Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells.

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

Associations between thalamocortical functional connectivity and sensory over-responsivity in infants at high likelihood for ASD.

Despite growing evidence implicating thalamic functional connectivity atypicalities in autism spectrum disorder (ASD), it remains unclear how such alterations emerge early in human development. Because the thalamus plays a critical role in sensory processing and neocortical organization early in life, its connectivity with other cortical regions could be key for studying the early onset of core ASD symptoms. Here, we investigated emerging thalamocortical functional connectivity in infants at high (HL) and typical (TL) familial likelihood for ASD in early and late infancy. We report significant thalamo-limbic hyperconnectivity in 1.5-month-old HL infants, and thalamo-cortical hypoconnectivity in prefrontal and motor regions in 9-month-old HL infants. Importantly, early sensory over-responsivity (SOR) symptoms in HL infants predicted a direct trade-off in thalamic connectivity whereby stronger thalamic connectivity with primary sensory regions and basal ganglia was inversely related to connectivity with higher order cortices. This trade-off suggests that ASD may be characterized by early differences in thalamic gating. The patterns reported here could directly underlie atypical sensory processing and attention to social vs. nonsocial stimuli observed in ASD. These findings lend support to a theoretical framework of ASD whereby early disruptions in sensorimotor processing and attentional biases early in life may cascade into core ASD symptomatology.

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs.

O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.

Requirement of the N-terminal activation domain of herpes simplex virus ICP4 for viral gene expression.

ICP4 is the major activator of herpes simplex virus (HSV) transcription. Previous studies have defined several regions of ICP4 that are important for viral gene expression, including a DNA binding domain and transactivation domains that are contained in the C-terminal and N-terminal 520 and 274 amino acids, respectively. Here we show that the N-terminal 210 amino acids of ICP4 are required for interactions with components of TFIID and mediator and, as a consequence, are necessary for the activation of viral genes. A mutant of ICP4 deleted for amino acids 30 to 210, d3-10, was unable to complement an ICP4 null virus at the level of viral replication. This was the result of a severe deficiency in viral gene and protein expression. The absence of viral gene expression coincided with a defect in the recruitment of RNA polymerase II to a representative early promoter (thymidine kinase [TK]). Affinity purification experiments demonstrated that d3-10 ICP4 was not found in complexes with components of TFIID and mediator, suggesting that the defect in RNA polymerase II (Pol II) recruitment was the result of ablated interactions between d3-10 and TFIID and mediator. Complementation assays suggested that the N-terminal and C-terminal regions of ICP4 cooperate to mediate gene expression. The complementation was the result of the formation of more functional heterodimers, which restored the ability of the d3-10-containing molecules to interact with TFIID. Together, these studies suggest that the N terminus contains a true activation domain, mediating interactions with TFIID, mediator, and perhaps other transcription factors, and that the C terminus of the molecule contains activities that augment the functions of the activation domain.

The N terminus and C terminus of herpes simplex virus 1 ICP4 cooperate to activate viral gene expression.

Infected cell polypeptide 4 (ICP4) activates transcription from most viral promoters. Two transactivation domains, one N-terminal and one C terminal, are largely responsible for the activation functions of ICP4. A mutant ICP4 molecule lacking the C-terminal activation domain (n208) efficiently activates many early genes, whereas late genes are poorly activated, and virus growth is severely impaired. The regions within the N terminus of ICP4 (amino acids 1 to 210) that contribute to activation were investigated by analysis of deletion mutants in the presence or absence of the C-terminal activation domain. The mutants were assessed for their abilities to support viral replication and to regulate gene expression. Several deletions in regions conserved in other alphaherpesviruses resulted in impaired activation and viral growth, without affecting DNA binding. The single small deletion that had the greatest effect on activation in the absence of the C terminus corresponded to a highly conserved stretch of amino acids between 81 and 96, rendering the molecule nonfunctional. However, when the C terminus was present, the same deletion had a minimal effect on activity. The amino terminus of ICP4 was predicted to be relatively disordered compared to the DNA-binding domain and the C-terminal 500 amino acids. Moreover, the amino terminus appears to be in a relatively extended conformation as determined by the hydrodynamic properties of several mutants. The data support a model where the amino terminus is an extended and possibly flexible region of the protein, allowing it to efficiently interact with multiple transcription factors at a distance from where it is bound to DNA, thereby enabling ICP4 to function as a general activator of polymerase II transcription. The C terminus of ICP4 can compensate for some of the mutations in the N terminus, suggesting that it either specifies redundant interactions or enables the amino terminus to function more efficiently.

A natural language fMRI dataset for voxelwise encoding models.

