Central tolerance shapes the neutralizing B cell repertoire against a persisting virus in its natural host.

Viral mimicry of host cell structures has been postulated to curtail the B cell receptor (BCR) repertoire against persisting viruses through tolerance mechanisms. This concept awaits, however, experimental testing in a setting of natural virus-host relationship. We engineered mouse models expressing a monoclonal BCR specific for the envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV), a naturally persisting mouse pathogen. When the heavy chain of the LCMV-neutralizing antibody KL25 was paired with its unmutated ancestor light chain, most B cells underwent receptor editing, a behavior reminiscent of autoreactive clones. In contrast, monoclonal B cells expressing the same heavy chain in conjunction with the hypermutated KL25 light chain did not undergo receptor editing but exhibited low levels of surface IgM, suggesting that light chain hypermutation had lessened KL25 autoreactivity. Upon viral challenge, these IgMlow cells were not anergic but up-regulated IgM, participated in germinal center reactions, produced antiviral antibodies, and underwent immunoglobulin class switch as well as further affinity maturation. These studies on a persisting virus in its natural host species suggest that central tolerance mechanisms prune the protective antiviral B cell repertoire.

TT2023 meeting report on the 18th Transgenic Technology meeting in Houston, United States.

The 18th Transgenic Technology Meeting, held in Houston, Texas from November 12-15, 2023, was a vibrant international forum. It brought together nearly 400 delegates to discuss advances in transgenic technologies and the science these technologies support. Among them were 329 in-person and 70 remote delegates, representing 26 countries from 5 continents. The event, hosted by the International Society for Transgenic Technologies (ISTT), was set against the backdrop of the Hyatt Regency's panoramic views, reflecting the innovative spirit of the conference. A notable precursor to the main conference was the Allele Design Pre-conference Workshop, which fostered in-depth discussions on state-of-the-art methodologies. The main conference encompassed ten sessions, delving into diverse topics from Precision Animal Models of Human Disease to the use of transgenic animals in Space Biology. Eighty posters provided for a lively exchange of ideas, while the ISTT Prize and other awards highlighted the event's commitment to excellence. Beyond the conference halls, attendees had the opportunity to venture into Houston's Museum District, home to 19 museums in the downtown area, or indulge in unique dining experiences.

Biofilm Production and Antimicrobial Resistance of Clinical and Food Isolates of Pseudomonas spp.

Due to its ubiquity, ability to form biofilms, and acquire resistance mechanisms, Pseudomonas spp. become one of the major challenge for healthcare settings and food industry. The aims of this study were to assess the biofilm production of Pseudomonas spp. recovered from clinical and food specimens and to evaluate their antimicrobial resistance. A total of 108 isolates of Pseudomonas spp. were included in the study, 48 being clinical isolates recovered from patients admitted to four tertiary care hospitals throughout Serbia and 60 were isolated from the bulk tank milk samples and meat carcasses. Biofilm production was analyzed by microtiter plate assay. Antimicrobial susceptibility was evaluated by disk diffusion method according to the CLSI guidelines, while class A and B ß-lactamases encoding genes were screened by PCR. A total of 98 (90.7%) strains were biofilm producers (moderate producer: 68, 69.4%; strong producer: 8, 8.2%). Although a slightly higher percentage of clinical isolates were biofilm producers (91.7%) compared to food isolates (90%), statistical significance was not observed (P > 0.05). The proportion of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates was significantly higher among clinical (42%) isolates compared to food (1.7%) Pseudomonads (P < 0.05). The blaPER and blaNDM genes were found in ESBL (seven isolates) and MBL (two isolates) production, respectively. In the present study, we confirmed that biofilm formation was highly present in both clinical and food Pseudomonas spp. irrespective of the prior existence of resistance genes. Additionally, clinical settings pose a major reservoir of MDR Pseudomonas spp. and especially CRPA isolates.

ARTD2 activity is stimulated by RNA.

