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A small library of stereochemically-defined α,β-diamino acids has been prepared to generate non-native monobactams through fermentation, highlighting the potential of this platform for directed biosynthesis.

BACKGROUND: In addition to its intended purpose of perforator mapping, computed tomography angiography can also identify incidental findings that may require further evaluation. In this multi-institutional study, the authors evaluated the frequency of incidental findings and their significance and effects on treatment course and aimed to identify risk factors for detecting such findings. METHODS: A retrospective review of patients who underwent perforator mapping with computed tomography angiography was performed over a 5-year period from three academic institutions. Relevant sociodemographic and clinicopathologic information, computed tomography angiography reports, follow-up visits, and treatment outcomes were reviewed. Univariate and multivariate analyses were performed to assess the relationship between risk factors and incidental findings. RESULTS: From January of 2015 to July of 2020, a total of 656 patients were identified who met inclusion criteria. Overall, 342 incidental findings were found, 76 of which required additional imaging or consultation. Ultimately, 10 patients (1.5 percent) had findings that altered reconstructive management, including five patients (0.8 percent) having severe disease that resulted in the cancellation of their reconstruction. Advanced age and immediate reconstruction timing were independent risk factors for incidental findings. CONCLUSIONS: Incidental findings are commonly identified on preoperative computed tomography angiography for deep inferior epigastric perforator flap breast reconstruction. Suspicious findings should be investigated thoroughly because they can alter the reconstructive course. Understanding of high-risk groups for incidental findings can further advance patient education during initial consultation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Prediction of treatment responses is essential to move forward translational science. Our question was to identify patient-based variables that predicted responses to treatments. We conducted secondary analyses on pooled data from 2 randomized Phase 3 clinical trials (NCT02697773 and NCT02709486) conducted in participants with moderate-to-severe osteoarthritis randomized to subcutaneous placebo (n=514) or tanezumab 2.5 mg (n=514). We used gradient boosted regression trees to identify variables that predicted Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain subscale scores at week 16 and marginal plots to determine the directional relationship between each variable category and responses to placebo or tanezumab within the models. We also used Virtual Twins models to identify potential subgroups of response to the active treatment versus placebo. We found that responses to placebo were predicted by baseline WOMAC Physical Function, baseline WOMAC Pain, the radiographic classification of the index joint, and the standard deviation of diary pain scores at baseline. In contrast, baseline WOMAC Pain along with failure of prior medications, duration of disease and standard deviation of diary pain scores at baseline were predictive of tanezumab responses as expressed by the WOMAC Pain scores at week 16. Those who responded to tanezumab versus placebo were identified based on the radiographic classification of the index joint and either age or smoking status. These secondary-data analyses identified distinct and common patient-based variables to predict response to placebo or tanezumab. These findings will inform the design of future clinical trials helping to move forward clinical pharmacology and translational science.

Alzheimer's disease (AD) is one of the most prevalent and devastating neurodegenerative disorders which affects tens of millions of patients. It imposes significant physical, emotional, and financial burdens on patients, families, and society. Formation of Amyloid-β (Aβ) plaques from the accumulation of Aβ peptides in the cortex and hippocampal region is considered one of the fundamental neuropathological pillars of the disease. Pathological Aβ peptides are generated from its precursor protein through proteolysis. Four small molecule medications were approved by the U. S. Food and Drug Administration (FDA) since 1968 to treat the cognitive symptoms of AD, but they are not curative and have no effect on the development of AD. At least four anti-Aβ antibodies and dozens of other anti-Aβ small molecule agents have been evaluated in clinical trials in the past decades but none of them showed consistent clinical efficacy, presumably because they cannot remove Aβ plaques effectively enough or other reasons such as the patient selection and trial design. Despite the failures, more anti-Aβ antibodies are under development, aiming for more efficient Aβ plaques removal through selectively targeting Aβ oligomers. Among those aducanumab was approved by FDA in 2021 as the first drug to address the underlying biology of AD rather than the symptoms.