Speech comprehension is a complex process that draws on humans' abilities to extract lexical information, parse syntax, and form semantic understanding. These sub-processes have traditionally been studied using separate neuroimaging experiments that attempt to isolate specific effects of interest. More recently it has become possible to study all stages of language comprehension in a single neuroimaging experiment using narrative natural language stimuli. The resulting data are richly varied at every level, enabling analyses that can probe everything from spectral representations to high-level representations of semantic meaning. We provide a dataset containing BOLD fMRI responses recorded while 8 participants each listened to 27 complete, natural, narrative stories (~6 hours). This dataset includes pre-processed and raw MRIs, as well as hand-constructed 3D cortical surfaces for each participant. To address the challenges of analyzing naturalistic data, this dataset is accompanied by a python library containing basic code for creating voxelwise encoding models. Altogether, this dataset provides a large and novel resource for understanding speech and language processing in the human brain.

'Giving back' through mobility trajectories: motivations for engaging in development encounters in Ghana among transnational youth.

Literature on diaspora engagement in development activities has centred on the contributions of migrating adults to the 'homeland', which range from private transfers to single households, to community development projects. While such studies often focus on the impact of such activities on the country of origin, relatively few have focused on what transpires during development encounters and how this affects migrants', and especially young people's, motivation to engage transnationally over time. This paper combines migration and development, transnational migration studies and second generation 'returns' literature, to address these gaps. It studies the motivations of transnational youth to engage in development encounters, which they referred to as 'giving back', in the context of their mobility trajectories. Drawing on 17 months of multi-sited ethnographic fieldwork in the Netherlands and accompanying young people during trips to Ghana, we show that giving back contributes to a sense of purpose that connects them transnationally. Young people's expectations of giving back were embedded in community narratives, which framed this as a means to 'become successful' in culturally valued ways. While young people sometimes encountered unexpected surprises, emotions experienced during development encounters led to learning that ultimately resulted in enhanced intentions of transnational engagement.

Challenge of diagnosing splenic torsion in a paediatric patient with gastroschisis.

Gastroschisis is an uncommon congenital defect of the abdominal wall resulting in intestinal prolapse, most commonly associated with short gut syndrome or bowel obstruction. Wandering spleen, movement of the spleen due to the underdevelopment of splenic ligaments, has a prevalence of 0.25% and is asymptomatic in 15% of paediatric cases. An 11-year-old patient, admitted with a history of gastroschisis repaired at birth, presents with 18 months of intermittent, worsening abdominal pain. Imaging demonstrated splenomegaly and tortuosity of the splenic vein with abnormal positioning of the superior mesenteric artery and vein. The patient was found to have a wandering spleen with subacute splenic infarct secondary to splenic torsion, necessitating emergent surgical intervention. This patient experienced an extremely rare complication of gastroschisis that has not previously been reported. This complication is caused by a lack of appropriate abdominal fixation points for the spleen.

What Do Kayaking and a Hog Have in Common? Two Cases of Aeromonas Hydrophila infection in Georgia.

We highlight 2 cases of immunocompetent pediatric patients diagnosed with osteomyelitis and cellulitis attributed to Aeromonas hydrophila. One case had a direct water source; however, the second case occurred following a wild hog bite. Both required fluoroquinolone treatment and demonstrated the need to consider A. hydrophila in patients with water exposure, animal bites and initial antibiotic failure.

Orexin mediates initiation of sexual behavior in sexually naive male rats, but is not critical for sexual performance.

The hypothalamic neuropeptide orexin mediates arousal, sleep, and naturally rewarding behaviors, including food intake. Male sexual behavior is altered by orexin receptor-1 agonists or antagonists, suggesting a role for orexin-A in this naturally rewarding behavior. However, the specific role of endogenous orexin-A or B in different elements of male sexual behavior is currently unclear. Therefore, the current studies utilized markers for neural activation and orexin cell-specific lesions to test the hypothesis that orexin is critical for sexual motivation and performance in male rats. First, cFos expression in orexin neurons was demonstrated following presentation of a receptive or non-receptive female without further activation by different elements of mating. Next, the functional role of orexin was tested utilizing orexin-B conjugated saporin, resulting in orexin cell body lesions in the hypothalamus. Lesions were conducted in sexually naive males and subsequent sexual behavior was recorded during four mating trials. Lesion males showed shortened latencies to mount and intromit during the first, but not subsequent mating trials, suggesting lesions facilitated initiation of sexual behavior in sexually naive, but not experienced males. Likewise, lesions did not affect sexual motivation in experienced males, determined by runway tests. Finally, elevated plus maze tests demonstrated reduced anxiety-like behaviors in lesioned males, supporting a role for orexin in anxiety associated with initial exposure to the female in naive animals. Overall, these findings show that orexin is not critical for male sexual performance or motivation, but may play a role in arousal and anxiety related to sexual behavior in naive animals.

Social and neuromolecular phenotypes are programmed by prenatal exposures to endocrine-disrupting chemicals.

