Attenuation of a DNA cruciform by a conserved regulator directs T3SS1 mediated virulence in Vibrio parahaemolyticus.

Pathogenic Vibrio species account for 3-5 million annual life-threatening human infections. Virulence is driven by bacterial hemolysin and toxin gene expression often positively regulated by the winged helix-turn-helix (wHTH) HlyU transcriptional regulator family and silenced by histone-like nucleoid structural protein (H-NS). In the case of Vibrio parahaemolyticus, HlyU is required for virulence gene expression associated with type 3 Secretion System-1 (T3SS1) although its mechanism of action is not understood. Here, we provide evidence for DNA cruciform attenuation mediated by HlyU binding to support concomitant virulence gene expression. Genetic and biochemical experiments revealed that upon HlyU mediated DNA cruciform attenuation, an intergenic cryptic promoter became accessible allowing for exsA mRNA expression and initiation of an ExsA autoactivation feedback loop at a separate ExsA-dependent promoter. Using a heterologous E. coli expression system, we reconstituted the dual promoter elements which revealed that HlyU binding and DNA cruciform attenuation were strictly required to initiate the ExsA autoactivation loop. The data indicate that HlyU acts to attenuate a transcriptional repressive DNA cruciform to support T3SS1 virulence gene expression and reveals a non-canonical extricating gene regulation mechanism in pathogenic Vibrio species.

Safe and Scalable Syntheses of N,N-Dimethyltrifluoromethanesulfonamide (DMTMSA) and Other Trifluoromethanesulfonamide Solvents for High Energy Density Battery Applications.

A simple, scalable synthetic methodology for the synthesis of N,N-dimethyltrifluoromethanesulfonamide (DMTMSA) and other trifluoromethanesulfonamide solvents is described. No specialized glassware is required, water is the solvent, and an ice bath is used for cooling. Up to 155 g of DMTMSA is synthesized in a single batch in 92% yield. The optimized process is highly mass efficient (PMI = 9.1), and excess dimethylamine may be recovered (93% recovery, 51% decrease in waste) and recycled via a simple short-path distillation.

VALSALVA RETINOPATHY: Clinical Features and Treatment Outcomes.

PURPOSE: To report the clinical presentation and outcomes in patients with Valsalva retinopathy. METHODS: This was a retrospective case series of patients diagnosed with Valsalva retinopathy between June 1, 2010, and May 31, 2020. Clinical notes, operative reports, fundus photography, and optical coherence tomography images were reviewed. RESULTS: This study comprised 58 eyes of 58 patients. The most common causes were lifting (34.4%), vomiting (20.6%), straining (20.6%), and coughing (17.2%). Mean best-corrected visual acuity at diagnosis was 20/163. The most frequently involved vitreoretinal compartment was the subhyaloid space (42.3%) followed by the intraretinal (32.7%), intravitreal (23.1%), and subretinal (13.4%) spaces. Mean best-corrected visual acuity of all patients was 20/59 at 3 months, 20/48 at 6 months, and 20/22 at 1 year. Mean time to clearance of hemorrhage on clinical examination was 99.0 ± 18.7 days in patients who underwent observation and 4.5 ± 3.5 days after surgery in patients who received pars plana vitrectomy. CONCLUSION: Valsalva retinopathy is generally associated with a favorable visual prognosis. Most eyes perform well with observation although pars plana vitrectomy may be indicated in patients requiring rapid resolution of hemorrhage.

H3 K27-altered diffuse midline glioma presenting as massive cerebellopontine hemorrhage.

We report the case of a 14-year-old boy who presented with extensive cerebellar and brainstem hemorrhage. Our presumptive diagnosis was a ruptured arteriovenous malformation (AVM), but two cerebral angiograms showed no significant vascular abnormalities. The patient underwent posterior fossa craniotomy and microsurgical evacuation of the hematoma. Pathological analysis of the hemorrhagic tissue made the diagnosis of diffuse midline glioma, H3 K27-altered (WHO grade 4), based on immunohistochemistry. He subsequently developed diffuse craniospinal leptomeningeal disease and progressed rapidly, with respiratory failure followed by severe neurologic decline without further hemorrhage. He was compassionately extubated at the request of the family and died before initiation of adjuvant therapy. This unusual case of a diffuse midline glioma presenting with massive hemorrhage underscores the importance of searching for an underlying etiology of hemorrhage in a child when a vascular lesion cannot be identified.

Addressing the dark matter of gene therapy: technical and ethical barriers to clinical application.

