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This study investigated potential associations between ectropion and various chronic and systemic inflammatory diseases affecting the eyelids and ocular surface. This retrospective case-control study, spanning 2001-2022, used electronic medical records from Clalit Health Services (CHS) in Israel. Patients diagnosed with involutional ectropion served as the case group, while two control groups were used for comparison: one comprising patients diagnosed with senile cataract and the other with various ophthalmic diseases. Matching for the control groups was performed at a 1:3 ratio based on year of birth, sex, and ethnicity. A total of 17,292 patients with involutional ectropion and 51,876 matched controls in each group were included. The average age of the patients with involutional ectropion was 78 ± 10 years and 58% were men. Significant associations were found between involutional ectropion and several local inflammatory eyelid diseases: blepharitis (OR 4.66), chalazion (OR 2.94), hordeolum (OR 2.26), and dermatitis of the eyelid (OR 2.46). Ocular surface diseases associated with ectropion included chronic conjunctivitis (OR2.89 ) and pterygium (OR 2.48). Systemic diseases such as hypertension (OR 1.46), dyslipidemia (OR 1.45), and autoimmune rheumatic diseases (OR 1.43) were also associated with involutional ectropion. This study found that localized chronic inflammatory conditions affecting the periocular tissues and ocular surface are significantly associated with the development of involutional ectropion., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: This retrospective, case-control study was approved by the CHS Research Ethics Committee and the Soroka Medical Center Institutional Review Board (approval number: 0335-22-SOR). The study was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was waived as the data were obtained retrospectively and anonymized., (© 2025. The Author(s).)

Background: Children with Down syndrome (DS) often experience recurrent and prolonged hospitalizations from respiratory infections. While Azithromycin has been increasingly used for lower-respiratory tract infections (LRTIs) in children, its potential benefits for DS patients are unexplored. This study evaluates the effect of chronic azithromycin treatment on respiratory morbidity in children with DS., Methods: In this retrospective cohort study, we analyzed data from children with DS aged 0-6 years treated with Azithromycin for at least 6 weeks (10 mg/kg, thrice weekly). Respiratory morbidity indicators, such as primary care visits, medication consumption, emergency department visits, hospitalizations, and hospital length of stay (LOS), were assessed and compared six months before and after the Azithromycin treatment., Results: Twenty-three episodes of Azithromycin treatment (≥ 6 weeks) in eighteen children with DS (mean age of 2.3 years, 78% males) during 2016-2023 were included. A significant reduction in mean respiratory LOS was observed (13.6 vs. 4.7 days, p = 0.05) when comparing pre to post-Azithromycin treatment. Other secondary respiratory outcomes showed no significant differences., Conclusion: The significant reduction in respiratory LOS suggests the potential benefits of Azithromycin in children with DS, and emphasizes the need for larger clinical trials to determine optimal use and long-term effects in this vulnerable population., Competing Interests: Declarations. Ethics approval and consent to participate: The study was approved by the “institutional review board of Soroka University Medical Center”, in accordance with the Helsinki Declaration (approval No. 052 − 21 SOR). Informed consent was deemed unnecessary according to the retrospective nature of the study. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background : Prone positioning is a standard intervention in managing patients with severe acute respiratory distress syndrome (ARDS) and is known to improve oxygenation. However, its effects on other organs, particularly the kidneys, are less well understood. This study aimed to assess the association between prone positioning and the development of acute kidney injury (AKI), specifically in overweight and obese patients. Methods : A retrospective pre-post study was conducted on a cohort of 60 critically ill ARDS patients who were placed in the prone position during hospitalization. The development of AKI was assessed using the Acute Kidney Injury Network (AKIN) criteria, with AKI measured by both creatinine levels (AKIN Cr ) and urine output (AKIN UO ). Patients were divided into two groups based on body mass index (BMI): overweight/obese (BMI ≥ 25) and non-obese (BMI < 25). Data were collected before and after prone positioning. Results : In overweight/obese patients (n = 39, 57 cases), both the median AKIN Cr and AKIN UO scores increased significantly following prone positioning (from 0 to 1, median p < 0.01, and from 0 to 2, median p < 0.01, respectively). No statistically significant changes in AKIN scores were observed in non-obese patients nor were significant differences found in either group after repositioning to supine. Conclusions : Prone positioning is associated with an increased risk of acute kidney injury in overweight and obese ARDS patients. This may be due to the kidneys' susceptibility to intra-abdominal hypertension in these patients. Further research is needed to explore optimal proning strategies for overweight and obese populations.