ADP-ribosyltransferases (ARTs) are important enzymes that regulate the genotoxic stress response and the maintenance of genome integrity. ARTD1 (PARP1) and ARTD2 (PARP2) are homologous proteins that modify themselves and target proteins by the addition of mono- and poly-ADP-ribose (PAR) moieties. Both enzymes have been described to be involved in the genotoxic stress response. Here, we characterize cellular PAR formation on hydrogen peroxide (H2O2) or N-methyl-N'-methyl-nitro-N-nitrosoguanidine (MNNG) stress, in combination with application of the RNA polymerase I inhibitor Actinomycin D (ActD), known to cause accumulation of short RNA polymerase I-dependent rRNA transcripts. Intriguingly, co-treatment with ActD substantially increased H2O2- or MNNG-induced PAR formation. In cells, this enhancement was predominantly mediated by ARTD2 and not ARTD1. In vitro experiments confirmed that ARTD2 is strongly activated by RNA and that the N-terminal SAP domain is important for the binding to RNA. Thus, our findings identify a new activator of ARTD2-dependent ADP-ribosylation, which has important implications for the future analysis of the biological role of ARTD2 in the nucleus.

Misdiagnosis of amyotrophic lateral sclerosis in clinical practice in Europe and the USA: a patient chart review and physician survey.

OBJECTIVE: Delays in amyotrophic lateral sclerosis (ALS) diagnosis can result in compromised disease management and unnecessary costs. We examined the extent of ALS misdiagnosis in the US and Europe. METHODS: Data were collected via the Adelphi ALS Disease Specific Programme™, a cross-sectional survey of physicians and a medical chart review of their consulting patients with ALS in France, Germany, Italy, Spain, the UK (EU5), and the US. Between July 2020 and March 2021, eligible physicians (primary speciality neurology, active involvement in managing patients with ALS) abstracted data from patients (≥18 years old) with confirmed ALS. RESULTS: Overall, 138 physicians completed the survey (EU5 107, US 31), with data reviewed from 795 patient medical charts (EU5 568, US 227); 278 (35.0%) patients (EU5 183 [32.2%], US 95 [41.9%]) had received ≥1 initial misdiagnosis based on symptoms later attributed to ALS. Mean (SD) time from symptom onset to first healthcare professional consultation was 3.8 (5.2) months (EU5 4.3 [4.8] months, US 2.6 [5.8] months). Mean (SD) time from symptom onset to ALS diagnosis was 8.2 (12.5) months (EU5 9.6 [14.0] months, US 5.0 [6.8] months) and increased to 10.4 (17.9) for patients with a misdiagnosis (compared with 6.9 [7.2] for patients with no misdiagnosis). Physician-identified barriers to timely ALS diagnosis included the similarity of symptoms to other conditions and delayed referral to neurologists. CONCLUSIONS: Misdiagnosis of ALS is frequent, with a protracted diagnostic pathway. Targeted education of patients and physicians about signs and symptoms and benefits of prompt referral to multidisciplinary care are needed.

Improvement of fatigue in generalised myasthenia gravis with zilucoplan.

BACKGROUND: Fatigue is a debilitating symptom of myasthenia gravis (MG). The impact of fatigue on MG can be assessed by Quality of Life in Neurological Disorders (Neuro-QoL) Short Form Fatigue scale. Transformation of raw Neuro-QoL fatigue scores to T-scores is a known approach for facilitating clinical interpretation of clinically meaningful and fatigue severity thresholds. METHODS: In the Phase 3, double-blind, placebo-controlled RAISE study (NCT04115293), adults with acetylcholine receptor autoantibody-positive generalised MG (MG Foundation of America Disease Class II-IV) were randomised 1:1 to daily subcutaneous zilucoplan 0.3 mg/kg or placebo for 12 weeks. Patients completing RAISE could opt to receive zilucoplan 0.3 mg/kg in an ongoing, open-label extension study, RAISE-XT (NCT04225871). In this post-hoc analysis, we evaluated the long-term effect of zilucoplan on fatigue in RAISE patients who entered RAISE-XT. We report change in Neuro-QoL Short Form Fatigue T-scores and fatigue severity levels from RAISE baseline to Week 60. RESULTS: Mean Neuro-QoL Short Form Fatigue T-scores improved from baseline to Week 12 in the zilucoplan group (n = 86) with a clinically meaningful difference versus placebo (n = 88; least squares mean difference: - 3.61 (nominal p-value = 0.0060]), and these improvements continued further to Week 60. At Week 12, more patients on zilucoplan (n = 34, 47.2%) experienced improvements in ≥ 1 fatigue severity level from baseline versus placebo (n = 23, 28.4%; p = 0.017). At Week 60, most (n = 55, 65.5%) patients had mild fatigue or none. CONCLUSION: Treatment with zilucoplan demonstrated statistical and clinically meaningful improvements in fatigue scores and severity versus placebo during RAISE, which were sustained to Week 60 in RAISE-XT.

