Polymerase theta is a synthetic lethal target for killing Epstein-Barr virus lymphomas.

Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.

The role of pandemic planning in the management of COVID-19 in England from an infection prevention and control perspective: results of a national survey.

OBJECTIVES: This national survey aimed to explore how existing pandemic preparedness plans (PPP) accounted for the demands placed on infection prevention and control (IPC) services in acute and community settings in England during the first wave of the COVID-19 pandemic. STUDY DESIGN: This was a cross-sectional survey of IPC leaders working within National Health Service Trusts or clinical commissioning groups/integrated care systems in England. METHODS: The survey questions related to organisational COVID-19 preparedness pre-pandemic and the response provided during the first wave of the pandemic (January to July 2020). The survey ran from September to November 2021, and participation was voluntary. RESULTS: In total, 50 organisations responded. Seventy-one percent (n = 34/48) reported having a current PPP in December 2019, with 81% (n = 21/26) indicating their plan was updated within the previous 3 years. Around half of IPC teams were involved in previous testing of these plans via internal and multi-agency tabletop exercises. Successful aspects of pandemic planning were identified as command structures, clear channels of communication, COVID-19 testing, and patient pathways. Key deficiencies were lack of personal protective equipment, difficulties with fit testing, keeping up to date with guidance, and insufficient staffing. CONCLUSIONS: Pandemic plans need to consider the capability and capacity of IPC services to ensure they can contribute their critical knowledge and expertise to the pandemic response. This survey provides a detailed evaluation of how IPC services were impacted during the first wave of the pandemic and identifies key areas, which need to be included in future PPP to better manage the impact on IPC services.

Correction: Mitochondrial oxodicarboxylate carrier deficiency is associated with mitochondrial DNA depletion and spinal muscular atrophy-like disease.

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Changes in intracuff pressure of cuffed endotracheal tubes while positioning for adenotonsillectomy in children.

BACKGROUND: When using cuffed endotracheal tubes (cETTs), changes in head and neck position can lead to changes in intracuff pressure. AIM: The aim of this study was to assess the combined effect of neck extension, shoulder roll placement, and Crowe-Davis retractor use during adenotonsillectomy on the intracuff pressure of cETTs in children. METHODS: Patients <18 years of age undergoing adenotonsillectomy under general anesthesia following the placement of a cETT were included in the study. After inflation of the cuff to seal the trachea, using the leak test, baseline intracuff pressure was recorded and then continuously monitored. After neck extension, placement of a shoulder roll, insertion of the Crow-Davis retractor, suspension from a Mayo stand, and positioning for surgery, the intracuff pressure was recorded again. RESULTS: The study cohort included 84 patients, ranging in age from 0.9 to 17 years (5.7 ± 3.9 years). In 46 patients (54.8%), the intracuff pressure increased from baseline after positioning for adenotonsillectomy. In 12 of these patients (14.3%), the intracuff pressure was >30 cm H2O. The intracuff pressure decreased in 28 patients (33.3%), while no change was noted in 10 patients (11.9%). Overall, the general trend was an increase in intracuff pressure from 15.9 ± 7.8 cm H2O to 18.9 ± 11.6 cm H2O. CONCLUSION: Both increases and decreases in the intracuff pressure may occur following positioning of the pediatric patient for adenotonsillectomy. An increase in intracuff pressure may result in a higher risk of damage to the tracheal mucosa. A decrease in the intracuff pressure can result in an air leak resulting in inadequate ventilation, increased risk of aspiration, and even predispose to airway fire if oxygen-enriched gases are used. Continuous intracuff pressure monitoring or rechecking the intracuff pressure after positioning for adenotonsillectomy may be indicated.

A novel de novo STXBP1 mutation is associated with mitochondrial complex I deficiency and late-onset juvenile-onset parkinsonism.

Mutations in STXBP1 have recently been identified as a cause of infantile epileptic encephalopathy. The underlying mechanism of the disorder remains unclear and, recently, several case reports have described broad and progressive neurological phenotypes in addition to early-onset epilepsy. Herein, we describe a patient with early-onset epilepsy who subsequently developed a progressive neurological phenotype including parkinsonism in her early teens. A de novo mutation in STXBP1 (c.416C>T, p.(Pro139Leu)) was detected with exome sequencing together with profound impairment of complex I of the mitochondrial respiratory chain on muscle biopsy. These findings implicate a secondary impairment of mitochondrial function in the progressive nature of the disease phenotype.