Background: Non-arteritic anterior ischemic optic neuropathy (NAION) is a leading cause of acute optic nerve damage. While cardiovascular risk factors such as hypertension, diabetes mellitus, and obstructive sleep apnea are well-established, the association between NAION and states of hypoperfusion is underexplored. This study investigated this potential association., Methods: This retrospective case-control study analyzed all electronic medical records of Clalit Health Services' patients from 2001 to 2022. Patients diagnosed with NAION were matched in a 1:4 ratio by year of birth and sex, using propensity score analysis to adjust for various comorbidities. Events of hypoperfusion occurring in the month prior to the diagnosis of NAION were categorized into two physiological mechanisms: a decrease in SVR and a decrease in cardiac output due to cardiac dysfunction or diminished preload (attributed to hypovolemia). Conditional logistic regression was used to explore differences between the groups., Results: A total of 1,374 patients diagnosed with NAION and 5,496 matched controls were included in the study. We found a nearly 6.5-fold increase in the likelihood of NAION in association with events of hypoperfusion that occurred in the month period preceding the diagnosis of NAION (odds ratio [OR] 6.48; 95% confidence interval [CI]: 5.05-8.32). In particular, the group of patients with cardiac dysfunction (OR 6.47; 95% CI: 4.63-9.04) and the group with hypovolemia (OR 6.1; 95% CI: 4.08-9.13) emerged as having the most substantial risk factors. The group with decreased Systemic Vascular Resistance (SVR) (OR 4.64; 95% CI: 2.84-7.59) was also strongly related with NAION. Cerebrovascular accident emerged as an independent significant risk factor for NAION (OR 16.1; 95% CI: 10.8-24)., Conclusion: Hypoperfusion states are significant, independent risk factors for NAION., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gabbay et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and is associated with an increased risk of asthma development. Palivizumab, an RSV prophylactic, reduces RSV-related hospitalizations in high-risk infants, but its impact on long-term asthma outcomes remains unclear. This study compares asthma-related healthcare utilization in preschool children born prematurely between those who received Palivizumab (the Prophylaxis (+) group) and those who did not (the Prophylaxis (-) group)., Methods: This nationwide, population-based retrospective cohort study utilized data from Clalit Healthcare Services in Israel. The study included children born between 32 + 6 and 34 + 6 weeks of gestational age from 2011 to 2018. Descriptive analysis, univariate analysis, and multivariate logistic regression were performed to compare the Prophylaxis (+) and the Prophylaxis (-) groups., Results: In total, 4503 children were included, with 3287 in the Prophylaxis (+) group and 1216 in the Prophylaxis (-) group. Palivizumab administration was associated with reduced hospitalizations for RSV bronchiolitis (1.8% vs. 3.3%, p = 0.003). However, no significant differences were observed in multivariate analysis for long-term asthma outcomes, including asthma diagnosis (OR = 1.04, CI = 0.84-1.30, p = 0.7) or emergency department visits for asthma (OR = 0.79, CI = 0.54-1.17, p = 0.2). Similarly, Palivizumab administration was not associated with the purchase of short-acting beta-agonists (OR = 1.14, 95% CI 0.98-1.32, p = 0.084), inhaled corticosteroids (OR = 1.1, CI = 0.93-1.32, p = 0.3), or oral corticosteroids (OR = 1.09, CI = 0.94-1.26, p = 0.3)., Conclusions: While Palivizumab effectively reduces RSV acute bronchiolitis in preterm infants, it does not significantly impact long-term preschool asthma-related healthcare utilization.

Objectives: To evaluate the impact of the pneumococcal conjugate vaccines (PCVs) introduction on the orbital complications of acute rhino-sinusitis (OC-ARS)., Methods: A retrospective cohort study of all pediatric patients with OC-ARS during the period 2002-2019. Data included clinical, demographic, laboratory, and microbiology findings. Patients were divided into three groups: before PCV7 introduction (group 1), after PCV7 and before PCV13 (group 2), and after PCV13 (group 3)., Results: Of 265 enrolled patients, 117, 39, and 109 were assigned to groups 1, 2, and 3. During the study period, a significant decrease was recorded in the percentages of patients in Chandler classification severity category 1, with an increase in patients in category 3 (P = 0.011). The yearly incidence of OC-ARS decreased from 12.64 cases per 100,000 population in 2002 to 5.56 per 100,000 in 2008, and 2.99 per 100,000 in 2019 (P < 0.001). Patients aged 0-4 years showed a dramatic decrease from 29 cases per 100,000 population in 2002 to 4.27 per 100,000 in 2019 (P < 0.001). The pathogens retrieved from all cultures performed were Streptococcus pneumoniae (32.5%), non-typeable Haemophilus influenzae (27.5%), Streptococcus Species, (12.5%), and Staphylococcus aureus (20%), with no changes in distribution during the study periods. Surgery was performed in 28 (10.6%) patients., Conclusions: A significant decrease was seen in the overall incidence of OC-ARS, mainly attributable to the decrease in patients aged 0-4 years. An increase was recorded in the severity of the disease following PCVs introduction., Competing Interests: Declaration of competing interest No competing interests to declare., (Copyright © 2024 Taiwan Pediatric Association. Published by Elsevier B.V. All rights reserved.)

