Search

Background and Aims: A substantial minority of autistic individuals score within typical ranges on standard language tests, suggesting that autism does not necessarily affect language acquisition. This idea is reflected in current diagnostic criteria for autism, wherein language impairment is no longer included. However, some work has suggested that probing autistic speakers' language carefully may reveal subtle differences between autistic and nonautistic people's language that cannot be captured by standardized language testing. The current study aims to test this idea, by determining whether a group of autistic and nonautistic individuals who score similarly on a standardized test show differences in the number of unconventional and erroneous language features they produce in a spontaneous language sample., Methods: Thirty-eight older children and adolescents (19 autistic; 19 nonautistic), between the ages of 10 and 18, were recruited. Both participant groups scored within normal ranges on standardized language and IQ tests. Participants engaged in a "double interview" with an experimenter, during which they were first asked questions by the experimenter about themselves, and then they switched roles, so that it was the participant's turn to ask the experimenter questions. Participants' language during the interview was transcribed and analyzed for linguistic irregularities, including both semantic anomalies and morphosyntactic errors., Results: Group membership accounted for significant variance in irregularity frequency; autistic participants produced more linguistic irregularities than nonautistic participants. Scores on a standardized language test did not improve model fit. Secondary analyses involving irregularity type (semantic vs. morphosyntactic) showed that group differences were primarily driven by relatively high numbers of semantic unconventionalities produced by the autistic group. While the autistic group made more morphosyntactic errors than the nonautistic group, differences in these numbers were only marginally significant., Conclusions and Implications: These findings suggest that a commonly used standardized language test does not adequately predict the number and perhaps type of language irregularities produced by some older autistic children and adolescents during spontaneous discourse. Results also suggest that differences in language use, especially semantic differences, may characterize autistic language, even the language produced by people who score within normal ranges on standardized language tests. It is debatable whether differences reflect underlying language impairments and/or a linguistic style adopted/preferred by autistic speakers. In this paper, we discuss both possibilities and offer suggestions to future research for teasing these possibilities apart., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

N -Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1- exo -acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H · abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H · from C7 ∼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2 H 2 O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.

Objectives: Despite evidence for the validity of using client-led outcome measures in gender-affirming voice training (GAVT), the existing body of research on voice feminization relies heavily on acoustic-perceptual measures without additional qualitative exploration of client experience. Additionally, the authors are not aware of any existing studies prompting client input on the voice feminization methods they find most helpful in achieving their voice goals. The current study focuses on crucial client perceptions of GAVT for voice feminization and identifies the methods clients find most helpful., Methods: Using a mixed-methods approach, we gathered numeric and qualitative survey data from 21 individuals who engaged in GAVT supporting voice feminization. We conducted follow-up interviews with five survey participants to gather additional qualitative data on client experiences and perceptions of GAVT., Results: Quantitative and qualitative data reveal that clients are satisfied with GAVT supporting voice feminization, perceive their clinicians as being culturally inclusive, and identify oral/forward resonance work as one of the most helpful voice training methods. Interestingly, however, frequency of work on oral/forward resonance during training did not predict voice satisfaction post-training., Conclusions: Clients identified oral/forward resonance as one of the most helpful methods of voice feminization and found their GAVT experiences to be supportive of their voice goals. These findings emphasize the importance and value of client-led outcome measures when investigating effective approaches to GAVT., Competing Interests: Declaration of Competing Interest The first and third authors of this study declare a professional involvement in providing gender-affirming voice services to three of the study participants. This disclosure is made to ensure transparency and acknowledge our role in supporting individuals in their journey towards gender affirmation. We affirm that this potential conflict of interest has been managed appropriately throughout the research process, adhering to ethical guidelines, and maintaining the confidentiality and anonymity of the participants. Additionally, when selecting participants for the study interviews, conducted by the first author, the five participants who received services from the first and third author were excluded from consideration to minimize potential bias in interview responses stemming from this prior relationship., (Copyright © 2024 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Four previous papers reported the isolation and structural determination of 10 polycyclic polyprenylated acylphloroglucinols (PPAPs), uraliones F, G, K, and O, attenuatumiones E and F, and scabrumiones A-D, from Hypericum species. Their structures were identified as type B PPAPs that featured not only the characteristic acyl group at C-3 of the bicyclo[3.3.1]nonane core but also a partly reduced furan ring fused to the C-1-C-2-O-2 atoms of the core. However, the 1D and 2D NMR data of these compounds were more consistent with type A PPAPs that featured not only the acyl group at C-1 but also a partially reduced furan ring fused to the C-3-C-2-O-2 atoms of the core. Now we revise these 10 previously proposed structures to the corresponding type A PPAPs via NMR analysis. Additionally, we propose a rule that uses NMR data to determine whether a particular PPAP that is fused to a partly reduced furan ring at C-3-C-2-O-2 or C-1-C-2-O-2 is type A or type B, respectively. We also propose a rule to assign the relative configurations of corresponding type A PPAPs at C-18 and revise the configurations of sampsonione N, hypericumoxides A-C, and hyperscabin G.

