Identification of high-performing antibodies for the reliable detection of Tau proteoforms by Western blotting and immunohistochemistry.

Antibodies are essential research tools whose performance directly impacts research conclusions and reproducibility. Owing to its central role in Alzheimer's disease and other dementias, hundreds of distinct antibody clones have been developed against the microtubule-associated protein Tau and its multiple proteoforms. Despite this breadth of offer, limited understanding of their performance and poor antibody selectivity have hindered research progress. Here, we validate a large panel of Tau antibodies by Western blot (79 reagents) and immunohistochemistry (35 reagents). We address the reagents' ability to detect the target proteoform, selectivity, the impact of protein phosphorylation on antibody binding and performance in human brain samples. While most antibodies detected Tau at high levels, many failed to detect it at lower, endogenous levels. By WB, non-selective binding to other proteins affected over half of the antibodies tested, with several cross-reacting with the related MAP2 protein, whereas the "oligomeric Tau" T22 antibody reacted with monomeric Tau by WB, thus calling into question its specificity to Tau oligomers. Despite the presumption that "total" Tau antibodies are agnostic to post-translational modifications, we found that phosphorylation partially inhibits binding for many such antibodies, including the popular Tau-5 clone. We further combine high-sensitivity reagents, mass-spectrometry proteomics and cDNA sequencing to demonstrate that presumptive Tau "knockout" human cells continue to express residual protein arising through exon skipping, providing evidence of previously unappreciated gene plasticity. Finally, probing of human brain samples with a large panel of antibodies revealed the presence of C-term-truncated versions of all main Tau brain isoforms in both control and tauopathy donors. Ultimately, we identify a validated panel of Tau antibodies that can be employed in Western blotting and/or immunohistochemistry to reliably detect even low levels of Tau expression with high selectivity. This work represents an extensive resource that will enable the re-interpretation of published data, improve reproducibility in Tau research, and overall accelerate scientific progress.

Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites.

Introduction: Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. Methods: To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. Results: A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Discussion: Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

Digitalization of toxicology: improving preclinical to clinical translation.

Though the portfolio of medicines that are extending and improving the lives of patients continues to grow, drug discovery and development remains a challenging business on its best day. Safety liabilities are a significant contributor to development attrition where the costliest liabilities to both drug developers and patients emerge in late development or post-marketing. Animal studies are an important and influential contributor to the current drug discovery and development paradigm intending to provide evidence that a novel drug candidate can be used safely and effectively in human volunteers and patients. However, translational gaps-such as toxicity in patients not predicted by animal studies-have prompted efforts to improve their effectiveness, especially in safety assessment. More holistic monitoring and "digitalization" of animal studies has the potential to enrich study outcomes leading to datasets that are more computationally accessible, translationally relevant, replicable, and technically efficient. Continuous monitoring of animal behavior and physiology enables longitudinal assessment of drug effects, detection of effects during the animal's sleep and wake cycles and the opportunity to detect health or welfare events earlier. Automated measures can also mitigate human biases and reduce subjectivity. Reinventing a conservative, standardized, and traditional paradigm like drug safety assessment requires the collaboration and contributions of a broad and multi-disciplinary stakeholder group. In this perspective, we review the current state of the field and discuss opportunities to improve current approaches by more fully leveraging the power of sensor technologies, artificial intelligence (AI), and animal behavior in a home cage environment.

A platform for predicting mechanism of action based on bacterial transcriptional responses identifies an unusual DNA gyrase inhibitor.

In the search for much-needed new antibacterial chemical matter, a myriad of compounds have been reported in academic and pharmaceutical screening endeavors. Only a small fraction of these, however, are characterized with respect to mechanism of action (MOA). Here, we describe a pipeline that categorizes transcriptional responses to antibiotics and provides hypotheses for MOA. 3D-printed imaging hardware PFIboxes) profiles responses of Escherichia coli promoter-GFP fusions to more than 100 antibiotics. Notably, metergoline, a semi-synthetic ergot alkaloid, mimics a DNA replication inhibitor. In vitro supercoiling assays confirm this prediction, and a potent analog thereof (MLEB-1934) inhibits growth at 0.25 µg/mL and is highly active against quinolone-resistant strains of methicillin-resistant Staphylococcus aureus. Spontaneous suppressor mutants map to a seldom explored allosteric binding pocket, suggesting a mechanism distinct from DNA gyrase inhibitors used in the clinic. In all, the work highlights the potential of this platform to rapidly assess MOA of new antibacterial compounds.