Summary: Total testosterone, which is peripherally converted to its biologically active form dihydrotestosterone (DHT), is the first-line hormone investigation in hyperandrogenic states and infertility in premenopausal women. Polycystic ovary syndrome (PCOS), the most common cause of hyperandrogenism and infertility in young women, is often associated with mild elevations of total testosterone. Whereas very high levels of total testosterone (>2-3 SD of normal reference), are most often associated with hyperandrogenic signs, menstrual irregularity, rapid onset of virilization, and demand a prompt investigation. Herein, we report a case of a 32-year-old woman who was referred to the endocrinology outpatient clinic due to secondary amenorrhea and extremely high testosterone levels without any virilization signs. We initially suspected pitfalls in the testosterone laboratory test. Total serum testosterone decreased after a diethyl-ether extraction procedure was done prior to the immunoassay, but testosterone levels were still elevated. An ovarian steroid-cell tumor (SCT) was then revealed, which was thereby resected. Twenty-four hours post surgery, the total testosterone level returned to normal, and a month later menstruation resumed. This case emphasizes that any discrepancy between laboratory tests and the clinical scenario deserves a rigorous evaluation to minimize misinterpretation and errors in diagnosis and therapeutic approach. Additionally, we describe a possible mechanism of disease: a selective peripheral target-tissue response to high testosterone levels that did not cause virilization but did suppress ovulation and menstruation., Learning Points: Total testosterone is the most clinically relevant hormone in investigating hyperandrogenic states and infertility in premenopausal women. Very high total testosterone levels in women (>2-3 SD of normal reference) are most often associated with hyperandrogenic signs, menstrual irregularities, and a rapid onset of virilization. In women with very elevated testosterone levels and the absence of clinical manifestations, laboratory interference should be suspected, and diethyl ether extraction is a useful technique when other methods fail to detect it. Ovarian steroid cell tumors (SCT) encompass a rare subgroup of sex cord-stromal tumors and usually secrete androgen hormones. SCTs are clinically malignant in 25-43% of cases. A selective response of peripheral target tissues to testosterone levels, with clinical manifestations in some tissues and no expression in others, may reflect differences in the conformation of tumor-produced testosterone molecules.

Purpose: Point-of-care ultrasound (POCUS) is a sensitive, safe, and efficient tool used in many clinical settings and is an essential part of medical education in the United States. Numerous studies present improved diagnostic performances and positive clinical outcomes among POCUS users. However, others stress the degree to which the modality is user-dependent, rendering high-quality POCUS training necessary in medical education. In this study, the authors aimed to investigate the potential of an artificial intelligence (AI) based quality indicator tool as a teaching device for cardiac POCUS performance., Methods: The authors integrated the quality indicator tool into the pre-clinical cardiac ultrasound course for 4th-year medical students and analyzed their performances. The analysis included 60 students who were assigned to one of two groups as follows: the intervention group using the AI-based quality indicator tool and the control group. Quality indicator users utilized the tool during both the course and the final test. At the end of the course, the authors tested the standard echocardiographic views, and an experienced clinician blindly graded the recorded clips. Results were analyzed and compared between the groups., Results: The results showed an advantage in quality indictor users' median overall scores (P = 0.002) with a relative risk of 2.3 (95% CI: 1.10, 4.93, P = 0.03) for obtaining correct cardiac views. In addition, quality indicator users also had a statistically significant advantage in the overall image quality in various cardiac views., Conclusions: The AI-based quality indicator improved cardiac ultrasound performances among medical students who were trained with it compared to the control group, even in cardiac views in which the indicator was inactive. Performance scores, as well as image quality, were better in the AI-based group. Such tools can potentially enhance ultrasound training, warranting the expansion of the application to more views and prompting further studies on long-term learning effects., Competing Interests: I have reviewed the journal’s policy, and the authors of this manuscript have the following competing interests: GE Healthcare© provided the POCUS devices used in this study. Lior Fuchs declares that he is a consultant for GE Healthcare. However, it’s important to note that the company had no access to the idea, to the study’s primary objective, nor to its design, data analysis, or writing. This affiliation does not affect our adherence to PLOS ONE policies regarding data and material sharing. The remaining authors declare that they have no competing interests., (Copyright: © 2024 Aronovitz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Histiocytoses are inflammatory myeloid neoplasms often driven by somatic activating mutations in mitogen-activated protein kinase (MAPK) cascade genes. H syndrome is an inflammatory genetic disorder caused by germ line loss-of-function mutations in SLC29A3, encoding the lysosomal equilibrative nucleoside transporter 3 (ENT3). Patients with H syndrome are predisposed to develop histiocytosis, yet the mechanism is unclear. Here, through phenotypic, molecular, and functional analysis of primary cells from a cohort of patients with H syndrome, we reveal the molecular pathway leading to histiocytosis and inflammation in this genetic disorder. We show that loss of function of ENT3 activates nucleoside-sensing toll-like receptors (TLR) and downstream MAPK signaling, inducing cytokine secretion and inflammation. Importantly, MEK inhibitor therapy led to resolution of histiocytosis and inflammation in a patient with H syndrome. These results demonstrate a yet-unrecognized link between a defect in a lysosomal transporter and pathological activation of MAPK signaling, establishing a novel pathway leading to histiocytosis and inflammation., (© 2023 by The American Society of Hematology.)