Pulmonary renal syndrome in a child with coexistence of anti-neutrophil cytoplasmic antibodies and anti-glomerular basement membrane disease: case report and literature review.

BACKGROUND: Pulmonary renal syndrome (PRS), denoting the presence of diffuse alveolar hemorrhage and glomerulonephritis as manifestations of systemic autoimmune disease, is very rare in childhood. The coexistence of circulating anti-neutrophil cytoplasmic antibody (ANCA) and anti-glomerular basement membrane (GBM) disease in children affected by this syndrome is exceptional, with unfavorable outcome in five out of seven patients reported to date. We describe a child with PRS associated with both circulating anti-myeloperoxidase (anti-MPO) ANCA and anti-GBM disease on renal biopsy who was successfully treated with immunosuppressive therapy. CASE PRESENTATION: A 10-year old girl presented with fever, fatigue, malaise, and pallor followed by hemoptysis and severe anemia. Diffuse alveolar hemorrhage was revealed on fiberoptic bronchoscopy. Renal findings consisted of microscopic hematuria, moderate proteinuria, and anti-GBM disease on renal biopsy. ANCA with anti-MPO specificity were present whereas anti-GBM antibodies were on borderline for positivity. Methyl-prednisolone pulses followed by prednisone led to cessation of hemoptysis, marked improvement of lung fuction, and normal finding on chest x-ray within 10 days. An immunosuppressive regimen was then given consisting of prednisone daily for 4 weeks with subsequent taper on alternate day, i.v. cyclophosphamide pulses monthly for 6 doses, followed by mycophenolate mofetil that resulted in normal lung function tests, hemoglobin concentration, and anti-MPO level within four subsequent weeks. During 10-months of follow-up she remained well, her blood pressure and renal function tests were normal, and proteinuria and hematuria gradually resolved. CONCLUSION: We report a child with an exceptionally rare coexistence of circulating ANCA and anti-GBM disease manifesting as PRS in whom renal disease was not the prominent part of clinical presentation, contrary to other reported pediatric patients. A review of literature on disease with double positive antibodies is also presented. Evaluation of a patient with PRS should include testing for presence of different antibodies. An early diagnosis and rapid institution of aggressive immunosuppressive therapy can induce remission and preserve renal function. Renal prognosis depends on the extent of kidney injury at diagnosis and appropriate treatment.

Mapping analysis to predict SF-6D utilities from health outcomes in people with focal epilepsy.

BACKGROUND: Focal-onset seizures (FOS) are commonly experienced by people with epilepsy and have a significant impact on quality of life (QoL). This study aimed to develop a mapping algorithm to predict SF-6D values in adults with FOS for use in economic evaluations of a new treatment, cenobamate. METHODS: An online survey, including questions on disease history, SF-36, and an epilepsy-specific measure (QOLIE-31-P) was administered to people with FOS in the UK, France, Italy, Germany, and Spain. A range of regression models were fitted to SF-6D scores including direct and response mapping approaches. RESULTS: 361 individuals were included in the analysis. In the previous 28 days, the mean number of FOS experienced was 3, (range 0-43) and the mean longest period of consecutive days without experiencing a seizure was 14 days (range 1-28 days or more). Mean responses on all SF-36 dimensions were lower than general population norms. Mean SF-6D and QOLIE-31-P scores were 0.584 and 45.72, respectively. The best performing model was the ordinary least squares (OLS), with root mean squared error and mean absolute error values of 0.0977 and 0.0742, respectively. Explanatory variables which best predicted SF-6D included seizure frequency, severity, freedom, and age. CONCLUSION: People with uncontrolled FOS have poor QoL. The mapping algorithm enables the prediction of SF-6D values from clinical outcomes in people with FOS. It can be applied to outcome data from clinical trials to facilitate cost-utility analysis.

Epidemiology and economic burden of amyotrophic lateral sclerosis in the United States: a literature review.

