Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation.

The emerging discoveries on the link between polyadenylation and disease states underline the need to fully characterize genome-wide polyadenylation states. Here, we report comprehensive maps of global polyadenylation events in human and yeast generated using refinements to the Direct RNA Sequencing technology. This direct approach provides a quantitative view of genome-wide polyadenylation states in a strand-specific manner and requires only attomole RNA quantities. The polyadenylation profiles revealed an abundance of unannotated polyadenylation sites, alternative polyadenylation patterns, and regulatory element-associated poly(A)(+) RNAs. We observed differences in sequence composition surrounding canonical and noncanonical human polyadenylation sites, suggesting novel noncoding RNA-specific polyadenylation mechanisms in humans. Furthermore, we observed the correlation level between sense and antisense transcripts to depend on gene expression levels, supporting the view that overlapping transcription from opposite strands may play a regulatory role. Our data provide a comprehensive view of the polyadenylation state and overlapping transcription.

Meta-analysis of the influence of lifestyle changes for preoperative weight loss on surgical outcomes.

BACKGROUND: The aim was to investigate whether preoperative weight loss results in improved clinical outcomes in surgical patients with clinically significant obesity. METHODS: This was a systematic review and aggregate data meta-analysis of RCTs and cohort studies. PubMed, MEDLINE, Embase and CINAHL Plus databases were searched from inception to February 2018. Eligibility criteria were: studies assessing the effect of weight loss interventions (low-energy diets with or without an exercise component) on clinical outcomes in patients undergoing any surgical procedure. Data on 30-day or all-cause in-hospital mortality were extracted and synthesized in meta-analyses. Postoperative thromboembolic complications, duration of surgery, infection and duration of hospital stay were also assessed. RESULTS: A total of 6060 patients in four RCTs and 12 cohort studies, all from European and North American centres, were identified. Most were in the field of bariatric surgery and all had some methodological limitations. The pooled effect estimate suggested that preoperative weight loss programmes were effective, leading to significant weight reduction compared with controls: mean difference -7·42 (95 per cent c.i. -10·09 to -4·74) kg (P < 0·001). Preoperative weight loss interventions were not associated with a reduction in perioperative mortality (odds ratio 1·41, 95 per cent c.i. 0·24 to 8·40; I2 = 0 per cent, P = 0·66) but the event rate was low. The weight loss groups had shorter hospital stay (by 27 per cent). No differences were found for morbidity. CONCLUSION: This limited preoperative weight loss has advantages but may not alter the postoperative morbidity or mortality risk.

Skill sets required for the management of military head, face and neck trauma: a multidisciplinary consensus statement.

INTRODUCTION: The evolution of medical practice is resulting in increasing subspecialisation, with head, face and neck (HFN) trauma in a civilian environment usually managed by a combination of surgical specialties working as a team. However, the full combination of HFN specialties commonly available in the NHS may not be available in future UK military-led operations, necessitating the identification of a group of skill sets that could be delivered by one or more deployed surgeons. METHOD: A systematic review was undertaken to identify those surgical procedures performed to treat acute military head, face, neck and eye trauma. A multidisciplinary consensus group was convened following this with military HFN trauma expertise to define those procedures commonly required to conduct deployed, in-theatre HFN surgical combat trauma management. RESULTS: Head, face, neck and eye damage control surgical procedures were identified as comprising surgical cricothyroidotomy, cervico-facial haemorrhage control and decompression of orbital haemorrhage through lateral canthotomy. Acute in-theatre surgical skills required within 24 hours consist of wound debridement, surgical tracheostomy, decompressive craniectomy, intracranial pressure monitor placement, temporary facial fracture stabilisation for airway management or haemorrhage control and primary globe repair. Delayed in-theatre procedures required within 5 days prior to predicted evacuation encompass facial fracture fixation, delayed lateral canthotomy, evisceration, enucleation and eyelid repair. CONCLUSIONS: The identification of those skill sets required for deployment is in keeping with the General Medical Council's current drive towards credentialing consultants, by which a consultant surgeon's capabilities in particular practice areas would be defined. Limited opportunities currently exist for trainees and consultants to gain experience in the management of traumatic head, face, neck and eye injuries seen in a kinetic combat environment. Predeployment training requires that the surgical techniques described in this paper are covered and should form the curriculum of future military-specific surgical fellowships. Relevant continued professional development will be necessary to maintain required clinical competency.

Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells.

The highly conserved, multifunctional YB-1 is a powerful breast cancer prognostic indicator. We report on a pervasive role for YB-1 in which it associates with thousands of nonpolyadenylated short RNAs (shyRNAs) that are further processed into small RNAs (smyRNAs). Many of these RNAs have previously been identified as functional noncoding RNAs (http://www.johnlab.org/YB1). We identified a novel, abundant, 3'-modified short RNA antisense to Dicer1 (Shad1) that colocalizes with YB-1 to P-bodies and stress granules. The expression of Shad1 was shown to correlate with that of YB-1 and whose inhibition leads to an increase in cell proliferation. Additionally, Shad1 influences the expression of additional prognostic markers of cancer progression such as DLX2 and IGFBP2. We propose that the examination of these noncoding RNAs could lead to better understanding of prostate cancer progression.

New class of gene-termini-associated human RNAs suggests a novel RNA copying mechanism.

Small (<200 nucleotide) RNA (sRNA) profiling of human cells using various technologies demonstrates unexpected complexity of sRNAs with hundreds of thousands of sRNA species present. Genetic and in vitro studies show that these RNAs are not merely degradation products of longer transcripts but could indeed have a function. Furthermore, profiling of RNAs, including the sRNAs, can reveal not only novel transcripts, but also make clear predictions about the existence and properties of novel biochemical pathways operating in a cell. For example, sRNA profiling in human cells indicated the existence of an unknown capping mechanism operating on cleaved RNA, a biochemical component of which was later identified. Here we show that human cells contain a novel type of sRNA that has non-genomically encoded 5' poly(U) tails. The presence of these RNAs at the termini of genes, specifically at the very 3' ends of known mRNAs, strongly argues for the presence of a yet uncharacterized endogenous biochemical pathway in cells that can copy RNA. We show that this pathway can operate on multiple genes, with specific enrichment towards transcript-encoding components of the translational machinery. Finally, we show that genes are also flanked by sense, 3' polyadenylated sRNAs that are likely to be capped.

An in-depth map of polyadenylation sites in cancer.

We present a comprehensive map of over 1 million polyadenylation sites and quantify their usage in major cancers and tumor cell lines using direct RNA sequencing. We built the Expression and Polyadenylation Database to enable the visualization of the polyadenylation maps in various cancers and to facilitate the discovery of novel genes and gene isoforms that are potentially important to tumorigenesis. Analyses of polyadenylation sites indicate that a large fraction (∼30%) of mRNAs contain alternative polyadenylation sites in their 3' untranslated regions, independent of the cell type. The shortest 3' untranslated region isoforms are preferentially upregulated in cancer tissues, genome-wide. Candidate targets of alternative polyadenylation-mediated upregulation of short isoforms include POLR2K, and signaling cascades of cell-cell and cell-extracellular matrix contact, particularly involving regulators of Rho GTPases. Polyadenylation maps also helped to improve 3' untranslated region annotations and identify candidate regulatory marks such as sequence motifs, H3K36Me3 and Pabpc1 that are isoform dependent and occur in a position-specific manner. In summary, these results highlight the need to go beyond monitoring only the cumulative transcript levels for a gene, to separately analysing the expression of its RNA isoforms.

Serotypes and virulence profiles of atypical enteropathogenic Escherichia coli (EPEC) isolated from bovine farms and abattoirs.