E2-Loaded Microcapsules and Bone Marrow-Derived Mesenchymal Stem Cells with Injectable Scaffolds for Endometrial Regeneration Application.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been recognized as new candidates for the treatment of serious endometrial injuries. However, owing to the local microenvironment of damaged endometrium, transplantation of BMSCs yielded disappointing results. In this study, Pectin-Pluronic® F-127 hydrogel as scaffolds were fabricated to provide three-dimensional architecture for the attachment, growth, and migration of BMSCs. E2 was encapsulated into the W/O/W microspheres to construct pectin-based E2-loaded microcapsules (E2 MPs), which has the potential to serve as a long-term reliable source of E2 for endometrial regeneration. Then, the BMSCs/E2 MPs/scaffolds system was injected into the uterine cavity of mouse endometrial injury model for treatment. At 4 weeks after transplantation, the system increased proliferative abilities of uterine endometrial cells, facilitated microvasculature regeneration, and restored the ability of endometrium to receive an embryo, suggesting that the BMSCs/E2 MPs/scaffolds system is a promising treatment option for endometrial regeneration. Furthermore, the mechanism of E2 in promoting the repair of endometrial injury was also investigated. Exosomes are critical paracrine mediators that act as biochemical cues to direct stem cell differentiation. In this study, it was found that the expression of endometrial epithelial cell (EEC) markers was upregulated in BMSCs treated by exosomes secreted from endometrial stromal cells (ESCs-Exos). Exosomes derived from E2-stimulated ESCs further promoted the expression level of EECs markers in BMSCs, suggesting exosomes released from ESCs by E2 stimulation could enhance the differentiation efficiency of BMSCs. Therefore, exosomes derived from ESCs play paracrine roles in endometrial regeneration stimulated by E2 and provide optimal estrogenic response.

Higher protein diet for weight management in young overweight women: a 12-month randomized controlled trial.

UNLABELLED: Clinical research on weight management in young women is limited. This randomized controlled trial compared the efficacy of two iso-energetically restricted (5600 kJ) diets [higher protein (HP): 32% protein, 41% carbohydrate, 25% fat or higher carbohydrate (HC): 20, 58, 21%, respectively] in 71 (HP: n = 36; HC: n = 35) young healthy women (18-25 years; body mass index ≥ 27.5 kg/m2) for weight (kg; percent weight loss), body composition, metabolic and iron changes assessed at baseline, 6 and 12 months. DATA: mean (95% CI). In HP completers at 6 months, percent weight loss was higher [HP: 9.3 (5.6-13.1); HC: 5.1 (2.3-7.9)%; p = 0.06]; although, this did not reach statistical significance. Absolute weight [HP: 8.9 (5.3-12.5); HC: 4.6 (2.2-7.0) kg; p = 0.034] and fat loss [HP: 8.0 (4.4-11.5); HC: 3.4 (1.3-5.6) kg; p = 0.022] were significantly greater. No significant between-diet differences were observed at 12 months. Biochemistry remained within normal ranges with HP showing superior preservation of ferritin at 6 months [HP: 53 (40-66); HC: 46 (30-61) µg/l; p = 0.029]. Both diets supported clinically meaningful weight loss with HP tending to be more effective in the medium-term.

Identifying patients at increased risk of non-ventilator-associated pneumonia on admission to hospital: a pragmatic prognostic screening tool to trigger preventative action.