The COVID-19 pandemic notably influenced the transmission of infectious diseases across various age groups. In this study, we assessed its impact on pediatric acute conjunctivitis trends in southern Israel. We analyzed acute conjunctivitis diagnoses from 2017 to 2022, categorizing them into pre-lockdown, lockdown, and post-lockdown intervals. A control group of non-infectious dermatologic conditions was included. Time-series analysis, adjusted for seasonality, was employed. Pre-lockdown data indicated steady conjunctivitis diagnoses, primarily in winter. Post-lockdown interval exhibited an added summer peak before the regular winter surge. The lockdown saw a 56% decline in diagnoses, most pronounced in younger ages. Post-lockdown observed a 7% overall drop with age-specific variations. The acute conjunctivitis IRR was 0.44 (95% CI 0.39-0.49) during lockdowns and 0.93 (95% CI 0.86-1.02) post-lockdowns. Control group IRRs were 0.84 (95% CI 0.78-0.89) and 0.90 (95% CI 0.84-0.96), respectively, with the 0-5 age range demonstrating significant disparities. Pediatric acute conjunctivitis in southern Israel decreased significantly during the pandemic. Post-lockdown patterns varied by age group. An unusual summer peak in cases was observed post-lockdown; this peak may be influenced by a combination of altered behaviors in the summer and possibly increased susceptibility to infection., (© 2023. The Author(s).)

Background: In a large nationwide mass vaccination setting, the SARS-CoV-2 vaccine was recently linked to myocarditis, lymphadenopathy, herpes zoster infection and appendicitis. We aimed to examine the characteristics and management of SARS-CoV-2 vaccine-related acute appendicitis., Methods: We performed a retrospective cohort study in a large tertiary medical centre in Israel. All patients presenting with acute appendicitis within 21 days of receiving their SARS-CoV-2 vaccination (PCVAA group) were compared with patients who presented with acute appendicitis not related to the vaccination (N-PCVAA group)., Results: We reviewed the records of 421 patients with acute appendicitis from December 2020 to September 2021; 38 (9%) patients presented with acute appendicitis within 21 days of receiving their SARS-CoV-2 vaccination. Patients in the PCVAA group were older than those in the N-PCVAA group (mean 41 ± 19 yr v. 33 ± 15 yr, respectively, p = 0.008), with male predominance. More patients were managed nonsurgically during the pandemic than before the pandemic (24% v. 18%, p = 0.03)., Conclusion: With the exception of older age, the clinical characteristics of patients presenting with acute appendicitis within 21 days of receiving the SARS-CoV-2 vaccination did not differ from those of patients who presented with acute appendicitis not related to the vaccination. This finding suggests that vaccine-related acute appendicitis is similar to "classic" acute appendicitis., Competing Interests: Competing interests: None declared., (© 2023 CMA Impact Inc. or its licensors.)