AIMS: The objective of this study was to examine the prevalence of enteropathogenic Escherichia coli (EPEC) on beef and dairy farms and in beef abattoirs and to characterize the isolates in terms of serogroup and virulence markers. METHODS AND RESULTS: Bovine faecal samples (n = 1200), farm soil samples (n = 600), hide samples (n = 450) and carcass samples (n = 450) were collected from 20 farms and three abattoirs throughout Ireland over a 12-month period. After selective enrichment, samples testing positive for the intimin gene (eae) using PCR screening were cultured, and colonies were examined for the presence of the eae, vt(1) and vt(2) genes. Colonies that were positive for the intimin gene and negative for the verotoxin genes were further screened using PCR for a range of virulence factors including tir, espA, espB katP, espP, etpD, saa, sab, toxB, iha, lpfA(O157/OI-141) , lpfA(O113) and lpfA(O157/OI-154) . PCR screening was also used to screen for variations in the intimin gene (eae). Of the 2700 source samples analysed, 3.9% (47 of 1200) of faecal, 2% (12 of 600) of soil, 6.4% (29 of 450) of hide and 0.7% (3 of 450) of carcass samples were PCR positive (for the presence of the eae gene). All 140 isolates obtained were atypical EPEC (aEPEC), while θ and ß intimin types were common. The virulence factors hlyA, tir, lpfA (O113) , lpfA (O157/OI-154) , and iha were frequently detected, while lpfA(O157/OI-141) , saa, espA, espB and toxB were also present but to a lesser extent. CONCLUSIONS: It was concluded that cattle are a source of aEPEC, many of which have the virulence machinery necessary to be pathogenic to humans. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest the need for increased research on aEPEC with particular emphasis on food safety and public health risk.

Cognition and freezing of gait in Parkinson's disease: A systematic review and meta-analysis.

Freezing of gait (FOG) is a common and disabling symptom in people with Parkinson's Disease (PwPD). Although cognition is thought to be worse in PwPD who freeze, a comprehensive analysis of this relationship will inform future research and clinical care. This systematic review and meta-analysis compared cognition between PwPD who do and do not exhibit FOG across a range of cognitive domains and assessed the impact of disease severity and medication status on this relationship. 145 papers (n = 9010 participants) were included in the analysis, with 144 and 138 articles meeting the criteria to assess moderating effects of disease severity and medication status, respectively. PwPD who freeze exhibited worse cognition than PwPD without FOG across global cognition, executive function/attention, language, memory, and visuospatial domains. Greater disease severity and "ON" levodopa medication status moderated the FOG status-cognition relationship in global cognitive performance but not in other cognitive domains. This meta-analysis confirmed that cognition is worse in PwPD with FOG and highlights the importance of disease severity and medication status in this relationship.

A sensitive non-radioactive northern blot method to detect small RNAs.

The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (approximately 15-40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to approximately 1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data.

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil.

AIMS: This study estimated the incidence of non-O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non-O157 VTEC in clay and sandy loam soils. METHODS AND RESULTS: Twenty farms were tested over a 12-month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR-positive samples were plated onto Chromocult tryptone bile X-glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non-O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non-O157 serotypes had highest D-values in the sandy loam soil with D-values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60-48·25 days. CONCLUSIONS: This study shows that non-O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first such study of non-O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non-O157 VTEC should be seen as an emerging risk to be controlled within the food chain.

The relationship between plantar sensation and muscle onset during automatic postural responses in people with multiple sclerosis and healthy controls.

BACKGROUND: Plantar sensation is critical for balance control in people with multiple sclerosis (PwMS). While previous research has described its impact on standing balance, the influence of plantar sensation during automatic postural responses (APRs) is not well understood in PwMS. The purpose of this study was to characterize the relationship between plantar sensation and APRs in PwMS and controls. A secondary aim was to determine whether the relationship between plantar sensation and APRs is different across PwMS and control groups. METHODS: 122 PwMS and 48 age-matched controls underwent forward and backward support-surface perturbations from stance. The onset of the tibialis anterior (TA) and medial gastrocnemius (MG) were the primary reactive balance outcome measures for backward and forward losses of balance, respectively. Plantar sensation was measured as the vibration sensation threshold (VT). RESULTS: As expected, PwMS had significantly higher (i.e., worse) VT (p<0.001) and an increased MG and TA onset latency (TA: p<0.001, MG: p = 0.01) compared to the control group. A higher VT was related to increased MG (p<0.001) and TA latency (p<0.001) across all participants. However, no moderating effect of group (control or PwMS) was observed for the relationship between VT and muscle onset (MG: p = 0.14; TA: p = 0.34). CONCLUSION: PwMS demonstrated poorer plantar sensation and delayed muscle onset during APRs compared to controls. Plantar sensation was also related to muscle onset after perturbations in all participants. Although this relationship was not moderated by group, this may be related to the lack of dynamic range of VT scores in controls. These results indicate that plantar sensation may be related to reactive balance and provides insight into a potential contributing factor of delayed automatic postural responses in people with MS.