Exposures to endocrine-disrupting chemicals (EDCs) affect the development of hormone-sensitive neural circuits, the proper organization of which are necessary for the manifestation of appropriate adult social and sexual behaviors. We examined whether prenatal exposure to polychlorinated biphenyls (PCBs), a family of ubiquitous industrial contaminants detectable in virtually all humans and wildlife, caused changes in sexually-dimorphic social interactions and communications, and profiled the underlying neuromolecular phenotype. Rats were treated with a PCB commercial mixture, Aroclor 1221 (A1221), estradiol benzoate (EB) as a positive control for estrogenic effects of A1221, or the vehicle (4% DMSO), on embryonic day (E) 16 and 18. In adult F1 offspring, we first conducted tests of ultrasonic vocalization (USV) calls in a sociosexual context as a measure of motivated communications. Numbers of certain USV call types were significantly increased by prenatal treatment with A1221 in males, and decreased by EB in females. In a test of sociosexual preference for a hormone-vs. a non-hormone-primed opposite sex conspecific, male (but not female) nose-touching with opposite-sex rats was significantly diminished by EDCs. Gene expression profiling was conducted in two brain regions that are part of the social decision-making network in the brain: the medial preoptic nucleus (MPN) and the ventromedial nucleus (VMN). In both regions, many more genes were affected by A1221 or EB in females than males. In female MPN, A1221 changed expression of steroid hormone receptor and neuropeptide genes (e.g., Ar, Esr1, Esr2, and Kiss1). In male MPN, only Per2 was affected by A1221. The VMN had a number of genes affected by EB compared to vehicle (females: Kiss1, Kiss1r, Pgr; males: Crh) but not A1221. These differences between EB and A1221 indicate that the mechanism of action of A1221 goes beyond estrogenic pathways. These data show sex-specific effects of prenatal PCBs on adult behaviors and the neuromolecular phenotype.

Quality Improvement Project to Improve Screening for Tobacco Use in Adolescent Inpatients at a Children's Hospital.

Tobacco use begins in adolescence for the majority of smokers. The purpose of this study was to increase screening and reporting of tobacco use in hospitalized adolescents at a tertiary care children's hospital. We completed a nursing focus group to understand challenges and completed four iterative Plan-Do-Study-Act cycles, which included: (1) in-person nursing education regarding tobacco use screening, (2) addition of an e-cigarette-specific screening question, (3) the creation and dissemination of an educational video for nursing, and (4) adding the video as a mandatory component of nursing orientation. Run charts of the percentage of patients screened who reported tobacco use were created. Absolute counts of tobacco products used were also captured. From January 2016 to September 2018, 12,999 patients ≥13 years of age were admitted to the hospital. At baseline, 90.1% of patients were screened and 4.8% reported tobacco use. While the absolute number of adolescents reporting e-cigarette use increased from zero patients per month at baseline to five, the percentage of patients screened and reporting tobacco use was unchanged; the majority of e-cigarette users reported use of other tobacco products. This study demonstrates that adding e-cigarettes to screening increases reporting and suggests systems level changes are needed to improve tobacco use reporting.

A best-worst scaling experiment to prioritize caregiver concerns about ADHD medication for children.

OBJECTIVE: The objective of this feasibility study was to develop and pilot an instrument to elicit caregivers' priorities when initiating attention-deficit hyperactivity disorder (ADHD) medication for their child. METHODS: A best-worst scaling experiment was used to rank competing priorities when initiating ADHD medicine. Forty-six participants were recruited for a two-phase study involving survey development (phase 1, N=21) and the survey pilot (phase 2, N=25). Best-worst scores and 95% confidence intervals indicating the relative importance of 16 concerns were determined, and t tests were used to determine the scores' significance. RESULTS: The significance of best-worst scores for most concerns indicated that the choices were purposeful. Concerns about helping the child become a successful adult, having a doctor who addresses caregivers' concerns, and improving school behavior were ranked highest. CONCLUSIONS: The best-worst scaling method can elicit priorities for children's mental health treatment. Future work using this method will guide family-centered care.

Radiopacity and hounsfield attenuation of cystine urolithiasis: case series and review of the literature.