Asthma is the most common chronic respiratory disease in children. The neutrophil-to-lymphocyte ratio (NLR) is a marker of a chronic inflammatory state; however, data on the association of NLR with acute asthma exacerbations in children is lacking. In this cross-sectional study, between 2016 and 2021, children aged 2-18 years who were referred to the emergency department (ED) due to asthma exacerbation, were included. NLR, calculated from complete blood count upon arrival, was assessed as a continuous variable and was classified into four groups according to quartiles. The association between severity parameters and NLR quartiles was examined. A total of 831 ED visits for asthma exacerbation were included in the study. The median NLR was 1.6, 3.8, 6.7, and 12.9 in quartiles 1-4, respectively ( p < 0.001). Demographic parameters, background diseases, and chronic medications were similar between the quartiles. Higher heart rate, body temperature, systolic blood pressure, and respiratory rate were observed in the higher NLR quartiles, as well as lower oxygen saturation. Higher urgency scale and higher rates of intravenous magnesium sulfate were observed in the higher NLR quartiles, with higher admission rates and prolonged hospitalizations. In summary, NLR upon admission is associated with the severity of asthma exacerbation and higher chances of hospitalization among children in the ED.

Introduction: Inborn errors of metabolism (IEM) are scarce, and their diagnosis is often made after birth. This has led to the perception that most fetuses affected by these disorders do not become clinically apparent during pregnancy . Our aim was to determine the obstetrical characteristics of women with an offspring affected by IEM. Methods: This population-based retrospective cohort study included all women who delivered at the Soroka University Medical Center (SUMC) from 1988 to 2017 who met the inclusion criteria. Mothers who had an offspring with IEM were included in the study group, and those who had offsprings without IEM comprised the comparison group. Results: A total of 388,813 pregnancies were included in the study, and 184 of them were complicated by a fetus with IEM. The number of Bedouin women was higher in the IEM-affected infant group than in the comparison group (90.8% vs . 53.3%, p < 0.001); women who had a fetus with IEM had a higher rate of polyhydramnios (7.1% vs . 3.2%, p = 0.005), HELLP syndrome (3.3% vs . 1.1%, p = 0.014), and preterm birth (20.7% vs . 10.1%, p < 0.001); neonates with IEM had lower mean birth weight ( p < 0.001), lower Apgar scores at 1' and 5' minutes ( p < 0.001), and a higher rate of fetal growth restriction (FGR) ( p < 0.001), postpartum death <28 days ( p < 0.001), and neonatal death ( p < 0.001) than those in the comparison group. Pregnancies with IEM fetuses were independently associated with preterm birth (OR 2.00; CI 1.4-3), polyhydramnios (OR 2.08; CI 1.17-3.71), and FGR (OR 2.24; CI 1.2-4.19). Each family of metabolic diseases is independently associated with specific pregnancy complications (i.e., mitochondrial diseases are associated with HELLP syndrome (OR 5.6; CI 1.8-17), and lysosomal storage disease are associated with nonimmune hydrops fetalis (OR 26.4; CI 3.39-206). Conclusion: This study reports for the first time, an independent association of IEM with specific complications of pregnancy. This observation has clinical implications, as the identification of specific pregnancy complications in a population at risk for IEM can assist in the prenatal diagnosis of an affected fetus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Epstein Weiss, Erez, Hazan, Babiev and Staretz Chacham.)

Objective: To investigate whether there is a linear association between the degree of prematurity and the risk for long-term ophthalmic morbidity among preterm infants. Study design: A population-based, retrospective cohort study, which included all singleton deliveries occurring between 1991 and 2014 at a single tertiary medical center. All infants were divided into four groups according to gestational age categories: extremely preterm births, very preterm births, moderate to late preterm births and term deliveries (reference group). Hospitalizations of offspring up to 18 years of age involving ophthalmic morbidity were evaluated. Survival curves compared cumulative hospitalizations and regression models controlled for confounding variables. Results: During the study period, 243,363 deliveries met the inclusion criteria. Ophthalmic-related hospitalization rates were lower among children born at term (0.9%) as compared with extremely preterm (3.6%), very preterm (2%), and moderate to late preterm (1.4%) born offspring (p < 0.01; using the chi-square test for trends). The survival curve demonstrated significantly different hospitalization rates between the gestational ages (p < 0.001). The regression demonstrated an independent risk for ophthalmic morbidity among extremely preterm born offspring (adjusted hazard ratio 3.8, confidence interval 1.6−9.2, p < 0.01), as well as very preterm and moderate to late preterm (adjusted hazard ratio 2.2 and 1.5, respectively) as compared with term deliveries. Conclusions: The risk for long-term ophthalmic-related hospitalization of preterm offspring gradually decreases as the gestational age increases.