Formation of Tau protein aggregates in neurons is a pathological hallmark of several neurodegenerative diseases, including Alzheimer’s disease. Fluorescently-labeled Tau protein is therefore useful to study the aggregation of these pathological proteins and to identify potential therapeutic targets. Conventionally, cysteine residues are used for labeling Tau proteins, however, the full-length Tau isoform contains two cysteine residues in the microtubule-binding region, which are implicated in Tau aggregation by forming intermolecular disulfide bonds. In order to prevent the fluorescent label from disturbing the microtubule binding region, we developed a strategy to fluorescently label Tau at its C-terminus while leaving cysteine residues unperturbed. We took advantage of a Sortase A-mediated transpeptidation approach to bind a short peptide (GGGH6-Alexa647) with a His-tag and a covalently attached Alexa 647 fluorophore to the C-terminus of Tau. This reaction relies on the presence of a Sortase recognition motif (LPXTG), which we attached to the C-terminus of recombinantly expressed Tau. We determined the possible effects of the resulting C-terminal modification on the secondary structure of Tau protein, its aggregation kinetics, and fibril morphology compared to the unlabeled native Tau protein in vitro. The results showed no significant differences between the native and C-terminally labeled Tau monomer with regard to aggregation kinetics, secondary structure, and morphology.

Purpose This research project examined the vocabulary growth patterns of typically developing (TD) Cantonese–English dual language learners (DLLs) and their peers who are at risk for language impairment. Method Two studies were done. Study 1 examined the concurrent validity of a pilot vocabulary screening measure, the Kai Ming Vocabulary Test, that was designed for Cantonese–English preschool DLLs. Participants were 53 preschool children who learned Cantonese as their first language (L1) and English as a second language (L2). Their scores on the Kai Ming Vocabulary Test were examined in relation to their language sample measures, including number of different words and mean length of utterance, in L1 and in L2. Study 2 examined the vocabulary growth patterns of 24 TD Cantonese–English DLL preschoolers and 24 DLLs who are at risk for language impairment (At-Risk group). Each child was tested 3 times during an academic year. Results Results of Study 1 showed that children's vocabulary scores ( n = 53) were significantly correlated with their language sample measures in each language. In Study 2, hierarchical linear models were used to compare the TD and At-Risk groups. Results showed that children in the At-Risk group had lower L1 receptive vocabulary scores and children in the TD group had relatively higher L2 expressive vocabulary gains. Conclusions Children who are at risk are likely to continue to have lower vocabulary knowledge compared to their TD peers. The growth trajectories of DLLs provide valuable information for early identification and intervention.

Background: A number of guidelines have been developed to improve standardization of the terminology and criteria for cytologic specimens obtained from the thyroid, pancreas, lung, and salivary glands. A major goal of these guidelines is to improve reproducibility and understanding of the reporting of diagnostic results among cytopathologists and between cytopathologists and clinicians. The International Academy of Cytology Yokohama System for Reporting Breast Fine-Needle Aspiration Biopsy Cytopathology (IAC YSRB) is the most recent of these guidelines. The value of this system is, in part, dependent upon interobserver reproducibility. Design: Ninety consecutive fine-needle aspiration biopsies (FNAB) of the breast, performed over a 6-year period, were independently evaluated by 4 board-certified pathologists blinded to the original diagnoses. The 5 diagnostic categories used were those of the IAC YSRB according to published criteria for these categories. Observed agreement and chance corrected agreement (Fliess κ) were calculated. Differences in κ values were evaluated using the T statistic of Gwent. Statistical calculations were performed using STATA v16.0 (STATA Corp., College Station, TX, USA). Results: Overall agreement between observers was good. Observed unweighted agreement was 69% and weighted agreement was 91%. The majority of diagnoses were concordant (68.6%). Conclusions: Interobserver agreement of 4 cytopathologists was good using the 5 categories of the IAC YRSB (69%). Agreement was greater among pathologists with more years of experience. The IAC YSRB system appears to provide greater agreement among viewers than guidelines for cytologic specimens obtained from some other body sites (salivary gland and lung). Most discrepancies were only by a single category, with only 22/113 (19%) differing by more than one category.