BACKGROUND: Non-ventilator healthcare-associated pneumonia (NV-HAP) is an important healthcare-associated infection. This study tested the feasibility of using routine admission data to identify those patients at high risk of NV-HAP who could benefit from targeted, preventive interventions. METHODS: Patients aged ≥64 years who developed NV-HAP five days or more after admission to elderly-care wards, were identified by retrospective case note review together with matched controls. Data on potential predictors of NV-HAP were captured from admission records. Multi-variate analysis was used to build a prognostic screening tool (PRHAPs); acceptability and feasibility of the tool was evaluated. RESULTS: A total of 382 cases/381 control patients were included in the analysis. Ten predictors were included in the final model; nine increased the risk of NV-HAP (OR between 1.68 and 2.42) and one (independent mobility) was protective (OR 0.48; 95% CI 0.30-0.75). The model correctly predicted 68% of the patients with and without NV-HAP; sensitivity 77%; specificity 61%. The PRHAPs tool risk score was 60% or more if two predictors were present and over 70% if three were present. An expert consensus group supported incorporating the PRHAPs tool into electronic logic systems as an efficient mechanism to identify patients at risk of NV-HAP and target preventative strategies. CONCLUSIONS: This prognostic screening (PRHAPs) tool, applied to data routinely collected when a patient is admitted to hospital, could enable staff to identify patients at greatest risk of NV-HAP, target scarce resources in implementing a prevention care bundle, and reduce the use of antimicrobial agents.

Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco.

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

Awakening and Breathing Coordination: A Mixed-Methods Analysis of Determinants of Implementation.

Rationale: Routine spontaneous awakening and breathing trial coordination (SAT/SBT) improves outcomes for mechanically ventilated patients, but adherence varies. Understanding barriers to and facilitators of consistent daily use of SAT/SBT (implementation determinants) can guide the development of implementation strategies to increase adherence to these evidence-based interventions. Objectives: We conducted an explanatory, sequential mixed-methods study to measure variation in the routine daily use of SAT/SBT and to identify implementation determinants that might explain variation in SAT/SBT use across 15 intensive care units (ICUs) in urban and rural locations within an integrated, community-based health system. Methods: We described the patient population and measured adherence to daily use of coordinated SAT/SBT from January to June 2021, selecting four sites with varied adherence levels for semistructured field interviews. We conducted key informant interviews with critical care nurses, respiratory therapists, and physicians/advanced practice clinicians (n = 55) from these four sites between October and December 2021 and performed content analysis to identify implementation determinants of SAT/SBT use. Results: The 15 sites had 1,901 ICU admissions receiving invasive mechanical ventilation (IMV) for ⩾24 hours during the measurement period. The mean IMV patient age was 58 years, and the median IMV duration was 5.3 days (interquartile range, 2.5-11.9). Coordinated SAT/SBT adherence (within 2 h) was estimated at 21% systemwide (site range, 9-68%). ICU clinicians were generally familiar with SAT/SBT but varied in their knowledge and beliefs about what constituted an evidence-based SAT/SBT. Clinicians reported that SAT/SBT coordination was difficult in the context of existing ICU workflows, and existing protocols did not explicitly define how coordination should be performed. The lack of an agreed-upon system-level measure for tracking daily use of SAT/SBT led to uncertainty regarding what constituted adherence. The effects of the COVID-19 pandemic increased clinician workloads, impacting performance. Conclusions: Coordinated SAT/SBT adherence varied substantially across 15 ICUs within an integrated, community-based health system. Implementation strategies that address barriers identified by this study, including knowledge deficits, challenges regarding workflow coordination, and the lack of performance measurement, should be tested in future hybrid implementation-effectiveness trials to increase adherence to daily use of coordinated SAT/SBT and minimize harm related to the prolonged use of mechanical ventilation and sedation.

Mechanical evaluation of a tapered thread-run-out half-pin designed for external skeletal fixation in small animals.

An in vitro mechanical study was performed to compare the stiffness, maximum load, and cyclic load-to-failure of a new external fixation half-pin design utilizing a tapered thread-run-out (TRO) feature with currently available positive profile thread (PP) half-pins. Five different sizes of each of the two pin types were evaluated. Under static loading, TRO pins were significantly stiffer and had a higher maximum load compared to the similar sized PP pins (p <0.0001 for all comparisons). In cyclic fatigue testing, TRO pins lasted 2.3- to 4.9-fold more cycles than PP pins of similar size (p <0.0001 for all comparisons). The increased pin stiffness and improved cyclic lifespan provided by TRO pins may be especially valuable in the stabilization of biologically and mechanically challenging fracture cases where healing is prolonged.

The leptin to adiponectin ratio (LAR) is reduced by sleeve gastrectomy in adults with severe obesity: a prospective cohort study.