Background: SARS-CoV-2 variant Beta (B.1.351) was designated as a Variant of Concern (VoC) after becoming the dominant strain in South Africa and spreading internationally. BNT162b2 showed lower levels of neutralizing antibodies against Beta than against other strains raising concerns about effectiveness of vaccines against infections caused by Beta. We estimated BNT162b2 vaccine effectiveness (VE) against Beta infections in Israel, a country with high vaccine uptake., Methods: The Ministry of Health (MoH) identified Beta cases through mandatory reporting of SARS-CoV-2 cases and whole genome sequencing (WGS) of specimens from vaccination-breakthrough infections, reinfections, arriving international travelers, and a selection of other infected persons. A cohort analysis was conducted of exposure events of contacts of primary Beta cases. WGS was conducted on available PCR-positive specimens collected from contacts. VE estimates with 95% confidence intervals (CIs) against confirmed and probable Beta infections were determined by comparing infection risk between unvaccinated and fully-vaccinated (≥7 days after the second dose) contacts, and between unvaccinated and partially-vaccinated (<7 days after the second dose) contacts., Findings: MoH identified 310 Beta cases through Jun 27, 2021. During the study period (Dec 11, 2020 - Mar 25, 2021), 164 non-institutionalized primary Beta cases, with 552 contacts aged ≥16 years, were identified. 343/552 (62%) contacts were interviewed and tested. 71/343 (21%) contacts were PCR-positive. WGS was performed on 7/71 (10%) PCR-positive specimens; all were Beta. Among SARS-CoV-2-infected contacts, 48/71 (68%) were symptomatic, 10/71 (14%) hospitalized, and 2/71 (3%) died. Fully-vaccinated VE against confirmed or probable Beta infections was 72% (95% CI -5 - 97%; p=0·04) and against symptomatic confirmed or probable Beta infections was 100% (95% CI 19 - 100%; p=0·01). There was no evidence of protection in partially-vaccinated contacts., Interpretation: In a prospective observational study, two doses of BNT162b2 were effective against confirmed and probable Beta infections. Through the end of June 2021, introductions of Beta did not interrupt control of the pandemic in Israel., Funding: Israel Ministry of Health and Pfizer., Competing Interests: Frederick Angulo, David Swerdlow, John McLaughlin, Farid Khan, Gabriel Mircus, Kaijie Pan, Jo Southern, and Luis Jodar are employees of Pfizer Inc, and hold stock and stock options in Pfizer Inc. Marc Lipsitch has provided advice on COVID-19 free of charge to Janssen, Astra-Zeneca, Pfizer, and COVAXX (United Biomedical), as well as to the nonprofit One Day Sooner and has received consulting income or honoraria from Merck, Pfizer, Bristol Meyers Squibb, Janssen, and Sanofi, and institutional research support from Pfizer. He is on the Scientific Advisory Committee of the Coalition for Epidemic Preparedness and Innovations (CEPI). All other authors report no conflicts., (© 2021 The Authors.)

DNA double-strand breaks (DSBs) are deleterious and tumorigenic but could also be essential for DNA-based processes. Yet the landscape of physiological DSBs and their role and repair are still elusive. Here, we mapped DSBs at high resolution in cancer and non-tumorigenic cells and found a transcription-coupled repair mechanism at oncogenic super-enhancers. At these super-enhancers the transcription factor TEAD4, together with various transcription factors and co-factors, co-localizes with the repair factor RAD51 of the homologous recombination pathway. Depletion of TEAD4 or RAD51 increases DSBs at RAD51/TEAD4 common binding sites within super-enhancers and decreases expression of related genes, which are mostly oncogenes. Co-localization of RAD51 with transcription factors at super-enhancers occurs in various cell types, suggesting a broad phenomenon. Together, our findings uncover a coupling between transcription and repair mechanisms at oncogenic super-enhancers, to control the hyper-transcription of multiple cancer drivers., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The role of common fragile sites (CFSs) in cancer remains controversial. Two main views dominate the discussion: one suggests that CFS loci are hotspots of genomic instability leading to inactivation of genes encoded within them, while the other view proposes that CFSs are functional units and that loss of the encoded genes confers selective pressure, leading to cancer development. The latter view is supported by emerging evidence showing that expression of a given CFS is associated with genome integrity and that inactivation of CFS-resident tumor suppressor genes leads to dysregulation of the DNA damage response (DDR) and increased genomic instability. These two viewpoints of CFS function are not mutually exclusive but rather coexist; when breaks at CFSs are not repaired accurately, this can lead to deletions by which cells acquire growth advantage because of loss of tumor suppressor activities. Here, we review recent advances linking some CFS gene products with the DDR, genomic instability, and carcinogenesis and discuss how their inactivation might represent a selective advantage for cancer cells., Competing Interests: The authors have declared that no competing interests exist.