Diamine ligands are effective structural scaffolds for tuning the reactivity of transition-metal complexes for catalytic, materials, and phosphorescent applications and have been leveraged for biological use. In this work, we report the synthesis and characterization of a novel class of cyclometalated [C^N] Au(III) complexes bearing secondary diamines including a norbornane backbone, (2 R ,3 S )- N 2 , N 3 -dibenzylbicyclo[2.2.1]heptane-2,3-diamine, or a cyclohexane backbone, (1 R ,2 R )- N 1 , N 2 -dibenzylcyclohexane-1,2-diamine. X-ray crystallography confirms the square-planar geometry and chirality at nitrogen. The electronic character of the conformationally restricted norbornane backbone influences the electrochemical behavior with redox potentials of -0.8 to -1.1 V, atypical for Au(III) complexes. These compounds demonstrate promising anticancer activity, particularly, complex 1 , which bears a benzylpyridine organogold framework, and supported by the bicyclic conformationally restricted diaminonorbornane, shows good potency in A2780 cells. We further show that a cellular response to 1 evokes reactive oxygen species (ROS) production and does not induce mitochondrial dysfunction. This class of complexes provides significant stability and reactivity for different applications in protein modification, catalysis, and therapeutics.

Purpose: Stories told by autistic narrators often contain relatively frequent use of ambiguous references. However, it remains unclear whether this ambiguity is driven by ambiguous character establishment (e.g., "Once upon a time, she/the girl…") and/or ambiguous cohesion (e.g., "Two girls lived in a castle. She/The girl…"). In this study, we directly compared rates of each type of ambiguity within and between narratives told by autistic and non-autistic children, to determine which type of ambiguity is relatively more common in narratives told by autistic children., Method: Thirty-three 10- to 17-year-old autistic participants ( n = 17) and non-autistic peers ( n = 16), who were not statistically different in age, standardized language scores, and IQ scores ( p > .8 for all), watched two short animated videos alone and then described the videos' events to two listeners who were openly unfamiliar with the videos. We transcribed video recordings of narratives and coded all referential noun phrases (NPs) as either clear or ambiguous. We further categorized ambiguous NPs as either ineffective introduction or ineffective cohesion., Results: Autistic children produced significantly higher rates of ambiguous establishment than non-autistic peers, whereas between-group comparisons' rates of ambiguous cohesion were not statistically significant., Conclusions: Older children on the autism spectrum show differences in the way they introduce characters, selecting NP types that are only appropriate when their listener is already familiar with the referent. In contrast, once they have introduced characters, they show cohesive skills that are comparable to those of non-autistic peers. Findings support theories arguing that autistic children show differences in their application of social pragmatic principles (listener/context-specific pragmatic rules), whereas their use of linguistic pragmatics (context-independent rules) is similar to that of non-autistic peers.

Garcinielliptone FC (GFC) was assigned to be a type A polycyclic polyprenylated acylphloroglucinol (PPAP) and was found to exhibit diverse biological activities. Now we revise the structure of GFC to xanthochymol, a type B PPAP, via NMR and total synthesis methods. The total syntheses of (±)-xanthochymol and (±)-cycloxanthochymol were accomplished in 12 and 13 steps, respectively.