Identifying the Cerebral Physiologic Response to Aerobic Exercise Following Concussion: A Scoping Review.

OBJECTIVE: The purpose of this study was to identify the cerebral physiologic response to aerobic exercise in individuals with a symptomatic concussion, highlighting available knowledge and knowledge gaps in the literature. DESIGN: A systematic scoping review was conducted and reported in keeping with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) extension for scoping reviews. A search of EMBASE, MEDLINE, SCOPUS, BIOSIS, and Cochrane libraries was conducted on June 15, 2023 (from database inception). An online systematic/scoping review management system was used to remove duplicates, and the remaining articles were screened for inclusion by 2 researchers. Inclusion criteria required articles to be original research published in peer-reviewed journals. Additionally, studies were required to have an aerobic exercise component, include a measure of cerebral physiology during a bout of aerobic exercise, exclude moderate and/or severe traumatic brain injury (TBI) populations, and be in the English language. Both human and animal studies were included, with participants of any age who were diagnosed with a mild TBI/concussion only (ie, Glasgow Coma Scale score ≥ 13). Studies could be of any design as long as a measure of cerebral physiologic response to a bout of aerobic exercise was included. RESULTS: The search resulted in 1773 articles to be screened and data from 3 eligible studies were extracted. CONCLUSIONS: There are currently too few studies investigating the cerebral physiologic response to aerobic exercise following concussion or mild TBI to draw definitive conclusions. Further research on this topic is necessary since understanding the cerebral physiologic response to aerobic exercise in the concussion and mild TBI populations could assist in optimizing exercise-based treatment prescription and identifying other targeted therapies.

Intimate Partner Violence-Related Brain Injury: Unmasking and Addressing the Gaps.

Intimate partner violence (IPV) is a significant, global public health concern. Women, individuals with historically underrepresented identities, and disabilities are at high risk for IPV and tend to experience severe injuries. There has been growing concern about the risk of exposure to IPV-related head trauma, resulting in IPV-related brain injury (IPV-BI), and its health consequences. Past work suggests that a significant proportion of women exposed to IPV experience IPV-BI, likely representing a distinct phenotype compared with BI of other etiologies. An IPV-BI often co-occurs with psychological trauma and mental health complaints, leading to unique issues related to identifying, prognosticating, and managing IPV-BI outcomes. The goal of this review is to identify important gaps in research and clinical practice in IPV-BI and suggest potential solutions to address them. We summarize IPV research in five key priority areas: (1) unique considerations for IPV-BI study design; (2) understanding non-fatal strangulation as a form of BI; (3) identifying objective biomarkers of IPV-BI; (4) consideration of the chronicity, cumulative and late effects of IPV-BI; and (5) BI as a risk factor for IPV engagement. Our review concludes with a call to action to help investigators develop ecologically valid research studies addressing the identified clinical-research knowledge gaps and strategies to improve care in individuals exposed to IPV-BI. By reducing the current gaps and answering these calls to action, we will approach IPV-BI in a trauma-informed manner, ultimately improving outcomes and quality of life for those impacted by IPV-BI.

Dynamics of the Oral Microbiome During Initial Military Training at Fort Benning, Georgia.

INTRODUCTION: Military trainees are at increased risk for infectious disease outbreaks because of the unique circumstances of the training environment (e.g., close proximity areas and physiologic/psychologic stress). Standard medical countermeasures in military training settings include routine immunization (e.g., influenza and adenovirus) as well as chemoprophylaxis [e.g., benzathine penicillin G (Bicillin) for the prevention of group A streptococcal disease] for pathogens associated with outbreaks in these settings. In a population of U.S. Army Infantry trainees, we evaluated changes in the oral microbiome during a 14-week military training cycle. MATERIALS AND METHODS: Trainees were enrolled in an observational cohort study in 2015-2016. In 2015, Bicillin was administered to trainees to ameliorate the risk of group A Streptococcus outbreaks, whereas in 2016, trainees did not receive a Bicillin inoculation. Oropharyngeal swabs were collected from participants at days 0, 7, 14, 28, 56, and 90 of training. Swabs were collected, flash frozen, and stored. DNA was extracted from swabs, and amplicon sequencing of the 16s rRNA gene was performed. Microbiome dynamics were evaluated using the QIIME 2 workflow along with DADA2, SINA with SILVA, and an additional processing in R. RESULTS: We observed that microbiome samples from the baseline (day 0) visit were distinct from one another, whereas samples collected on day 14 exhibited significant microbiome convergence. Day 14 convergence was coincident with an increase in DNA sequences associated with Streptococcus, though there was not a significant difference between Streptococcus abundance over time between 2015 and 2016 (P = .07), suggesting that Bicillin prophylaxis did not significantly impact overall Streptococcus abundance. CONCLUSIONS: The temporary convergence of microbiomes is coincident with a rise in communicable infections in this population. The dynamic response of microbiomes during initial military training supports similar observations in the literature of transient convergence of the human microbiome under cohabitation in the time frame including in this experiment. This population and the associated longitudinal studies allow for controlled studies of human microbiome under diverse conditions.