Common fragile sites (CFSs) tend to break upon replication stress and have been suggested to be "hot spots" for genomic instability. Recent evidence, however, implies that in the wake of DNA damage, WW domain-containing oxidoreductase (WWOX, the gene product of the FRA16D fragile site), associates with ataxia telangiectasia-mutated (ATM) and regulates its activation to maintain genomic integrity.

Ikaros is a critical regulator of lymphocyte development and homeostasis; thus, understanding its transcriptional regulation is important from both developmental and clinical perspectives. Using a mouse transgenic reporter approach, we functionally characterized a network of highly conserved cis-acting elements at the Ikzf1 locus. We attribute B-cell and myeloid but not T-cell specificity to the main Ikzf1 promoter. Although this promoter was unable to counter local chromatin silencing effects, each of the 6 highly conserved Ikzf1 intronic enhancers alleviated silencing. Working together, the Ikzf1 enhancers provided locus control region activity, allowing reporter expression in a position and copy-independent manner. Only 1 of the Ikzf1 enhancers was responsible for the progressive upregulation of Ikaros expression from hematopoietic stem cells to lymphoid-primed multipotent progenitors to T-cell precursors, which are stages of differentiation dependent on Ikaros for normal outcome. Thus, Ikzf1 is regulated by both epigenetic and transcriptional factors that target its enhancers in both redundant and specific fashions to provide an expression profile supportive of normal lymphoid lineage progression and homeostasis. Mutations in the Ikzf1 regulatory elements and their interacting factors are likely to have adverse effects on lymphopoiesis and contribute to leukemogenesis.

Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci., Competing Interests: The authors have declared that no competing interests exist.

The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2beta of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2beta, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2beta in their regulation. Thus, Mi-2beta provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.

The human fungal pathogen Candida albicans can undergo a morphological transition from a unicellular yeast growth form to a multicellular hyphal growth form. During hyphal growth, cell division is asymmetric. Only the apical cell divides, whereas subapical cells remain in G(1), and cell surface growth is highly restricted to the tip of the apical cell. Hgc1, a hypha-specific, G(1) cyclin-like protein, is essential for hyphal development. Here, we report, using indirect immunofluorescence, that Hgc1 is preferentially localized to the dividing apical cells of hyphae. Hgc1 protein is rapidly degraded in a cell cycle-independent manner, and the protein turnover likely occurs in both the apical and the subapical cells of hyphae. In addition to rapid protein turnover, the HGC1 transcript is also dynamically regulated during cell cycle progression in hyphal growth. It is induced upon germ tube formation in early G(1); the transcript level is reduced during the G(1)/S transition and peaks again around the G(2)/M phase in the subsequent cell cycles. Transcription from the HGC1 promoter is essential for its apical cell localization, as Hgc1 no longer exhibits preferential apical localization when expressed under the MAL2 promoter. Using fluorescence in situ hybridization, the HGC1 transcript is detected only in the apical cells of hyphae, suggesting that HGC1 is transcribed in the apical cell. Therefore, the preferential localization of Hgc1 to the apical cells of hyphae results from the dynamic temporal and spatial control of HGC1 expression.

The rho-type GTPase Cdc42 is important for the establishment and maintenance of eukaryotic cell polarity. To examine whether Cdc42 is regulated during the yeast-to-hypha transition in Candida albicans, we constructed a green fluorescence protein (GFP)-Cdc42 fusion under the ACT1 promoter and observed its localization in live C. albicans cells. As in Saccharomyces cerevisiae, GFP-Cdc42 was observed around the entire periphery of the cell. In yeast-form cells of C. albicans, it clustered to the tips and sides of small buds as well as to the mother-daughter neck region of large-budded cells. Upon hyphal induction, GFP-Cdc42 clustered to the site of hyphal evagination and remained at the tips of the hyphae. This temporal and spatial localization of Cdc42 suggests that its activity is regulated during the yeast-to-hypha transition. In addition to the accumulation at the hyphal tip, GFP-Cdc42 was also seen as a band within the hyphal tube in cells that had undergone nuclear separation. With the F-actin-assembly inhibitor latrunculin A, we found that GFP-Cdc42 accumulation at the bud site in yeast-form cells is F-actin independent, whereas GFP-Cdc42 accumulation at the hyphal tip requires F-actin. Furthermore, disruption of the F-actin cytoskeleton impaired the transcriptional induction of hypha-specific genes. Therefore, hypha formation resembles mating in Saccharomyces cerevisiae in that both require F-actin for GFP-Cdc42 localization and efficient signaling.