Objective: This review sought to gain a comprehensive, up-to-date understanding of the epidemiology and cost and healthcare resource use (HCRU) burden of amyotrophic lateral sclerosis (ALS) in the US, at a patient and national level. Methods: A targeted literature review (TLR) to identify epidemiological evidence (prevalence, incidence, mortality, survival), and systematic literature review (SLR) to identify cost and HCRU data published since January 2016, were performed. MEDLINE databases and Embase searches were conducted in January 2021. Key congresses (2019-2020) and bibliographies of relevant SLRs were hand-searched. Two high-quality SLRs were reviewed for additional cost data published between January 2001-2015. Registry and database studies were prioritized for epidemiological evidence. To allow comparison between studies in this publication, only evidence from the US was considered, with costs inflated to the 2020/2021 cost-year and converted to US dollars. Results: Eight studies from the epidemiology TLR, and eighteen from the cost and HCRU SLR, were extracted. Reported ALS incidence in the US was ∼1.5 per 100,000 person-years, and point prevalence ranged from 3.84-5.56 per 100,000 population. Total US national costs spanned ∼$212 million-∼$1.4 billion USD/year, and variably consisted of direct costs associated with HCRU and indirect costs. Conclusions: The national cost of ∼$1.02 billion USD/year (estimated using a prevalence of 16,055 cases) best aligns with prevalence estimates found in the TLR (equating to ∼13,000-18,000 cases). However, large-scale, population-based studies are necessary to precisely assess US epidemiology of ALS and capture all costs needed to inform cost-effectiveness models and resource planning.

Bilateral Emphysematous Pyelonephritis Associated with COVID Pneumonia: A Case Report.

BACKGROUND 0ptviral pneumonia and bilateral emphysematous pyelonephritis create a rapid acute respiratory distress syndrome. CASE REPORT A 59-year-old diabetic man with altered awareness was admitted as an emergency due to fever, shivering, and pain in the lap. Based on the accurate diagnosis, we concluded that the patient had bilateral emphysematous pyelonephritis, as well as inflammatory changes in the lung parenchyma caused by coronavirus infection (SARS-CoV-2). Active therapy - nephrectomy - was ruled out due to the late detection of the gas collection in the kidneys, as well as the general condition caused by respiratory symptoms. With symptomatic, supportive, and antimicrobial therapy, such as percutaneous renal drainage, renal abnormalities improved. Unfortunately, the virus-induced parenchymal inflammation progressed and proved fatal. The inflammatory process in the urothelial cell is most likely where the linkage and potentiation of COVID-19 infection and emphysematous pyelonephritis begins. Local inflammation that obstructs the movement of the generated gas is one of the hypothesized processes of emphysematous pyelonephritis. The renal and urothelial tubular cells contain the angiotensin-converting enzyme II (ACE2) receptor, which is used by the SARS-CoV-2 virus to enter human cells and may be a risk factor for simultaneous and direct viral injury to urinary tract cells. Sepsis was most likely caused by viral pneumonia, based on the resolution of changes in the kidneys. CONCLUSIONS The combination of EPN and COVID-19 is difficult to treat. Despite multidisciplinary treatment, it has been linked to a worse prognosis and fatal outcome.

Quality of life study for caregivers of people with uncontrolled focal-onset seizures.

AIM: The aim of this study was to capture and measure the impact of caregiving for an adult with uncontrolled drug-resistant focal-onset seizures (FOS) on the caregivers' quality of life (QoL), and to quantify the costs of productivity losses associated with providing informal care in this patient population. METHODS: An online survey, which included the EQ-5D-5L, CarerQol-7D and the Work Productivity and Activity Impairment: Specific-Health Problem (WPAI:SHP) questionnaires, was administered to caregivers of individuals with uncontrolled drug-resistant FOS in the United Kingdom (UK), France, Spain, Germany, Italy, and Sweden. RESULTS: The study included 345 caregivers. Most were males, aged between 25 and 34 years old whose caring responsibilities took between 15 and 24 h per week. The caregivers' mean EQ-5D-5L score was 0.6, with 95% confidence intervals (CI) of [0.58, 0.63], whilst the mean CarerQol-7D score was 72.61 [70.46, 74.76]. Caregivers' mental health was the most substantially affected aspect of their QoL. In addition, most caregivers reported deriving some or a lot of fulfilment out of their caregiving tasks. The WPAI:SHP showed that the mean percentage of work impairment due to caregiving responsibilities was 63%, [59.75, 66.26]. The mean annualised costs of productivity losses per caregiver were estimated at €14,872 [€11,908; €17,888]. LIMITATIONS: One limitation consisted in the use of an online survey instead of a face-to-face interview. However, the medical terms were clearly explained, and examples were provided to help participants to give accurate responses. Another limitation was that the respondents self-reported as caregivers. Efforts were made to mitigate this weakness by using screener questions. CONCLUSION: This study found that providing informal care for people with uncontrolled drug-resistant FOS had a negative impact on caregivers' QoL, with mental health being affected the most. However, caregivers found their role fulfilling and had support with their caring tasks.