Profiling of serum factors associated with Staphylococcus aureus skin and soft tissue infections as a foundation for biomarker identification.

Background: People living in close quarters, such as military trainees, are at increased risk for skin and soft tissue infections (SSTI), especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). The serum immune factors associated with the onset of SSTI are not well understood. Methods: We conducted a longitudinal study of SSTIs, enrolling US Army trainees before starting military training and following up for 14 weeks. Samples were collected on Day 0, 56, and 90. Serum chemokines and cytokines among 16 SSTI cases and 51 healthy controls were evaluated using an electro-chemiluminescence based multiplex assay platform. Results: Of 54 tested cytokines, 12 were significantly higher among SSTI cases as compared to controls. Among the cases, there were correlations between factors associated with vascular injury (i.e., VCAM-1, ICAM-1, and Flt1), the angiogenetic factor VEGF, and IL-10. Unsupervised machine learning (Principal Component Analysis) revealed that IL10, IL17A, C-reactive protein, ICAM1, VCAM1, SAA, Flt1, and VGEF were indicative of SSTI. Conclusion: The study demonstrates the power of immunoprofiling for identifying factors predictive of pre-illness state of SSTI thereby identifying early stages of an infection and individuals susceptible to SSTI.

Supervisor Cultural Humility and Supervisee Nondisclosure: The Supervisory Working Alliance Matters.

Given that half or more of supervisees (therapist trainees) never have their clinical work monitored or observed, supervisees who withhold salient information in clinical supervision compromise supervisors' ability to monitor client welfare and promote supervisees' professional development. Attempting to further understand the factors explaining supervisee nondisclosure, we tested the supervisory working alliance as a mediator of the hypothesized inverse relations of cultural humility and collaborative supervision with supervisee nondisclosure (supervision-related and clinically-related nondisclosure) among a diverse sample of 214 supervisees in applied psychology and allied mental health programs. Results supported the hypotheses that (1) descriptively, supervision-related nondisclosure was more prominent than clinically-related nondisclosure, (2) cultural humility substantially inversely predicted supervisee nondisclosure, and (3) the supervisory working alliance fully mediated the inverse relations of cultural humility and collaborative supervision with supervisee nondisclosure. Understanding the mechanisms underlying supervisee nondisclosure have broad implications for clinicians and researchers alike.

p53 inhibitor iASPP is an unexpected suppressor of KRAS and inflammation-driven pancreatic cancer.

Oncogenic KRAS activation, inflammation and p53 mutation are key drivers of pancreatic cancer (PC) development. Here we report iASPP, an inhibitor of p53, as a paradoxical suppressor of inflammation and oncogenic KRASG12D-driven PC tumorigenesis. iASPP suppresses PC onset driven by KRASG12D alone or KRASG12D in combination with mutant p53R172H. iASPP deletion limits acinar-to-ductal metaplasia (ADM) in vitro but accelerates inflammation and KRASG12D-induced ADM, pancreatitis and PC tumorigenesis in vivo. KRASG12D/iASPPΔ8/Δ8 tumours are well-differentiated classical PCs and their derivative cell lines form subcutaneous tumours in syngeneic and nude mice. Transcriptomically, either iASPP deletion or p53 mutation in the KRASG12D background altered the expression of an extensively overlapping gene set, comprised primarily of NF-κB and AP1-regulated inflammatory genes. All these identify iASPP as a suppressor of inflammation and a p53-independent oncosuppressor of PC tumorigenesis.

Shear wave ultrasound elastography of the biceps brachii can be used as a precise proxy for passive elbow torque in individuals with hemiparetic stroke.