Bariatric surgery is known to reduce leptin and increase adiponectin levels, but the influence of sleeve gastrectomy on the leptin: adiponectin ratio (LAR), a measure of insulin sensitivity and cardiovascular risk, has not previously been described. We sought to determine the influence of sleeve gastrectomy on LAR in adults with severe obesity.In a single centre prospective cohort study of adults undergoing laparoscopic sleeve gastrectomy over a four-month period in our unit, we measured LAR preoperatively and 12 months after surgery. Of 22 patients undergoing sleeve gastrectomy, 17 (12 females, 12 with type 2 diabetes) had follow-up LAR measured at 12.1 ± 1 months. Mean body weight decreased from 130.6 ± 30.8 kg to 97.6 ± 21.6 kg, body mass index (BMI) from 46.9 ± 7.8 to 35.3 ± 7.2 kg m-2 and excess body weight from 87.5 ± 31.3 to 41.3 ± 28.8% (all p < 0.001). The reduction in leptin from 40.7 ± 24.9 to 30.9 ± 30.5 ng/ml was not significant (p = 0.11), but adiponectin increased from 4.49 ± 1.6 to 8.93 ± 6.36 µg/ml (p = 0.005) and LAR decreased from 8.89 ± 4.8 to 5.26 ± 6.52 ng/µg (p = 0.001), equivalent to a 70.9% increase in insulin sensitivity. The correlation with the amount of weight lost was stronger for LAR than it was for leptin or adiponectin alone. In this single-centre, interventional prospective cohort, patients undergoing laparoscopic sleeve gastrectomy had a substantial reduction in their LAR after 12 months which was proportional to the amount of weight lost. This may indicate an improvement in insulin sensitivity and a reduction in cardiovascular risk.

The factors that induce or overcome freezing of gait in Parkinson's disease.

Freezing of gait (FoG), a transient halt in walking, is a major mobility problem for patients with Parkinson's disease (PD). This study examined the factors that induce FoG, and identified the cues and strategies that help overcome it through a postal survey of 130 PD patients. 72% reported FoG. The factors that commonly induced FoG were turning, fatigue, confined spaces and stressful situations, in addition to emotional factors. FoG was also ameliorated by various attentional and external cueing strategies. The concept of paradoxical kinesis, the potential neural substrates of such external cueing effects, and their importance for rehabilitation in PD are discussed.

The use of ultrasound to assess fetal growth in a guinea pig model of fetal growth restriction.

Fetal growth restriction (FGR) is a common and potentially severe pregnancy complication. Currently there is no treatment available. The guinea pig is an attractive model of human pregnancy as placentation is morphologically very similar between the species. Nutrient restriction of the dam creates growth-restricted fetuses while leaving an intact uteroplacental circulation, vital for evaluating novel therapies for FGR. Growth-restricted fetuses were generated by feeding Dunkin Hartley guinea pig dams 70% of ad libitum intake from four weeks before and throughout pregnancy. The effect of maternal nutrient restriction (MNR) on dams and fetuses was carefully monitored, and ultrasound measurements of pups collected. There was no difference in maternal weight at conception, however by five weeks post conception MNR dams were significantly lighter ( P < 0.05). MNR resulted in significantly smaller pup size from 0.6-0.66 gestation. Ultrasound is a powerful non-invasive tool for assessing the effect of therapeutic interventions on fetal growth, allowing longitudinal measurement of fetuses. This model and method yield data applicable to the human condition without the need for animal sacrifice and will be useful in the translation of therapies for FGR into the clinic.

Novel HSPB1 mutation causes both motor neuronopathy and distal myopathy.

OBJECTIVE: To identify the cause of isolated distal weakness in a family with both neuropathic and myopathic features on EMG and muscle histology. METHODS: Case study with exome sequencing in 2 affected individuals, bioinformatic prioritization of genetic variants, and segregation analysis of the likely causal mutation. Functional studies included Western blot analysis of the candidate protein before and after heat shock treatment of primary skin fibroblasts. RESULTS: A novel HSPB1 variant (c.387C>G, p.Asp129Glu) segregated with the phenotype and was predicted to alter the conserved α-crystallin domain common to small heat shock proteins. At baseline, there was no difference in HSPB1 protein levels nor its binding partner αB-crystallin. Heat shock treatment increased HSPB1 protein levels in both patient-derived and control fibroblasts, but the associated increase in αB-crystallin expression was greater in patient-derived than control fibroblasts. CONCLUSIONS: The HSPB1 variant (c.387C>G, p.Asp129Glu) is the likely cause of distal neuromyopathy in this pedigree with pathogenic effects mediated through binding to its partner heat shock protein αB-crystallin. Mutations in HSBP1 classically cause a motor axonopathy, but this family shows that the distal weakness can be both myopathic and neuropathic. The traditional clinical classification of distal weakness into "myopathic" or "neuropathic" forms may be misleading in some instances, and future treatments need to address the pathology in both tissues.