Gene therapies for genetic diseases have been sought for decades, and the relatively recent development of the CRISPR/Cas9 gene-editing system has encouraged a new wave of interest in the field. There have nonetheless been significant setbacks to gene therapy, including unintended biological consequences, ethical scandals, and death. The major focus of research has been on technological problems such as delivery, potential immune responses, and both on and off-target effects in an effort to avoid negative clinical outcomes. While the field has concentrated on how we can better achieve gene therapies and gene editing techniques, there has been less focus on when and why we should use such technology. Here we combine discussion of both the technical and ethical barriers to the widespread clinical application of gene therapy and gene editing, providing a resource for gene therapy experts and novices alike. We discuss ethical problems and solutions, using cystic fibrosis and beta-thalassemia as case studies where gene therapy might be suitable, and provide examples of situations where human germline gene editing may be ethically permissible. Using such examples, we propose criteria to guide researchers and clinicians in deciding whether or not to pursue gene therapy as a treatment. Finally, we summarize how current progress in the field adheres to principles of biomedical ethics and highlight how this approach might fall short of ethical rigour using examples in the bioethics literature. Ultimately by addressing both the technical and ethical aspects of gene therapy and editing, new frameworks can be developed for the fair application of these potentially life-saving treatments.

Reexamining the Role of Postoperative ICU Admission for Patients Undergoing Elective Craniotomy: A Systematic Review.

OBJECTIVES: The standard-of-care for postoperative care following elective craniotomy has historically been ICU admission. However, recent literature interrogating complications and interventions during this postoperative ICU stay suggests that all patients may not require this level of care. Thus, hospitals began implementing non-ICU postoperative care pathways for elective craniotomy. This systematic review aims to summarize and evaluate the existing literature regarding outcomes and costs for patients receiving non-ICU care after elective craniotomy. DATA SOURCES: A systematic review of the PubMed database was performed following PRISMA guidelines from database inception to August 2021. STUDY SELECTION: Included studies were published in peer-reviewed journals, in English, and described outcomes for patients undergoing elective craniotomies without postoperative ICU care. DATA EXTRACTION: Data regarding study design, patient characteristics, and postoperative care pathways were extracted independently by two authors. Quality and risk of bias were evaluated using the Oxford Centre for Evidence-Based Medicine Levels of Evidence tool and Risk Of Bias In Non-Randomized Studies-of Interventions tool, respectively. DATA SYNTHESIS: In total, 1,131 unique articles were identified through the database search, with 27 meeting inclusion criteria. Included articles were published from 2001 to 2021 and included non-ICU inpatient care and same-day discharge pathways. Overall, the studies demonstrated that postoperative non-ICU care for elective craniotomies led to length of stay reduction ranging from 6 hours to 4 days and notable cost reductions. Across 13 studies, 53 of the 2,469 patients (2.1%) intended for postoperative management in a non-ICU setting required subsequent care escalation. CONCLUSIONS: Overall, these studies suggest that non-ICU care pathways for appropriately selected postcraniotomy patients may represent a meaningful opportunity to improve care value. However, included studies varied greatly in patient selection, postoperative care protocol, and outcomes reporting. Standardization and multi-institutional collaboration are needed to draw definitive conclusions regarding non-ICU postoperative care for elective craniotomy.

EVALUATION OF RETINAL DISPLACEMENT FOLLOWING PRIMARY SCLERAL BUCKLING FOR MACULA-INVOLVING RHEGMATOGENOUS RETINAL DETACHMENT.

PURPOSE: To evaluate the incidence and degree of retinal displacement following scleral buckling surgery for macula-involving rhegmatogenous retinal detachment. METHODS: Retrospective interventional case series comprised of patients treated with primary scleral buckling procedure without gas tamponade for macula-involving rhegmatogenous retinal detachment and imaged postoperatively with fundus autofluorescence imaging between June 1, 2016 and July 25, 2021. Clinical notes, operative reports, fundus autofluorescence photographs, and optical coherence tomography images were reviewed. The presence and degree of retinal displacement were recorded. RESULTS: Twelve eyes of 11 patients were included. One (8%) eye with an epiretinal membrane demonstrated 0.1 mm of retinal displacement along the superior arcade and in the superotemporal periphery. The remainder of eyes (92%) did not show any identifiable signs of retinal displacement. CONCLUSION: Retinal displacement does not seem to be a frequent complication of primary scleral buckling surgery for macula-involving rhegmatogenous retinal detachment.

TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry.

Protein synthesis is central to maintaining cellular homeostasis and its study is critical to understanding the function and dysfunction of eukaryotic systems. Here we report L-2-tellurienylalanine (TePhe) as a noncanonical amino acid for direct measurement of protein synthesis. TePhe is synthetically accessible, nontoxic, stable under biological conditions, and the tellurium atom allows its direct detection with mass cytometry, without postexperiment labeling. TePhe labeling is competitive with phenylalanine but not other large and aromatic amino acids, demonstrating its molecular specificity as a phenylalanine mimic; labeling is also abrogated in vitro and in vivo by the protein synthesis inhibitor cycloheximide, validating TePhe as a translation reporter. In vivo, imaging mass cytometry with TePhe visualizes translation dynamics in the mouse gut, brain, and tumor. The strong performance of TePhe as a probe for protein synthesis, coupled with the operational simplicity of its use, suggests TePhe could become a broadly applied molecule for measuring translation in vitro and in vivo.

Microperimetry as a diagnostic tool for the detection of early, subclinical retinal damage and visual impairment in multiple sclerosis.

BACKGROUND: A majority of multiple sclerosis patients experience visual impairment, often as the initial presenting symptom of the disease. While structural changes in the retinal nerve fiber layer and optic nerve have demonstrated correlations with brain atrophy in multiple sclerosis using magnetic resonance imaging, a non-invasive, cost-effective, and clinically efficacious modality to identify early damage and facilitate prompt therapeutic intervention to slow the progression of multiple sclerosis and its ocular manifestations, is still urgently needed. In this study, we sought to determine the role of macular sensitivity measured by microperimetry in the detection of subclinical multiple sclerosis-related retinal damage and visual dysfunction. METHODS: This cross-sectional observational case-control study involved population-based samples of multiple sclerosis patients and age-, race-, and gender-matched healthy control subjects. Among the key criteria for the multiple sclerosis patients were diagnosis by the McDonald criteria, visual acuity greater than 20/25, and no history of optic neuritis. Macular sensitivity and average macular thickness were measured in all subjects using microperimetry and spectral-domain optical coherence tomography, respectively. Pearson correlation coefficients were measured using bivariate correlations. Sample means, mean differences, and 95% confidence intervals were calculated using independent sample t-tests. RESULTS: Twenty-eight eyes from 14 MS patients and 18 eyes from 9 control subjects were included. Mean macular sensitivity of control subjects and multiple sclerosis patients in decibels was 18.2 ± 0.4 and 16.5 ± 0.4, respectively, corresponding to a mean difference of 1.7 (95% CI, 1.1-2.4; P < 0.001). Macular sensitivity was positively correlated with macular thickness in multiple sclerosis patients (r = 0.49, P = 0.01) but not control subjects (r = 0.15, P = 0.55). CONCLUSIONS: Macular sensitivity as measured by microperimetry was decreased in multiple sclerosis patients with normal visual acuity and no history of optic neuritis. Furthermore, macular sensitivity demonstrated a positive correlation with macular thickness as measured by optical coherence tomography. As such, microperimetry may represent a non-invasive and efficient method to identify signs of subclinical visual dysfunction that correspond with early macular architectural changes characteristic of multiple sclerosis.

Back to Basics: Application of the Principles of Bioethics to Heritable Genome Interventions.

Prior to their announcement of the birth of gene-edited twins in China, Dr. He Jiankui and colleagues published a set of draft ethical principles for discussing the legal, social, and ethical aspects of heritable genome interventions. Within this document, He and colleagues made it clear that their goal with these principles was to "clarify for the public the clinical future of early-in-life genetic surgeries" or heritable genome editing. In light of He's widely criticized gene editing experiments it is of interest to place these draft principles in the larger ethical debate surrounding heritable genome editing. Here we examine the principles proposed by He and colleagues through the lens of Beauchamp and Childress' Principles of Biomedical Ethics. We also analyze the stated goal that the "clinical future" of heritable genome editing was clarified by He and colleagues' proposed principles. Finally, we highlight what might be done to help prevent individual actors from pushing forward ahead of broad societal consensus on heritable genome editing.

Tyrosine Phosphorylation as a Widespread Regulatory Mechanism in Prokaryotes.