The mechanism for apical growth during hyphal morphogenesis in Candida albicans is unknown. Studies from Saccharomyces cerevisiae indicate that cell morphogenesis may involve cell cycle regulation by cyclin-dependent kinase. To examine whether this is the mechanism for hyphal morphogenesis, the temporal appearance of different spindle pole body and spindle structures, the cell cycle-regulated rearrangements of the actin cytoskeleton, and the phosphorylation state of the conserved Tyr19 of Cdc28 during the cell cycle were compared and found to be similar between yeast and serum-induced hyphal apical cells. These data suggest that hyphal elongation is not mediated by altering cell cycle progression or through phosphorylation of Tyr19 of Cdc28. We have also shown that germ tubes can evaginate before spindle pole body duplication, chitin ring formation, and DNA replication. Similarly, tip-associated actin polarization in each hypha occurs before the events of the G(1)/S transition and persists throughout the cell cycle, whereas cell cycle-regulated actin assemblies come and go. We have also shown that cells in phases other than G(1) can be induced to form hyphae. Hyphae induced from G(1) cells have no constrictions, and the first chitin ring is positioned in the germ tube at various distances from the base. Hyphae induced from budded cells have a constriction and a chitin ring at the bud neck, beyond which the hyphae continue to elongate with no further constrictions. Our data suggest that hyphal elongation and cell cycle morphogenesis programs are uncoupled, and each contributes to different aspects of cell morphogenesis.

We undertook a prospective study to evaluate the accuracy of PCR of serum (aimed at the pneumococcal pneumolysin gene) at detecting pneumococcal infections in infants and children. The assay was positive for all blood and cerebrospinal fluid culture-positive samples and for 38 and 44% of patients with lobar pneumonia and acute otitis media, respectively. It was positive for 17% of healthy controls. There was a marked effect of age on the rate of positivity among healthy controls, with the highest rate (33%) being in 2-year-old children, the age group with the highest rate of nasopharyngeal (NP) carriage; the lowest rate was found among infants <2 months of age (13%) and adults ages 18 to 50 years (0%), age groups with the lowest NP pneumococcal carriage rates. Carriers of pneumococci in the nasopharynges had a higher rate of positivity than noncarriers of pneumococci in the nasopharynges for all groups. Our results suggest that although PCR of serum is a sensitive test for the detection of Streptococcus pneumoniae in sterile fluids, its high rate of positivity for healthy controls, related to NP pneumococcal carriage, might exclude it from being useful in detecting deep-seated pneumococcal infections.

The role of cytosolic phospholipase A2 (cPLA2) and its mode of activation by opsonized zymosan (OZ) was studied in human neutrophils in comparison with activation by PMA. The activation of cPLA2 by 1 mg/ml OZ or 50 ng/ml PMA is evidenced by its translocation to the membrane fractions on stimulation. This translocation is consistent with dithiothreitol (DTT)-resistant phospholipase A2 (PLA2) activity detected in the membranes of activated cells. Neutrophils stimulated by either OZ or PMA exhibited an immediate stimulation of extracellular-signal-regulated kinases (ERKs). The inhibition of ERKs, DTT-resistant PLA2 and NADPH oxidase activities by the MAP kinase kinase inhibitor PD-98059 indicates that ERKs mediate the activation of cPLA2 and NADPH oxidase stimulated by either OZ or PMA. The protein kinase C (PKC) inhibitor GF-109203X inhibited epidermal growth factor receptor peptide kinase activity, the release of [3H]arachidonic acid, DTT-resistant PLA2 activity and superoxide generation induced by PMA, but did not inhibit any of these activities induced by OZ. PKC activity was similarly inhibited by GF-109203X in membrane fractions separated from neutrophils stimulated by either PMA or OZ. In the presence of the tyrosine kinase inhibit orgenistein, ERKs, PLA2 and NADPH oxidase activities were inhibited in cells stimulated by OZ, whereas they were hardly affected in cells stimulated by PMA. The results suggest that the activation of cPLA2 by PMA or OZ is mediated by ERKs. Whereas PMA stimulates ERKs activity through a PKC-dependent pathway, signal transduction stimulated by OZ involves tyrosine kinase activity leading to activation of ERKs via a PKC-independent pathway.