Glycosylphosphatidylinositol (GPI) is a glycolipid membrane anchor found on surface proteins in all eukaryotes. It is synthesized in the ER membrane. Each GPI anchor requires three molecules of ethanolamine phosphate (P-Etn), which are derived from phosphatidylethanolamine (PE). We found that efficient GPI anchor synthesis in Saccharomyces cerevisiae requires Csf1; cells lacking Csf1 accumulate GPI precursors lacking P-Etn. Structure predictions suggest Csf1 is a tube-forming lipid transport protein like Vps13. Csf1 is found at contact sites between the ER and other organelles. It interacts with the ER protein Mcd4, an enzyme that adds P-Etn to nascent GPI anchors, suggesting Csf1 channels PE to Mcd4 in the ER at contact sites to support GPI anchor biosynthesis. CSF1 has orthologues in Caenorhabditis elegans (lpd-3) and humans (KIAA1109/TWEEK); mutations in KIAA1109 cause the autosomal recessive neurodevelopmental disorder Alkuraya-Kučinskas syndrome. Knockout of lpd-3 and knockdown of KIAA1109 reduced GPI-anchored proteins on the surface of cells, suggesting Csf1 orthologues in human cells support GPI anchor biosynthesis.

A computational platform, Boolean network explorer (BoNE), has recently been developed to infuse AI-enhanced precision into drug discovery; it enables invariant Boolean Implication Networks of disease maps for prioritizing high-value targets. Here we used BoNE to query an Inflammatory Bowel Disease (IBD)-map and prioritize a therapeutic strategy that involves dual agonism of two nuclear receptors, PPARα/γ. Balanced agonism of PPARα/γ was predicted to modulate macrophage processes, ameliorate colitis, ‘reset’ the gene expression network from disease to health. Predictions were validated using a balanced and potent PPARα/γ-dual-agonist (PAR5359) in Citrobacter rodentium- and DSS-induced murine colitis models. Using inhibitors and agonists, we show that balanced-dual agonism promotes bacterial clearance efficiently than individual agonists, both in vivo and in vitro. PPARα is required and sufficient to induce the pro-inflammatory cytokines and cellular ROS, which are essential for bacterial clearance and immunity, whereas PPARγ-agonism blunts these responses, delays microbial clearance; balanced dual agonism achieved controlled inflammation while protecting the gut barrier and ‘reversal’ of the transcriptomic network. Furthermore, dual agonism reversed the defective bacterial clearance observed in PBMCs derived from IBD patients. These findings not only deliver a macrophage modulator for use as barrier-protective therapy in IBD, but also highlight the potential of BoNE to rationalize combination therapy.

Here we present a protocol for collecting large-volume, four-color, single-molecule localization imaging data from neural tissue. We have applied this technique to map the location and identities of chemical synapses across whole cells in mouse retinae. Our sample preparation approach improves 3D STORM image quality by reducing tissue scattering, photobleaching, and optical distortions associated with deep imaging. This approach can be extended for use on other tissue types enabling life scientists to perform volumetric super-resolution imaging in diverse biological models. For a detailed application of this protocol, please refer to Sigal et al., 2015.