OBJECTIVES: Given the high recurrence rate of cystine urolithiasis, understanding of the radiographic stone characteristics is important in following cystine stone formers over their lifetime. However, due to their infrequent incidence, in vivo radiographic properties of cystine stones have not been well characterized. The purpose of our study is to characterize the in vivo radiographic properties of cystine urolithiasis. METHODS: Patients with a cystine stone analysis and noncontrast computed tomography (NCCT) were extracted from our stone clinic database. Stone attenuation in Hounsfield units (HU) was measured for each stone and plain abdominal films (kidney, ureter, and bladder radiograph [KUB]) within 30 days of the NCCT prior to any intervention were reviewed by a blinded radiologist to assess whether urolithiasis could be visualized. RESULTS: Twenty patients met our study inclusion criteria. When plotted by attenuation, two distinct groups of stone attenuation were noted for cystine stone formers (p<0.001). The largest group (n=16) had an attenuation of <550 HU (424±106 HU), while a distinct second group (n=4) was >850 HU (972±134 HU). Sixteen patients had a KUB, with 88% of the stones being visualized by a blinded radiologist. Stone size and attenuation were not significantly different between visualized and nonvisualized stones via KUB, however, the body mass index was significantly higher in the nonvisualized group (34.4 vs 26.9 kg/m(2), p=0.03). CONCLUSIONS: Cystine stones were visualized by KUB, which has implications in post-treatment follow-up imaging. Though most cystine stones had an attenuation of <550 HU, a second distinct group of cystine stones were noted to have a high attenuation of >850 HU. HU measurements alone are not sufficient to differentiate cystine stones from other stone compositions.

Temporal association of herpes simplex virus ICP4 with cellular complexes functioning at multiple steps in PolII transcription.

The herpes simplex virus type 1 (HSV-1) immediate early protein, ICP4, participates in the regulation of viral gene expression by both activating and repressing RNA polII transcription. We used affinity purification of ICP4 expressed in infected cells followed by mass spectrometry and western blot analysis to determine the composition of cellular complexes associated with ICP4 throughout infection. ICP4 was associated with TFIID complexes containing a distinct set of TAFs. These complexes were most abundant early, but were detected throughout infection, whereas Mediator was found in ICP4 containing complexes later in infection, indicating a temporal pattern for the utilization of these complexes for the transcription of the viral genome. The form of Mediator copurifying with ICP4 was enriched for the kinase domain and also lacked the activator-specific component, Med26, suggesting that Mediator-ICP4 interactions may be involved in repression of viral transcription. The N-terminal 774 amino acids of ICP4, which retains partial function, were sufficient to form complexes with TFIID and Mediator, although these interactions were not as strong as with full-length ICP4. Additionally, components involved in transcription elongation, chromatin remodeling, and mRNA processing were isolated with ICP4. Together our data indicate that ICP4 plays a more integrated role in mediating HSV transcription, possibly affecting multiple steps in transcription and gene expression.

Socioeconomic variation in recall and perceived effectiveness of campaign advertisements to promote smoking cessation.

There are large disparities in cigarette smoking rates by socioeconomic status (SES) in many countries. There is mixed evidence about the relative effectiveness of smoking cessation media campaigns in promoting quitting between lower and higher SES populations, and studies suggest that some types of ad content may have differential effects by SES. We analyzed data from five waves of the New York Media Tracking Survey Online (MTSO), a web survey involving over 7000 adult smokers conducted between 2007 and 2009, to assess SES variation in response to smoking cessation ads. Smokers with low levels of education and income less often recalled ads focused on how to quit, and perceived them as less effective, than ads using graphic imagery or personal testimonials to convey why to quit. Contrary to predictions offered by the Stages of Change Model, we found no evidence that variation in readiness to quit smoking explained patterns of response by education. Results offer guidance for theorists and campaign planners in developing campaigns that are likely to promote cessation among less educated populations.

Women's perceptions about the etiology of urinary incontinence.

OBJECTIVE: Incontinent women have low rates of care seeking and treatment, some of which may be explained by their beliefs about the causes of their own urine loss. As little is known about these beliefs, our aim was to qualitatively assess what women perceive as the etiology of their urinary incontinence (UI). METHODS: In a written survey on urinary symptoms administered to female HMO enrollees aged 30-90 years, incontinent women were asked the open-ended question: "Why do you think you lose urine?" Qualitative analyses of the responses identified themes, which were quantified and organized into major categories. Subjects were assigned multiple themes/categories as indicated. Relationships between major categories and subject/incontinence characteristics were explored. RESULTS: Of the 1458 women with incontinence who completed the survey, 1192 (82%) responded to the open-ended question. Qualitative analyses identified 23 themes, with 5 themes cited by >or=10% of subjects: pelvic floor/bladder muscles (31%), pregnancy/childbirth (18%), age (14%), exertional triggers (12%), and waiting too long to void (10%). The 23 themes were organized into 5 major categories: pelvic floor/bladder related (53%), uncontrollable factors (23%), part of being female (21%), personal/lifestyle attributes (21%), and don't know (12%). Subjects expressed a range of 1-4 themes/categories. Major categories differed by age, and significant associations were seen between major categories and incontinence severity. CONCLUSIONS: Women attribute their UI to a number of causes, which can be condensed into workable themes and categories. By understanding women's beliefs about the etiology of incontinence, clinicians may improve their ability to educate, counsel, and treat women with incontinence.