Phosphorylation events modify bacterial and archaeal proteomes, imparting cells with rapid and reversible responses to specific environmental stimuli or niches. Phosphorylated proteins are generally modified at one or more serine, threonine, or tyrosine residues. Within the last ten years, increasing numbers of global phosphoproteomic surveys of prokaryote species have revealed an abundance of tyrosine-phosphorylated proteins. In some cases, novel phosphorylation-dependent regulatory paradigms for cell division, gene transcription, and protein translation have been identified, suggesting that a wide scope of prokaryotic physiology remains to be characterized. Recent observations of bacterial proteins with putative phosphotyrosine binding pockets or Src homology 2 (SH2)-like domains suggest the presence of phosphotyrosine-dependent protein interaction networks. Here in this minireview, we focus on protein tyrosine phosphorylation, a posttranslational modification once thought to be rare in prokaryotes but which has emerged as an important regulatory facet in microbial biology.

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence.

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.

The Transcriptional Regulator HlyU Positively Regulates Expression of exsA, Leading to Type III Secretion System 1 Activation in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a marine bacterium that is globally recognized as the leading cause of seafood-borne gastroenteritis. V. parahaemolyticus uses various toxins and two type 3 secretion systems (T3SS-1 and T3SS-2) to subvert host cells during infection. We previously determined that V. parahaemolyticus T3SS-1 activity is upregulated by increasing the expression level of the master regulator ExsA under specific growth conditions. In this study, we set out to identify V. parahaemolyticus genes responsible for linking environmental and growth signals to exsA gene expression. Using transposon mutagenesis in combination with a sensitive and quantitative luminescence screen, we identify HlyU and H-NS as two antagonistic regulatory proteins controlling the expression of exsA and, hence, T3SS-1 in V. parahaemolyticus Disruption of hns leads to constitutive unregulated exsA gene expression, consistent with its known role in repressing exsA transcription. In contrast, genetic disruption of hlyU completely abrogated exsA expression and T3SS-1 activity. A V. parahaemolyticushlyU null mutant was significantly deficient for T3SS-1-mediated host cell death during in vitro infection. DNA footprinting studies with purified HlyU revealed a 56-bp protected DNA region within the exsA promoter that contains an inverted repeat sequence. Genetic evidence suggests that HlyU acts as a derepressor, likely by displacing H-NS from the exsA promoter, leading to exsA gene expression and appropriately regulated T3SS-1 activity. Overall, the data implicate HlyU as a critical positive regulator of V. parahaemolyticus T3SS-1-mediated pathogenesis.IMPORTANCE Many Vibrio species are zoonotic pathogens, infecting both animals and humans, resulting in significant morbidity and, in extreme cases, mortality. While many Vibrio species virulence genes are known, their associated regulation is often modestly understood. We set out to identify genetic factors of V. parahaemolyticus that are involved in activating exsA gene expression, a process linked to a type III secretion system involved in host cytotoxicity. We discover that V. parahaemolyticus employs a genetic regulatory switch involving H-NS and HlyU to control exsA promoter activity. While HlyU is a well-known positive regulator of Vibrio species virulence genes, this is the first report linking it to a transcriptional master regulator and type III secretion system paradigm.

Reactive Oxygen Species-Mediated Damage of Retinal Neurons: Drug Development Targets for Therapies of Chronic Neurodegeneration of the Retina.

The significance of oxidative stress in the development of chronic neurodegenerative diseases of the retina has become increasingly apparent in recent years. Reactive oxygen species (ROS) are free radicals produced at low levels as a result of normal cellular metabolism that are ultimately metabolized and detoxified by endogenous and exogenous mechanisms. In the presence of oxidative cellular stress, ROS are produced in excess, resulting in cellular injury and death and ultimately leading to tissue and organ dysfunction. Recent studies have investigated the role of excess ROS in the pathogenesis and development of chronic neurodegenerative diseases of the retina including glaucoma, diabetic retinopathy, and age-related macular degeneration. Findings from these studies are promising insofar as they provide clear rationales for innovative treatment and prevention strategies of these prevalent and disabling diseases where currently therapeutic options are limited. Here, we briefly outline recent developments that have contributed to our understanding of the role of ROS in the pathogenesis of chronic neurodegenerative diseases of the retina. We then examine and analyze the peer-reviewed evidence in support of ROS as targets for therapy development in the area of chronic neurodegeneration of the retina.

Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.

In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells.

Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution.