The self-assembly of amyloid-β (Aβ) peptides into amyloid aggregates is a pathological hallmark of Alzheimer's Disease. We previously reported a fluorescent Aryl Cyano Amide (ARCAM) probe that exhibits an increase in fluorescence emission upon binding to Aβ aggregates in solution and in neuronal tissue. Here, we investigate the effect of introducing small aliphatic substituents on the spectroscopic properties of ARCAM both free in solution and when bound to aggregated Aβ. We found that introducing substituents designed to hinder the rotation of bonds between the electron donor and acceptor on these fluorophores can affect the overall brightness of fluorescence emission of the probes in amyloid-free solutions, but the relative fluorescence enhancement of these probes in amyloid-containing solutions is dependent on the location of the substituents on the ARCAM scaffold. We also observed the capability to tune the excitation or emission wavelength of these probes by introducing electron-donating or -withdrawing substituents that putatively affect either the energy required for photoexcitation or the stability of the photoexcited state. These studies reveal new design principles for developing ARCAM-based fluorescent Aβ-binding probes with an enhanced fluorescence signal compared to background and tunable spectroscopic properties, which may lead to improved chemical tools for aiding in the diagnosis of amyloid-associated neurodegenerative diseases.

Protein pores recently enabled a breakthrough in bioanalytics by making it possible to sequence individual DNA and RNA strands during their translocation through the lumen of the pore. Despite this success and the overall promise of nanopore-based single-molecule analytics, protein pores have not yet reached their full potential for the analysis and characterization of globular biomolecules such as natively folded proteins. One reason is that the diameters of available protein pores are too small for accommodating the translocation of most folded globular proteins through their lumen. The work presented here provides a step toward overcoming this limitation by programmed self-assembly of α-helical pore-forming peptides with covalently attached single-stranded DNA (ssDNA). Specifically, hybridization of the peptide ceratotoxin A (CtxA) with N-terminally attached ssDNA to a complementary DNA template strand with 4, 8, or 12 hybridization sites made it possible to trigger the assembly of pores with various diameters ranging from approximately 0.5 to 4 nm. Hybridization of additional DNA strands to these assemblies achieved extended functionality in a modular fashion without the need for modifying the amino acid sequence of the peptides. For instance, functionalization of these semisynthetic biological nanopores with DNA-cholesterol anchors increased their affinity to lipid membranes compared to pores formed by native CtxA, while charged transmembrane segments prolonged their open-state lifetime. Assembly of these hybrid DNA-peptides by a template increased their cytotoxic activity and made it possible to kill cancer cells at 20-fold lower total peptide concentrations than nontemplated CtxA.

Purpose To investigate the use of an amyloid-targeting fluorescent probe, ARCAM-1, to identify amyloid-containing deposits in the retina of a transgenic mouse model of Alzheimer's disease (AD) and in human postmortem AD patients. Methods Aged APP/PS1 transgenic AD and wild-type (WT) mice were given an intraperitoneal (IP) injection of ARCAM-1 and their retinas imaged in vivo using a fluorescence ophthalmoscope. Eyes were enucleated and dissected for ex vivo inspection of retinal amyloid deposits. Additionally, formalin-fixed eyes from human AD and control patients were dissected, and the retinas were stained using ARCAM-1 or with an anti-amyloid-β antibody. Confocal microscopy was used to image amyloid-containing deposits stained with ARCAM-1 or with immunostaining. Results Four out of eight APP/PS1 mice showed the presence of amyloid aggregates in the retina during antemortem imaging. Retinas from three human AD patients stained with ARCAM-1 showed an apparent increased density of fluorescently labeled amyloid-containing deposits compared to the retinas from two healthy, cognitively normal (CN) patients. Immunolabeling confirmed the presence of amyloid deposits in both the retinal neuronal layers and in retinal vasculature. Conclusions ARCAM-1 facilitates antemortem detection of amyloid aggregates in the retina of a mouse model for AD, and postmortem detection of amyloid-containing deposits in human retinal tissues from AD patients. These results support the hypothesis of AD pathology manifesting in the eye and highlight a novel area for fluorophore development for the optical detection of retinal amyloid in AD patients. Translational relevance This paper represents an initial examination for potential translation of an amyloid-targeting fluorescent probe to a retinal imaging agent for aiding in the diagnosis of Alzheimer's disease.