Muscle tissue is prone to changes in composition and architecture following stroke. Changes in muscle tissue of the extremities are thought to increase resistance to muscle elongation or joint torque under passive conditions. These effects likely compound neuromuscular impairments, exacerbating movement function. Unfortunately, conventional rehabilitation is devoid of precise measures and relies on subjective assessments of passive joint torques. Shear wave ultrasound elastography, a tool to measure muscle mechanical properties, may be readily available for use in the rehabilitation setting as a precise measure, albeit at the muscle-tissue level. To support this postulation, we evaluated the criterion validity of shear wave ultrasound elastography of the biceps brachii; we investigated its relationship with a laboratory-based criterion measure for quantifying elbow joint torque in individuals with moderate to severe chronic stroke. Additionally, we evaluated construct validity, with the specific sub-type of hypothesis testing of known groups, by testing the difference between arms. Measurements were performed under passive conditions at seven positions spanning the arc of elbow joint flexion-extension in both arms of nine individuals with hemiparetic stroke. Surface electromyography was utilized for threshold-based confirmation of muscle quiescence. A moderate relationship between the shear wave velocity and elbow joint torque was identified, and both metrics were greater in the paretic arm. Data supports the progression toward a clinical application of shear wave ultrasound elastography in evaluating altered muscle mechanical properties in stroke, while acknowledging that undetectable muscle activation or hypertonicity may contribute to the measurement. Shear wave ultrasound elastography may augment the conventional method of manually testing joint mobility by providing a high-resolution precise value. Tissue-level measurement may also assist in identifying new therapeutic targets for patient-specific impairment-based interventions.

Incidence of Subsequent Mental Health Disorders and Social Adversity Following Pediatric Concussion: A Longitudinal, Population-Based Study.

OBJECTIVE: To determine the long-term risk of new adverse psychosocial outcomes among adolescents diagnosed with a concussion compared with those not diagnosed. STUDY DESIGN: A retrospective, population-based cohort study was conducted. Adolescents (10-18 years) with a physician-diagnosed concussion between 2000 and 2005 were matched on neighborhood and age with 5 controls without concussion from the general population. New-onset mental health disorders, medication use, social, and justice outcomes were extracted using datasets linked to the population data repository. Adolescents were followed for 11-16 years. Adjusted hazard ratios (95% CIs) were estimated. RESULTS: In total, 2082 adolescents with a concussion were matched to 10 510 without. Adolescents with a concussion had an increased risk of any mental health disorder (HR 1.34; 95% CI 1.25-1.45), mood disorder (HR 1.30; 95% 1.18-1.43), psychosis (HR 1.43; 95% CI 1.18-1.74), substance abuse disorder (HR 1.67; 95% 1.31-2.14), and receiving a psychotropic prescription (HR 1.31; 95% CI 1.20-1.42). Female adolescents had an increased risk of ADHD following concussion (HR 1.89; 95% CI 1.17-3.05). Adolescents with a concussion had an increased risk of being accused (HR 1.22; 95% CI 1.11-1.34), victim (HR 1.29; 95% CI 1.11-1.48), or witness (HR 1.16; 95% CI 1.01-1.32) of a crime, or contact with Child and Family Services (HR 1.33; 95% CI 1.10-1.62). There was no association between concussion and attempting or completing suicide, receiving housing support, or collecting income support. CONCLUSIONS: Concussion was associated with an increased risk for multiple adverse psychosocial outcomes. Future work should focus on early identification of those at risk of these outcomes to help optimize longitudinal medical care and support.

Role of the Beta and Gamma Isoforms of the Adapter Protein SH2B1 in Regulating Energy Balance.

Human variants of the adapter protein SH2B1 are associated with severe childhood obesity, hyperphagia, and insulin resistance-phenotypes mimicked by mice lacking Sh2b1. SH2B1ß and γ isoforms are expressed ubiquitously, whereas SH2B1α and δ isoforms are expressed primarily in the brain. Restoring SH2B1ß driven by the neuron-specific enolase promoter largely reverses the metabolic phenotype of Sh2b1-null mice, suggesting crucial roles for neuronal SH2B1ß in energy balance control. Here we test this hypothesis by using CRISPR/Cas9 gene editing to delete the ß and γ isoforms from the neurons of mice (SH2B1ßγ neuron-specific knockout [NKO] mice) or throughout the body (SH2B1ßγ knockout [KO] mice). While parameters of energy balance were normal in both male and female SH2B1ßγ NKO mice, food intake, body weight, and adiposity were increased in male (but not female) SH2B1ßγ KO mice. Analysis of long-read single-cell RNA seq data from wild-type mouse brain revealed that neurons express almost exclusively the α and δ isoforms, whereas neuroglial cells express almost exclusively the ß and γ isoforms. Our work suggests that neuronal SH2B1ß and γ are not primary regulators of energy balance. Rather, non-neuronal SH2B1ß and γ in combination with neuronal SH2B1α and δ suffice for body weight maintenance. While SH2B1ß/γ and SH2B1α/δ share some functionality, SH2B1ß/γ appears to play a larger role in promoting leanness.