Changes in the oxygenation state of microenvironments within solid tumors are associated with the development of aggressive cancer phenotypes. Factors that influence cellular hypoxia have been characterized; however, methods for measuring the dynamics of oxygenation at a cellular level in vivo have been elusive. We report a series of tellurium-containing isotopologous probes for cellular hypoxia compatible with mass cytometry (MC)-technology that allows for highly parametric interrogation of single cells based on atomic mass spectrometry. Sequential labeling with the isotopologous probes (SLIP) in pancreatic tumor xenograft models revealed changes in cellular oxygenation over time which correlated with the distance from vasculature, the proliferation of cell populations, and proximity to necrosis. SLIP allows for capture of spatial and temporal dynamics in vivo using enzyme activated probes.

Organotellurium scaffolds for mass cytometry reagent development.

Mass cytometry (MC) is a powerful tool for studying heterogeneous cell populations. In previous work, our laboratory has developed an MC probe for hypoxia bearing a methyl telluride mass tag. The methyl telluride was unoptimized, displaying stability and toxicity limitations. Here, we investigate three classes of organotelluriums as MC mass tags: methyl tellurides, trifluoromethyl tellurides and 2-alkyl-tellurophenes. NMR was used to compare the stability of these compounds in aqueous and organic solutions and the compounds were analysed for toxicity in Jurkat cells. The methyl tellurides were moderately stable to aerobic oxidation in organic solution under dry ambient conditions. The trifluoromethyl tellurides were stable to aerobic oxidation in organic solution but decomposed in aqueous solution. The 2-alkyl-tellurophenes proved to be stable in both organic and aqueous solutions under ambient conditions and showed limited toxicity (IC50 > 200 µM) in cell based assays. The synthetic feasibility, chemically stability, and limited toxicity of tellurophenes suggests these groups will be good choices for MC reagent development.

Na,K-ATPase ß-subunit cis homo-oligomerization is necessary for epithelial lumen formation in mammalian cells.

Na,K-ATPase is a hetero-oligomer of an α- and a ß-subunit. The α-subunit (Na,K-α) possesses the catalytic function, whereas the ß-subunit (Na,K-ß) has cell-cell adhesion function and is localized to the apical junctional complex in polarized epithelial cells. Earlier, we identified two distinct conserved motifs on the Na,K-ß(1) transmembrane domain that mediate protein-protein interactions: a glycine zipper motif involved in the cis homo-oligomerization of Na,K-ß(1) and a heptad repeat motif that is involved in the hetero-oligomeric interaction with Na,K-α(1). We now provide evidence that knockdown of Na,K-ß(1) prevents lumen formation and induces activation of extracellular regulated kinases 1 and 2 (ERK1/2) mediated by phosphatidylinositol 3-kinase in MDCK cells grown in three-dimensional collagen cultures. These cells sustained cell proliferation in an ERK1/2-dependent manner and did not show contact inhibition at high cell densities, as revealed by parental MDCK cells. This phenotype could be rescued by wild-type Na,K-ß(1) or heptad repeat motif mutant of Na,K-ß(1), but not by the glycine zipper motif mutant that abrogates Na,K-ß(1) cis homo-oligomerization. These studies suggest that Na,K-ß(1) cis homo-oligomerization rather than hetero-oligomerization with Na,K-α(1) is involved in epithelial lumen formation. The relevance of these findings to pre-neoplastic lumen filling in epithelial cancer is discussed.

Flexible carbon dioxide laser-assisted endoscopic marsupialization and ablation of a laryngeal saccular cyst in a neonate.

OBJECTIVE: To report the occurrence of a neonate with a lateral saccular cyst that was successfully managed by flexible, carbon dioxide laser-assisted endoscopic marsupialization and ablation. CASE SUMMARY: A full-term, 14-day-old girl presented to the clinic for progressively worsening stridor since birth. On fiber optic laryngoscopy, she was found to have a large, right saccular cyst obstructing the laryngeal inlet. The patient was admitted and underwent microlaryngeal endoscopic CO2 laser marsupialization and ablation of the saccular cyst. She was observed overnight and was discharged without complication the next day. At that time, stridor was no longer present and she remained symptom free at both her 2-week and 3-month follow-up. DISCUSSION: The congenital saccular cyst is a rare, abnormal dilation of the laryngeal saccule that is known to be a cause of airway obstruction. A variety of contrasting treatment strategies for laryngeal saccular cysts in children have been reported in the literature. Determining the definitive treatment modality thus remains a challenge that is compounded further by the rarity of the lesion. CONCLUSION: We introduce a new surgical technique that employs a 30-degree angled telescope with a flexible laser fiber system that provides excellent visualization of saccular cysts, in particular, lateral lesions that are less visible using line-of-sight technology.