culling to assist in the control of bTB in GB and the Republic of Ireland (ROI). Our examination shows that failure of past measures to control bTB and the disparity in outcomes of badger culling experiments are the result of a complex relationship amongst the agent, host and environment, i.e. the episystem, of bTB. Here, we stress the role of distinctive bTB episystems and badger culling trial design in the ambiguity and resulting controversy associated with badger culling in GB and ROI. We argue this episystem perspective on bTB control measures in cattle and badger populations provides a useful and informative perspective on the design and implementation of future bTB management in GB, particularly at a time when both scientific and lay communities are concerned about the ongoing epidemic, the cost of current control measures and the execution of future control procedures.

Structural study of elements of Tetrahymena telomerase RNA stem-loop IV domain important for function.

Tetrahymena telomerase RNA (TER) contains several regions in addition to the template that are important for function. Central among these is the stem-loop IV domain, which is involved in both catalysis and RNP assembly, and includes binding sites for both the holoenzyme assembly protein p65 and telomerase reverse transcriptase (TERT). Stem-loop IV contains two regions with high evolutionary sequence conservation: a central GA bulge between helices, and a terminal loop. We solved the solution structure of loop IV and modeled the structure of the helical region containing the GA bulge, using NMR and residual dipolar couplings. The central GA bulge with flanking C-G base pairs induces a approximately 50 degrees semi-rigid bend in the helix. Loop IV is highly structured, and contains a conserved C-U base pair at the top of the helical stem. Analysis of new and previous biochemical data in light of the structure provides a rationale for some of the sequence conservation in this region of TER. The results suggest that during holoenzyme assembly the protein p65 recognizes a bend in stem IV, and this binding to central stem IV helps to position the structured loop IV for interaction with TERT and other region(s) of TER.

Two purified domains of telomerase reverse transcriptase reconstitute sequence-specific interactions with RNA.

Telomerase reverse transcriptase (TERT) and telomerase RNA (TER) function together to create a uniquely specialized polymerase. Here we have described for the first time domains of bacterially expressed Tetrahymena TERT that interacted directly with TER in the absence of assembly chaperones. We used quantitative binding assays to define TER sequence requirements for recognition by the high affinity RNA binding domain and an independent N-terminal RNA interaction domain. The TERT RNA binding domain and N-terminal RNA interaction domain had distinct, nonoverlapping requirements for TER sequence and structure that together accounted for all of the sites of TER contact inferred for full-length TERT. The TER residues important for TERT binding are only a subset of the residues required for catalytic activity. Our findings demonstrate telomerase functional specialization by an elaborate ribonucleoprotein architecture physically separable from the active site.