Injury primes mutation-bearing astrocytes for dedifferentiation in later life.

Despite their latent neurogenic potential, most normal parenchymal astrocytes fail to dedifferentiate to neural stem cells in response to injury. In contrast, aberrant lineage plasticity is a hallmark of gliomas, and this suggests that tumor suppressors may constrain astrocyte dedifferentiation. Here, we show that p53, one of the most commonly inactivated tumor suppressors in glioma, is a gatekeeper of astrocyte fate. In the context of stab-wound injury, p53 loss destabilized the identity of astrocytes, priming them to dedifferentiate in later life. This resulted from persistent and age-exacerbated neuroinflammation at the injury site and EGFR activation in periwound astrocytes. Mechanistically, dedifferentiation was driven by the synergistic upregulation of mTOR signaling downstream of p53 loss and EGFR, which reinstates stemness programs via increased translation of neurodevelopmental transcription factors. Thus, our findings suggest that first-hit mutations remove the barriers to injury-induced dedifferentiation by sensitizing somatic cells to inflammatory signals, with implications for tumorigenesis.

Current Use and Future Considerations for Concussion Telemedicine Healthcare in Canada.

OBJECTIVE: To examine the use of telemedicine among Canadian concussion providers and clinics before and after the COVID-19 pandemic onset and identify barriers and facilitators for future use. METHODS: Ninety-nine concussion clinics and healthcare providers across Canada that offered one or more clinical concussion-related service were identified using standardized online searches and approached to complete a cross-sectional online survey. RESULTS: Thirty clinics or providers completed the survey and two completed subsections of the survey (response rate of 32.3%). Only 28.1% of respondents indicated that they used telemedicine to provide care prior to the COVID-19 pandemic. Providers most commonly using telemedicine prior to the pandemic were occupational therapists and physicians, while the most commonly used services were in-person videoconferencing and eConsultation. Most respondents (87%) indicated their clinic's use of telemedicine changed following the onset of the COVID-19 pandemic including new use of in-person video-conferencing, telephone calls, and eConsultation. Ninety-three percent indicated that they would consider using telemedicine to provide care to their concussion patients once the pandemic was over. Barriers needed to be overcome to facilitate use or greater use of telemedicine-based services were the inability to conduct a complete physical examination, lack of appropriate reimbursement, lack of start-up, and maintenance funding and medico-legal risk. CONCLUSION: Telemedicine was used by a minority of Canadian concussion clinics and providers prior to the COVID-19 pandemic but was rapidly adopted by many facilities. This study provides important insight into the factors that must be considered to optimize use of telemedicine in concussion care in the future.

Animals, quality and the pursuit of relevance.

In 2021, the National Institutes of Health Advisory Committee to the Director (ACD) announced recommendations to improve the reproducibility of biomedical research using animals. In response, The Jackson Laboratory faculty and institutional leaders identified key strategies to further address this important issue. Taking inspiration from the evolution of clinical trials over recent decades in response to similar challenges, we identified opportunities for improvement, including establishment of common standards, use of genetically diverse populations, requirement for robust study design with appropriate statistical methods, and improvement in public databases to facilitate meta-analyses. In this Perspective, we share our response to ACD recommendations, with a specific focus on mouse models, with the aim of promoting continued active dialogue among researchers, using any animal system, worldwide. Such discussion will help to inform the biomedical community about these recommendations and further support their much-needed implementation.

Influence of high cyclic ambient temperature and water drinker design on growth performance and water disappearance of growing-finishing pigs.