Oral carcinoma cuniculatum in a young child.

From the Department of Dental Specialties, Birmingham Children's Hospital. This case study describes a rare case of oral carcinoma cuniculatum in a 7-year-old female. She presented with an enlarged mass of the anterior maxilla arising from the gingiva. An anterior maxillectomy with immediate prosthetic replacement and obturation of the residual defect were carried out. The management of this case was challenging given the rare nature of the disease, unclear etiology, the patient's young age and the mutilating effects of surgery. The treatment involved a large multidisciplinary team. The provision of obturators was particularly difficult due to poor patient compliance and the extent of surgery carried out in a growing child. Oral cancer in children under 15 years old is extremely rare and this is the youngest case of oral carcinoma cuniculatum reported in the literature.

Clinical strategies in the management of complex maxillofacial injuries sustained by British military personnel.

The maxillofacial injuries sustained by British troops requiring aeromedical evacuation to the United Kingdom are almost exclusively treated at The Royal Centre for Defence Medicine in Birmingham. As a result the Maxillofacial Department has collectively gained extensive experience in the management of ballistic injuries. In many cases the most successful outcomes have been achieved by using traditional strategies combined with contemporary techniques. This paper will highlight the types of injuries sustained and discuss some cases that typify those the department has managed.

Five months of surgery in the multinational field hospital in Afghanistan with an emphasis on oral and maxillofacial injuries.

The aim of this review was to assess the workload of theatres in the role 3 Multinational Field Hospital in Kandahar, Afghanistan and to identify what period of day most emergency admissions arrived. During the period 05 August 2006 to 21 December 2006, 288 operations were performed on 259 patients and comprised 393 individually quantifiable procedures. 98% of these operations were to treat acute injuries. Oral and Maxillofacial surgeons were involved in 24% of operations. 63% of procedures done at these operations involved upper or lower limbs, 19% the head and neck and 18% involved the torso. An analysis of emergency admissions in November 2006 showed that most occurred between 18.00 and midnight. Although theatre timetabling made provision for this, whenever possible, elective surgery was scheduled for the following morning when emergency injury admissions were at their lowest.

Sall1 regulates mitral cell development and olfactory nerve extension in the developing olfactory bulb.

Sall1 is a zinc finger containing transcription factor that is highly expressed during mammalian embryogenesis. In humans, the developmental disorder Townes Brocks Syndrome is associated with mutations in the SALL1 gene. Sall1-deficient animals die at birth due to kidney deficits; however, its function in the nervous system has not been characterized. We examined the role of Sall1 in the developing olfactory system. We demonstrate that Sall1 is expressed by cells in the olfactory epithelium and olfactory bulb (OB). Sall1-deficient OBs are reduced in size and exhibit alterations in neurogenesis and mitral cell production. In addition, the olfactory nerve failed to extend past the ventral-medial region of the OB in Sall1-deficient animals. We observed intrinsic patterns of neurogenesis during olfactory development in control animals. In Sall1-mutant animals, these patterns of neurogenesis were disrupted. These findings suggest a role for Sall1 in regulating neuronal differentiation and maturation in developing neural structures.

The potential impacts of 21st century climatic and population changes on human exposure to the virus vector mosquito Aedes aegypti.