Preeclampsia (PE) is a hypertensive disorder of pregnancy and one of the leading contributors to both maternal and perinatal morbidity and mortality. Reliable diagnostic parameters unique to the disorder that accurately define and diagnose PE are currently unavailable. Recent studies have revealed that PE is accompanied by the accumulation of amyloidogenic deposits in the placenta and the presence of congophilic amyloid-like protein aggregates in the urine. Here, we evaluate the capability of an amyloid-targeting aryl cyano amide (ARCAM-1) fluorophore to identify PE patients from analysis of urine samples. Our results reveal that this probe can distinguish patients with PE from gestationally healthy patients and patients suffering from non-PE hypertension, highlighting the potential for amyloid-targeting fluorophores to help identify PE patients during pregnancy.

Breast-conserving therapy (BCT) offers oncologic outcomes similar to those of mastectomy, yet many patients, when provided the option, choose mastectomy. This study aimed to evaluate the relationship between patient-reported distress and surgical decisions and to determine factors predictive of choosing BCT versus mastectomy. Patients with newly diagnosed breast cancer deemed candidates for BCT who completed a distress screen at their initial visit to an academic institution between 2016 and 2019 were retrospectively reviewed. This screening tool captures distress in emotional, social, health, and practical domains on a scale of 0 to 10. The distress scores were compared against surgical decisions using nonparametric Wilcoxon rank-sum tests. Patient factors associated with surgical choice were analyzed using chi-square, Fisher’s exact, and Student’s t tests. A two-sided p value lower than 0.05 was considered significant. Of 506 patients deemed eligible for BCT, 430 (85%) chose BCT and 76 (15%) pursued mastectomy. The distress levels did not differ significantly between the surgical options. The patients who underwent mastectomy were on the average younger (50.7 vs 60.4 years; p

Thermoacidophilic archaea are able to survive in low pH environments by maintaining large proton gradients across their cell membranes. The unique structure of the unipolar and bipolar lipids that compose these membranes, with branched isoprene chains and ether linkages, may contribute to their reduced permeability to protons at elevated temperature. Here we present a proton permeability assay that interrogates hundreds of surface-tethered single liposomes in parallel using total internal reflection fluorescence (TIRF) microscopy to determine a proton permeability value for each liposome. The large number of liposomes that can be individually interrogated by this microscopy platform allow for the collection and statistical analysis of large quantities of data within a single experiment, as opposed to the single, averaged result produced by a bulk fluorimetry assay. In addition, tethering individual liposomes to a surface prevents liposome aggregation in free solution, which is especially prevalent when performing studies at elevated temperatures. Using a temperature-controlled chamber, we demonstrated a decreased temperature-dependence of proton permeability for bipolar archaea-mimetic membranes in comparison to unipolar eukaryote-mimetic membranes. Eyring-Polanyi analysis revealed a high entropic cost associated with proton flux across the archaea-mimetic membranes. Furthermore, analysis of the distribution of proton permeability values within populations of liposomes revealed a positive correlation between size and permeability in the eukaryote-mimetic liposomes that was absent in the archaea-mimetic liposomes.

The COVID-19 pandemic has led to the implementation of unprecedented policy actions by central banks around the world. Along with the reduction of interest rates and the use of asset purchase and lending programs, central bank communications have been actively deployed as a policy tool.

Introduction: There is a lack of data reflecting the trend of neonatal pneumothorax in regional Australia. The aim of this study is to review the incidence and characteristics of neonates diagnosed with pneumothorax in Central Queensland, analyse outcomes in terms of the ability of local hospitals to manage this condition, and describe predictors for severe disease requiring transfer to a tertiary centre. Thus the role of regional health services in managing this condition will be reviewed. Methods: This was a retrospective observational study of all neonates born between 1 January 2008 and 31 December 2015 coded by hospital records with a diagnosis of neonatal pneumothorax in Central Queensland. Data for sex and birth gestation for all Central Queensland births of the same period were also obtained. Descriptive statistics were calculated for birth weight and gestation, and Apgar scores. Frequencies were calculated for sex, length of admission, age of diagnosis and risk factors including meconium aspiration syndrome (MAS), prolonged rupture of membranes (PROM) and positive pressure ventilation (PPV). The primary outcome measure was successful treatment at a Central Queensland hospital versus requirement for transfer to tertiary hospital or death prior to transfer. Statistical significance was calculated for binary and continuous variables. Results: During the study period, there were 31 cases of pneumothorax amongst 17 640 deliveries recorded by three Central Queensland hospitals, with a significant bias towards males (84%) amongst pneumothorax cases (p