Triple A syndrome: 32 years experience of a single centre (1977-2008).

Triple A syndrome is an autosomal recessive disorder characterized by alacrima, achalasia, ACTH-resistant adrenal insufficiency, autonomic dysfunction, and neurodegeneration. Mutations in the AAAS gene on chromosome 12q13 encoding the nuclear pore protein ALADIN have been reported in these patients. Over the period 1977-2008 we evaluated ten subjects with the clinical diagnosis of triple A syndrome. Molecular analysis was performed in seven patients and revealed that all except one are compound heterozygotes for two mutations in the AAAS gene. Two novel mutations were detected: c.123+2T>C resulted in splice defect while c.1261_1262insG mutation resulted in a truncated protein (p.V421fs), which most probably is not functional. Genotype-phenotype correlation could not be established. In all our patients, except one sibling of previously diagnosed brother and sister, genetic analysis was performed when at least two symptoms were present, usually alacrima and achalasia. Based on our experience, we recommend that in case of the presence of alacrima and at least one more symptom of triple A syndrome, adrenal function testing and molecular analysis should be performed. In all children with mutation in AAAS gene, regular follow up of adrenal function is necessary to avoid adrenal crisis and start substitution therapy as soon as adrenal insufficiency is noted.

In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells.

The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision. The preference of mammalian cells to repair DSBs by NHEJ rather than HDR, however, limits the potential of CRISPR-Cas9 for applications where precise editing is needed. To enhance the efficiency of DSB repair by HDR from donor templates, we recently engineered a CRISPR-Cas9 system where the template DNA is bound to the Cas9 enzyme. In short, single-stranded oligonucleotides were labeled with O6-benzylguanine (BG), and covalently linked to a Cas9-SNAP-tag fusion protein to form a ribonucleoprotein-DNA (RNPD) complex consisting of the Cas9 nuclease, the sgRNA, and the repair template. Here, we provide a detailed protocol how to generate O6-benzylguanine (BG)-linked DNA repair templates, produce recombinant Cas9-SNAP-tag fusion proteins, in vitro transcribe single guide RNAs, and transfect RNPDs into various mammalian cells.

Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair.

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.

Advances in therapeutic CRISPR/Cas9 genome editing.

Targeted nucleases are widely used as tools for genome editing. Two years ago the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease was used for the first time, and since then has largely revolutionized the field. The tremendous success of the CRISPR/Cas9 genome editing tool is powered by the ease design principle of the guide RNA that targets Cas9 to the desired DNA locus, and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks. Several studies recently used CRISPR/Cas9 to successfully modulate disease-causing alleles in vivo in animal models and ex vivo in somatic and induced pluripotent stem cells, raising hope for therapeutic genome editing in the clinics. In this review, we will summarize and discuss such preclinical CRISPR/Cas9 gene therapy reports.

Stenting versus non-stenting following uncomplicated ureteroscopic lithotripsy: Comparsion and evaluation of symptoms.

Background/Aim: Currently, ureterorenoscopic (URS) stone fragmentation and removal is the treatment of choice for managing ureteral stones, especially mid and distal ones and is advocated as initial management of ureteric stones. The aim of this work was to evaluate the symptoms, necessity, potential benefits and adverse effects of ureteral stent placement after uncomplicated ureteroscopic lithotripsy. Methods: This retrospective-prospective study evaluated a total of 125 patients who had underwent ureteroscopic lithotripsy (URSL). The patients were divided into two groups: stented (59 patients) and unstented (controls, 66 patients). The outcomes measured and compared between the two groups included: stone free rate, postoperative patient pain validated by scale, lower urinary tract symptoms (LUTS), the need for unplanned hospital care, stent related complications, and functional recovery in the form return to normal physical activities. Results: A successful outcome, defined as being stonefree after 12 weeks, was achieved in all 125 (100%) patients. The stone-free rate showed no significant differences between the two groups. LUTS was frequent complaint in the stented group, with statistically significant difference in the domain of frequency/urgency (p = 0.0314). There was a statistically significant difference between the groups in the mean operative time and mean hospitalization time, mean pain visual analog scale (VAS) score and in the use of nonnarcotic analgesic. On the day of the surgery and until postoperative day 3 (POD 3) and postoperative day 5 (POD 5), the pain score was much higher among stented patients than among the controls (p = 0.0001) and non-narcotic analgesic use (p = 0.001) was frequently required in the stented group. Conclusion: Routine placement of ureteral stent after URSL is not mandatory and may be associated with stent side effects. Uncomplicated URSL is safe without stent placement after the treatment.