The mosquito Aedes (Ae). aegypti transmits the viruses that cause dengue and chikungunya, two globally-important vector-borne diseases. We investigate how choosing alternate emissions and/or socioeconomic pathways may modulate future human exposure to Ae. aegypti. Occurrence patterns for Ae. aegypti for 2061-2080 are mapped globally using empirically downscaled air temperature and precipitation projections from the Community Earth System Model, for the Representative Concentration Pathway (RCP) 4.5 and 8.5 scenarios. Population growth is quantified using gridded global population projections consistent with two Shared Socioeconomic Pathways (SSPs), SSP3 and SSP5. Change scenarios are compared to a 1950-2000 reference period. A global land area of 56.9 M km2 is climatically suitable for Ae. aegypti during the reference period, and is projected to increase by 8% (RCP4.5) to 13% (RCP8.5) by 2061-2080. The annual average number of people exposed globally to Ae. aegypti for the reference period is 3794 M, a value projected to statistically significantly increase by 298-460 M (8-12%) by 2061-2080 if only climate change is considered, and by 4805-5084 M (127-134%) for SSP3 and 2232-2483 M (59-65%) for SSP5 considering both climate and population change (lower and upper values of each range represent RCP4.5 and RCP8.5 respectively). Thus, taking the lower-emissions RCP4.5 pathway instead of RCP8.5 may mitigate future human exposure to Ae. aegypti globally, but the effect of population growth on exposure will likely be larger. Regionally, Australia, Europe and North America are projected to have the largest percentage increases in human exposure to Ae. aegypti considering only climate change.

Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms.

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway (CBP), and are used for the prevention of cardiovascular disease. The anti-inflammatory effects of statins may also provide therapeutic benefits and have led to their use in clinical trials for preeclampsia, a pregnancy-associated inflammatory condition, despite their current classification as category X (i.e. contraindicated during pregnancy). In the developing neocortex, products of the CBP play essential roles in proliferation and differentiation of neural stem-progenitor cells (NSPCs). To understand how statins could impact the developing brain, we studied effects of pravastatin and simvastatin on primary embryonic NSPC survival, proliferation, global transcription, and cell fate in vitro. We found that statins dose dependently decrease NSPC expansion by promoting cell death and autophagy of NSPCs progressing through the G1 phase of the cell cycle. Genome-wide transcriptome analysis demonstrates an increase in expression of CBP genes following pravastatin treatment, through activation of the SREBP2 transcription factor. Co-treatment with farnesyl pyrophosphate (FPP), a CBP metabolite downstream of HMG-CoA reductase, reduces SREBP2 activation and pravastatin-induced PARP cleavage. Finally, pravastatin and simvastatin differentially alter NSPC cell fate and mRNA expression during differentiation, through a non-CBP dependent pathway.

Activating transcription factor 1 and CREB are important for cell survival during early mouse development.

Activating transcription factor 1 (ATF1), CREB, and the cyclic AMP (cAMP) response element modulatory protein (CREM), which constitute a subfamily of the basic leucine zipper transcription factors, activate gene expression by binding as homo- or heterodimers to the cAMP response element in regulatory regions of target genes. To investigate the function of ATF1 in vivo, we inactivated the corresponding gene by homologous recombination. In contrast to CREB-deficient mice, which suffer from perinatal lethality, mice lacking ATF1 do not exhibit any discernible phenotypic abnormalities. Since ATF1 and CREB but not CREM are strongly coexpressed during early mouse development, we generated mice deficient for both CREB and ATF1. ATF1(-/-) CREB(-/-) embryos die before implantation due to developmental arrest. ATF1(+/-) CREB(-/-) embryos display a phenotype of embryonic lethality around embryonic day 9.5 due to massive apoptosis. These results indicate that CREB and ATF1 act in concert to mediate signals essential for maintaining cell viability during early embryonic development.

Loss of the Sall3 gene leads to palate deficiency, abnormalities in cranial nerves, and perinatal lethality.

Members of the Spalt gene family encode putative transcription factors characterized by seven to nine C2H2 zinc finger motifs. Four genes have been identified in mice--Spalt1 to Spalt4 (Sall1 to Sall4). Spalt homologues are widely expressed in neural and mesodermal tissues during early embryogenesis. Sall3 is normally expressed in mice from embryonic day 7 (E7) in the neural ectoderm and primitive streak and subsequently in the brain, peripheral nerves, spinal cord, limb buds, palate, heart, and otic vesicles. We have generated a targeted disruption of Sall3 in mice. Homozygous mutant animals die on the first postnatal day and fail to feed. Examination of the oral structures of these animals revealed that abnormalities were present in the palate and epiglottis from E16.5. In E10.5 embryos, deficiencies in cranial nerves that normally innervate oral structures, particularly the glossopharyngeal nerve (IX), were observed. These studies indicate that Sall3 is required for the development of nerves that are derived from the hindbrain and for the formation of adjacent branchial arch derivatives.