We describe two new methods for the 1,2-diamination of alkenes. First, either an azidium ion (ArN═N + ═NAr) undergoes 1,3-dipolar cycloaddition with an alkene to give a 1,2,3-triazolinium ion directly, or an intramolecular azide-alkene cycloaddition followed by N-benzylation provides the same. Second, hydrogenation of the 1,2,3-triazolinium ion over Raney Ni excises the central N atom and gives the 1,2-diamine. The stereochemistry of the alkene is usually, but not always, preserved in the 1,2-diamine.

Many type B polycyclic polyprenylated acylphloroglucinols (PPAPs) bear a lavandulyl-derived substituent, and the configurational assignment of this side chain can be difficult and sometimes leads to erroneous conclusions. In this study, 21 PPAPs, including the new xanthochymusones A-I ( 1 - 9 ), have been isolated from the fruits of Garcinia xanthochymus and structurally characterized. The relative configuration of the C-30 stereocenter was assigned by a combination of chemical transformations, 1 H- 1 H coupling constants, conformational analysis, and NOE experiments. The configurational assignment of compound 7 indicates that the relative configuration at C-30 of PPAPs is not always the same. The absolute configurations of the new compounds were assigned by ECD and X-ray diffraction data, as well as by biosynthetic considerations. Analysis of NMR data enabled the configurational revision of garcicowins C and D. All the isolated PPAPs were tested for antiproliferative activity against three human hepatocellular carcinoma cell lines, including Huh-7, Hep 3B, and HepG2. Compounds 5 and 6 , 7- epi -isogarcinol ( 16 ), and coccinone C ( 17 ) exhibited moderate antiproliferative activity. Compounds 6 and 16 induced apoptosis and inhibited cell migration in Huh-7 cells, probably through downregulating the STAT3 signaling pathway. This study provides effective methods for configurational assignments of type B PPAPs.

Background & Aims: Discourse markers, such as well or like , serve a variety of functions to support conversational reciprocity: filling pauses, aiding word-finding, and modulating turn-taking by holding the conversational floor. Previous research shows that autistic individuals use discourse markers less frequently than non-autistic (NonAu) peers; however, the discourse marker like has not been included in that research, despite its ubiquitous use by NonAu individuals, and despite the fact that like serves important pragmatic functions that are not encoded by any other discourse marker. Specifically, like signals to the listener that the content of upcoming speech is 1) Important/new; 2) Loose/approximate; 3) Reformulative; or 4) Quotative. The current study addresses this gap in the literature by comparing the frequency of discourse marker like use between older autistic and non-autistic children as well as exploring patterns of usage between the four like functions., Methods: Twenty-one 10-to-17-year-old children on the autism spectrum and 20 NonAu peers-statistically matched on age, sex, IQ and language scores-engaged in a semi-structured interview with a researcher. Uses of discourse-marker like were identified from written transcripts of interviews and each use was categorized into one of the four functions., Results: There were no significant differences in like frequencies between groups, nor were there differences in relative proportions of functions used by each group., Conclusions: Research consistently indicates that autistic individuals use discourse markers significantly less often than their NonAu counterparts, but the findings from our study suggest that this pattern does not persist to all such markers. This group of older autistic children use like as often as their peers and use it to signify similar information about upcoming speech to their listener., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

LolO, a 2-oxoglutarate-dependent nonheme Fe oxygenase, catalyzes both the hydroxylation of 1-exo-acetamidopyrrolizidine (AcAP), a pathway intermediate in the biosynthesis of the loline alkaloids, and the cycloetherification of the resulting alcohol. We have prepared fluorinated AcAP analogues to aid in continued mechanistic investigation of the remarkable LolO-catalyzed cycloetherification step. LolO was able to hydroxylate 6,6-difluoro-AcAP (prepared from N,O-protected 4-oxoproline) and then cycloetherify the resulting alcohol, forming a difluorinated analogue of N-acetylnorloline and providing evidence for a cycloetherification mechanism involving a C(7) radical as opposed to a C(7) carbocation. By contrast, LolO was able to hydroxylate 7,7-difluoro-AcAP (prepared from 3-oxoproline) but failed to cycloetherify it, forming (1R,2R,8S)-7,7-difluoro-2-hydroxy-AcAP as the sole product. The divergent LolO-catalyzed reactions of the difluorinated AcAP analogues provide insight into the LolO cycloetherification mechanism and indicate that the 7,7-difluorinated compound, in particular, may be a useful tool to accumulate and characterize the iron intermediate that initiates the cycloetherification reaction., (© 2022 Wiley-VCH GmbH.)