Exome sequencing in dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is the second most common form of degenerative dementia. Siblings of affected individuals are at greater risk of developing DLB, but little is known about the underlying genetic basis of the disease. We set out to determine whether mutations in known highly penetrant neurodegenerative disease genes are found in patients with DLB. Whole-exome sequencing was performed on 91 neuropathologically confirmed cases of DLB, supplemented by independent APOE genotyping. Genetic variants were classified using established criteria, and additional neuropathological examination was performed for putative mutation carriers. Likely pathogenic variants previously described as causing monogenic forms of neurodegenerative disease were found in 4.4% of patients with DLB. The APOE ɛ4 allele increased the risk of disease (P=0.0001), conferred a shorter disease duration (P=0.043) and earlier age of death (P=0.0015). In conclusion, although known pathogenic mutations in neurodegenerative disease genes are uncommon in DLB, known genetic risk factors are present in >60% of cases. APOE ɛ4 not only modifies disease risk, but also modulates the rate of disease progression. The reduced penetrance of reported pathogenic alleles explains the lack of a family history in most patients, and the presence of variants previously described as causing frontotemporal dementia suggests a mechanistic overlap between DLB and other neurodegenerative diseases.

Primary Caregiver Perception of Pain Control following Pediatric Adenotonsillectomy: A Cross-Sectional Survey.

OBJECTIVES: To (1) review pain medications prescribed following pediatric adenotonsillectomy (T&A), (2) identify pain medications reported to be helpful, and (3) compare parent-reported outcomes among various combinations of pain medications. STUDY DESIGN: Case series with planned data collection. SETTING: Multihospital network. SUBJECTS AND METHODS: The primary caregivers of children aged 1 to 18 years who underwent isolated T&A from June to December 2014 were contacted 14 to 21 days after surgery. Data collected included pain medications prescribed, medications most helpful in controlling pain, and duration that pain medication was required. Parents rated their children's pain on postoperative days 2, 3, 7, and 14 and reported the time to resumption of normal diet/activity, as well as any hospital return visits. RESULTS: The study cohort included 672 subjects of 1444 potential participants (46% response rate). The mean age of the patients was 7.9 ± 3.6 years. Narcotics were prescribed in 71.9%, and 70.4% were told to use ibuprofen. Children who took ibuprofen alone were significantly younger (P < .001). Pain was significantly less on postoperative days 2 and 3 in the ibuprofen-only group as compared with the groups taking narcotics only (P < .001) and ibuprofen with narcotics (P = .002). Those taking ibuprofen alone returned to normal activity (P < .001) and diet (P = .026) sooner than those taking ibuprofen with narcotics. No difference was seen in pain control on subgroup analysis comparing oxycodone and hydrocodone. CONCLUSIONS: For pediatric T&A, significant variation exists in the management of postoperative pain. Parents of children given ibuprofen reported less pain than those given narcotics with and without ibuprofen. Further studies are needed to identify the optimal pain regimen for children after T&A.

Frequency of rare recessive mutations in unexplained late onset cerebellar ataxia.

Sporadic late onset cerebellar ataxia is a well-described clinical presentation with a broad differential diagnosis that adult neurologists should be familiar with. However, despite extensive clinical investigations, an acquired cause is identified in only a minority of cases. Thereafter, an underlying genetic basis is often considered, even in those without a family history. Here we apply whole exome sequencing to a cohort of 12 patients with late onset cerebellar ataxia. We show that 33% of 'idiopathic' cases harbor compound heterozygous mutations in known ataxia genes, including genes not included on multi-gene panels, or primarily associated with an ataxic presentation.