The aggregation of misfolded proteins into amyloids is a common characteristic of many neurodegenerative and non-neurologic diseases. Fluorescent amyloid-targeting probes that discriminate amyloids based on differences in protein composition can provide rapid information to aid in disease diagnosis. In this chapter, we present protocols for the synthesis and use of ANCA-11 as an environmentally-sensitive amyloid-targeting probe that can fluorescently discriminate between amyloids with different disease origin. We also present a protocol for preparing amyloid samples of synthetic Amyloid-β(1-42), as problems with amyloid preparations can be a large driver of time and cost for research. The methods presented here can be generalized for evaluation of other amyloid-targeting fluorescent probes with different aggregates of amyloidogenic proteins in solution or in tissue.

The aggregation of misfolded proteins into amyloids is a common characteristic of many neurodegenerative and non-neurologic diseases. Fluorescent amyloid-targeting probes that discriminate amyloids based on differences in protein composition can provide rapid information to aid in disease diagnosis. In this chapter, we present protocols for the synthesis and use of ANCA-11 as an environmentally-sensitive amyloid-targeting probe that can fluorescently discriminate between amyloids with different disease origin. We also present a protocol for preparing amyloid samples of synthetic Amyloid-β(1-42), as problems with amyloid preparations can be a large driver of time and cost for research. The methods presented here can be generalized for evaluation of other amyloid-targeting fluorescent probes with different aggregates of amyloidogenic proteins in solution or in tissue.

A major challenge in liposomal research is to minimize the leakage of encapsulated cargo from either uncontrolled passive permeability across the liposomal membrane or upon fusion with other membranes. We previously showed that liposomes made from pure Archaea-inspired bipolar tetraether lipids exhibit exceptionally low permeability of encapsulated small molecules due to their capability to form more tightly packed membranes compared to typical monopolar lipids. Here, we demonstrate that liposomes made of synthetic bipolar tetraether lipids can also undergo membrane fusion, which is commonly accompanied by content leakage of liposomes when using typical bilayer-forming lipids. Importantly, we demonstrate calcium-mediated fusion events between liposome made of glycerolmonoalkyl glycerol tetraether lipids with phosphatidic acid headgroups (GMGTPA) occur without liposome content release, which contrasts with liposomes made of bilayer-forming EggPA lipids that displayed ~80% of content release under the same fusogenic conditions. NMR spectroscopy studies of a deuterated analog of GMGTPA lipids reveal the presence of multiple rigid and dynamic conformations, which provide evidence for the possibility of these lipids to form intermediate states typically associated with membrane fusion events. The results support that biomimetic GMGT lipids possess several attractive properties (e.g., low permeability and non-leaky fusion capability) for further development in liposome-based technologies.

The current study examined whether the vocabulary skills of sequential bilingual children who learned Cantonese as a home language (L1) and English as a second language (L2) were predicted by the amount of L1 and L2 used at home. Ninety-two preschool children who learned Cantonese as L1 were recruited from a Head Start program. The amounts of L1 and L2 used at home were measured using parent questionnaires. Mixed patterns of L1 and L2 use were found across family members and home activities. After controlling for time spent in preschool, regression analyses showed that the amount of L1 and L2 used by individual family members, with the exception of older siblings, was not significantly linked to children's vocabulary skills. In contrast, the language used during some home activities such as dinner and book reading significantly predicted children's vocabulary knowledge. Implications for family involvement in facilitating children's vocabulary development are discussed.