Previously, Gao et al. reported the isolation and structural determination of three natural products, hyperibrin B (HB), hyperscabrone H (HH), and hyperscabrone I (HI), from Hypericum scabrum . HB and HH had different NMR spectroscopic data, but they were assigned identical structures. Furthermore, these compounds should be derived from bicyclic polyprenylated acylphloroglucinols (BPAPs) via degradation, but the assigned structural features of the prenyl and prenylmethyl groups being cis and meta-substituted on the cyclohexanone core were not consistent with their biosynthetic origin. In this note, we revise the structures of HB, HH, and HI via NMR and MS spectroscopic analyses and biosynthetic considerations. We also complete a total synthesis of the revised structure of HB as well as its analogue, hyperibrin A, to further confirm the revision. The revised structures of HB, HH, and HI have not been reported.

Lay Abstract: What is already known about the topic? Parents of children with autism experience enormous challenges managing the complex needs of caring for their children. This includes coordinating multiple and complex therapies and acting as partners in treatment. Parenting self-efficacy is the confidence a person has in their ability to manage the tasks that are part of raising a child. People who have more confidence, or greater parenting self-efficacy, often feel less stressed and are more able to manage the demands of family life. This is particularly important for parents with children who have autism spectrum disorder, since they experience more parenting pressures. Although a lot is known about parenting self-efficacy in parents of neurotypical children, we do not know enough about how to help parents of children with autism spectrum disorder develop greater parenting self-efficacy. What this paper adds? This study shows that parents gain a greater sense of parenting self-efficacy when they feel more involved in their child's therapy and are more satisfied with the training they receive as part of these therapies. We also find that feeling pressure related to being a caregiver of a child with autism spectrum disorder can undermine autism-specific parenting self-efficacy. However, parents' sense of confidence was not limited by the severity of their child's symptoms. Implications for practice, research, or policy The results suggest that there is an opportunity to help parents develop a greater sense of confidence in their ability to manage the complexities of raising a child with autism spectrum disorder by helping them feel more involved in treatment and by creating intervention-related training experiences that are more satisfying. Providers might also help by taking time to address the challenges and pressures that parents are experiencing, and helping them find ways to deal with these challenges. We suggest that there needs to be more research exploring how providers can best design interventions that support autism-specific parenting self-efficacy as a way of improving parental and child well-being.

This study investigates the use of structural and discourse contextual cues in the interpretation of third-person pronouns by children and adolescents with autism and their neurotypical peers. Results show that referent-biasing contextual information influences pronominal interpretation and modulates looking patterns in both groups compared to a context-neutral condition. These results go against the predictions of Weak Central Coherence and the notion that pragmatics in general is impaired in ASD, since the ASD group was able to use details in discourse context to influence the pronominal interpretation process. However, although discourse context influenced looking patterns in both groups, the groups nevertheless diverged in the nature of these patterns, suggesting that behavioral differences may emerge in more complicated discourse tasks.

Autism spectrum disorder (ASD) is characterized by deficits in social communication, and even children with ASD with preserved language are often perceived as socially awkward. We ask if linguistic patterns are associated with social perceptions of speakers. Twenty-one adolescents with ASD participated in conversations with an adult; each conversation was then rated for the social dimensions of likability, outgoingness, social skilfulness, responsiveness, and fluency. Conversations were analysed for responses to questions, pauses, and acoustic variables. Wide intonation ranges and more pauses within children's own conversational turn were predictors of more positive social ratings while failure to respond to one's conversational partner, faster syllable rate, and smaller quantity of speech were negative predictors of social perceptions.