Management of calculus anuria using ureteroscopic lithotripsy as a first line treatment: its efficacy and safety.

PURPOSE: To present our experience with emergency ureteroscopic lithotripsy (URSL) for ureteral calculi associated with acute kidney injury (AKI). MATERIALS AND METHODS: We retrospectively evaluated the 61 patients consisted of 90 ureteral units (UU), who underwent URSL. The cause of anuria was bilateral calculus obstructions in 29 cases, and unilateral calculus obstruction with, absent, nephrectomized contralateral kidney in 32 cases. In the case of bilateral synchronous ureteric calculi same-session bilateral ureteroscopy (SBBU) was done. The duration of anuria varied between 12 to 72 hours. At the end of the procedure, ureteral stent was systematically left in place in all patients. Surgery was performed 6-12 hours after admission to hospital. Patients were followed at least 1 month postoperatively. RESULTS: The stone free rates (SFR) were determined as baseline, on the first post-operative day, and as overall on the 30 days after procedure. The greatest success was achieved in the distal localization of stones up to 10 mm (93%). Renal function returned in 51 (83.6%) patients within 7 days. In 18 (29.5%) patients [18 (20%) UU] we performed second procedure as extracorporeal shockwave lithotripsy in 16.7% and open surgery in 2.2%. In 43 (70.5%) patients URSL was a successful therapeutic approach in dealing with pain, obstruction and calculus. CONCLUSION: Calculus anuria is a medical emergency that requires rapid diagnosis and prompt treatment for the purpose of decompression. URSL is the proper method of choice for selected patients and can be performed safely and has high success rates with minimal morbidity.

lncRNA maturation to initiate heterochromatin formation in the nucleolus is required for exit from pluripotency in ESCs.

The open chromatin of embryonic stem cells (ESCs) condenses into repressive heterochromatin as cells exit the pluripotent state. How the 3D genome organization is orchestrated and implicated in pluripotency and lineage specification is not understood. Here, we find that maturation of the long noncoding RNA (lncRNA) pRNA is required for establishment of heterochromatin at ribosomal RNA genes, the genetic component of nucleoli, and this process is inactivated in pluripotent ESCs. By using mature pRNA to tether heterochromatin at nucleoli of ESCs, we find that localized heterochromatin condensation of ribosomal RNA genes initiates establishment of highly condensed chromatin structures outside of the nucleolus. Moreover, we reveal that formation of such highly condensed, transcriptionally repressed heterochromatin promotes transcriptional activation of differentiation genes and loss of pluripotency. Our findings unravel the nucleolus as an active regulator of chromatin plasticity and pluripotency and challenge current views on heterochromatin regulation and function in ESCs.

Two seasons' experience with pandemic A H1N1 influenza infection in neonates.

There are only a few reports on influenza A H1N1 infection in neonates. In this paper, we present our additional experience on the clinical characteristics, epidemiology and treatment of influenza A H1N1 (2009) infection in 10 newborn infants (aged 9-24 days). Influenza A H1N1 infection was confirmed by real-time reverse transcription-polymerase chain reaction of the nasopharyngeal swab specimens. The majority of neonates presented with fever, respiratory symptoms and lethargy. The respiratory illness ranged from mild symptoms to severe pneumonia requiring mechanical ventilation. Antiviral treatment with oseltamivir was started in five patients (50%). One lethal outcome was observed, while nine patients (90%) had complete recovery. To our knowledge, this is the largest presented series of neonatal cases with different clinical symptoms. We discuss the necessity of initiation of oseltamivir in infants with different clinical features.