The objective of this study was to determine effects of room temperature and drinker design on growth and water disappearance of growing-finishing pigs (26.9 ±â€…3.67 to 130.9 ±â€…5.10 kg live body weight). A split-plot design was used with a 2 × 2 factorial arrangement of treatments: Room Temperature (RT) [Thermoneutral (TN) vs. High (HI); main plot], Drinker Design (DD; Nipple vs. Cup; sub-plot). A total of 316 pigs were used, housed in 32 pens in 4 rooms (8 pens/room; 7 pens of 10 pigs and 1 pen of 9 pigs). Two rooms were on each RT treatment. Room temperature for the TN treatment was constant throughout each day but decreased from 24°C at the start to 20°C and 18°C on d 14 and 45 of the study period, respectively. For the HI treatment, a single, cyclic RT protocol was used throughout the study (30°C from 08:00 to 19:00 h and 20°C from 20:00 to 07:00 h, with 1-h transition periods). Pens had fully-slatted concrete floors and 1 feeder and drinker (either nipple or cup); floor space was 0.67 m2/pig. Pigs had ad libitum access to standard corn-soybean diets, formulated to meet or exceed NRC (2012) nutrient requirements. Water disappearance was measured using a meter fitted to the water line supplying each drinker. There were no interactions (P > 0.05) between RT and DD treatments. Drinker Design did not affect (P > 0.05) growth performance; water disappearance was 7.3% greater (P ≤ 0.05) for Nipple than Cup drinkers. Compared to the TN treatment, the HI treatment had no effect (P > 0.05) on gain:feed ratio, but resulted in lower (P ≤ 0.05) average daily gain (6.5%) and average daily feed intake (5.5%) and greater (P ≤ 0.05) average daily water disappearance (16.8%). These results suggest that both drinker design and RT can affect water disappearance, and that the high, cyclic RT regime used reduced growth performance of growing-finishing pigs. Further research is needed to determine the contribution of water intake and wastage to treatment differences in water disappearance.

Synthetic Genetic Interactions Reveal a Dense and Cryptic Regulatory Network of Small Noncoding RNAs in Escherichia coli.

Over the past 20 years, we have learned that bacterial small noncoding RNAs (sRNAs) can rapidly effect changes in gene expression in response to stress. However, the broader role and impact of sRNA-mediated regulation in promoting bacterial survival has remained elusive. Indeed, there are few examples where disruption of sRNA-mediated gene regulation results in a discernible change in bacterial growth or survival. The lack of phenotypes attributable to loss of sRNA function suggests that either sRNAs are wholly dispensable or functional redundancies mask the impact of deleting a single sRNA. We investigated synthetic genetic interactions among sRNA genes in Escherichia coli by constructing pairwise deletions in 54 genes, including 52 sRNAs. Some 1,373 double deletion strains were studied for growth defects under 32 different nutrient stress conditions and revealed 1,131 genetic interactions. In one example, we identified a profound synthetic lethal interaction between ArcZ and CsrC when E. coli was grown on pyruvate, lactate, oxaloacetate, or d-/l-alanine, and we provide evidence that the expression of ppsA is dysregulated in the double deletion background, causing the conditionally lethal phenotype. This work employs a unique platform for studying sRNA-mediated gene regulation and sheds new light on the genetic network of sRNAs that underpins bacterial growth. IMPORTANCE sRNAs have long been purported to be a critical mechanism by which bacteria respond to stress; however, uncovering growth phenotypes for sRNA deletion strains in E. coli and related bacteria has proven particularly challenging. In contrast, the deletion of hfq, a chaperone required for the activity of many sRNAs in E. coli, results in striking growth defects in E. coli under a variety of medium conditions and chemical stressors. Here, we examined the importance of hfq and sRNA deletion strains for E. coli growth in nutrient-limited medium supplemented with 30 different carbon sources. We then systematically combined sRNA deletion mutations, creating a library of 1,373 sRNA double deletion strains, which we screened for growth under the same conditions, yielding 43,936 individual growth measurements. Our data uncovered more than 1,000 growth phenotypes for sRNA double deletion strains, shedding light on complicated networks of sRNA regulation that underpin bacterial survival under nutrient stress.

Hypotony maculopathy and choroidal detachment from repeated nocturnal ocular compression in a pediatric patient.

Purpose: To report clinical outcomes of a pediatric patient with unilateral reversible vision loss secondary to hypotony from repeated accidental nocturnal ocular compression from circumaural headphone wear. Observations: A 17-year-old male with pathologic myopia and history of retinopathy of prematurity previously treated with laser ablation in both eyes presented with reduced visual acuity in his right eye from choroidal detachment and hypotony maculopathy. In the absence of uveitis and intraocular pressure lowering medications, it was determined that repeated nocturnal ocular compression from circumaural headphones created episodes of hypotony. With avoidance of this behavior and in the absence of pathologic aqueous dynamics, intraocular pressure normalized with gradual resolution of choroidal thickening and restoration to baseline visual acuity. Conclusions and Importance: Persistent and prolonged ocular compression, even unintentionally, can create hypotony with risk for vision loss, maculopathy, and choroidal detachment.