Previously, Lin et al. reported the isolation and structural determination of two triterpenoids, garcinielliptin oxide (GO) and garcinielliptone E (GE). Their unusual structural features, which remained unparalleled in subsequent decades despite the intervening discovery of hundreds of other polycyclic polyprenylated acylphloroglucinols (PPAPs), caused us to question the originally assigned structures, so GO was reisolated from Garcinia subelliptica , and its NMR spectra were reacquired. In this Note, we revise the structures of GO and the related GE via NMR analysis, biosynthetic considerations, and chemical conversion. Garcinielliptone T, a new PPAP, was also isolated and characterized. GO exhibited weak inhibitory activity against acetylcholinesterase with an IC 50 value of 20.7 μM.

Recently, a paper in this journal reported the isolation and structure determination of hypatulone A. Several features of the proposed structure and biosynthesis induced us to reexamine the compound's NMR spectra. Now we propose a revised structure, confirm it with quantum computations, and suggest a reasonable radical-mediated biosynthetic pathway to the revised structure. Our work illustrates how both biosynthetic considerations and quantum computations can complement spectroscopic structure determination.

The title compound [systematic name: (1 R *, 8 S )-2-acetamidoocta-hydro-pyrrol-izin-4-ium chloride- N -[(1 R , 8 S )-hexa-hydro-1 H -pyrrolizin-2-yl)acetamide (1/1)], 2(C 9 H 16 N 2 O)·HCl or C 9 H 17 N 2 O + ·Cl - ·C 9 H 16 N 2 O, arose as an unexpected product when 1- exo -acetamido-pyrrolizidine (AcAP; C 9 H 16 N 2 O) was dissolved in CHCl 3 . Within the AcAP pyrrolizidine group, the unsubstituted five-membered ring is disordered over two orientations in a 0.897 (5):0.103 (5) ratio. Two AcAP mol-ecules related by a crystallographic twofold axis link to H + and Cl - ions lying on the rotation axis, thereby forming N-H⋯N and N-H⋯Cl⋯H-N hydrogen bonds. The first of these has an unusually short N⋯N separation of 2.616 (2) Å: refinement of different models against the present data set could not distinguish between a symmetrical hydrogen bond (H atom lying on the twofold axis and equidistant from the N atoms) or static or dynamic disorder models ( i.e. N-H⋯N + N⋯H-N). Computational studies suggest that the disorder model is slightly more stable, but the energy difference is very small., (© Bhardwaj et al. 2020.)

Iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases generate iron(IV)-oxo (ferryl) intermediates that can abstract hydrogen from aliphatic carbons (R-H). Hydroxylation proceeds by coupling of the resultant substrate radical (R•) and oxygen of the Fe(III)-OH complex ("oxygen rebound"). Nonhydroxylation outcomes result from different fates of the Fe(III)-OH/R• state; for example, halogenation results from R• coupling to a halogen ligand cis to the hydroxide. We previously suggested that halogenases control substrate-cofactor disposition to disfavor oxygen rebound and permit halogen coupling to prevail. Here, we explored the general implication that, when a ferryl intermediate can ambiguously target two substrate carbons for different outcomes, rebound to the site capable of the alternative outcome should be slower than to the adjacent, solely hydroxylated site. We evaluated this prediction for (i) the halogenase SyrB2, which exclusively hydroxylates C5 of norvaline appended to its carrier protein but can either chlorinate or hydroxylate C4 and (ii) two bifunctional enzymes that normally hydroxylate one carbon before coupling that oxygen to a second carbon (producing an oxacycle) but can, upon encountering deuterium at the first site, hydroxylate the second site instead. In all three cases, substrate hydroxylation incorporates a greater fraction of solvent-derived oxygen at the site that can also undergo the alternative outcome than at the other site, most likely reflecting an increased exchange of the initially O 2 -derived oxygen ligand in the longer-lived Fe(III)-OH/R• states. Suppression of rebound may thus be generally important for nonhydroxylation outcomes by these enzymes.

Atypical eye gaze to social stimuli is one of the most frequently reported and studied social behaviors affected by autism spectrum disorder (ASD). The vast majority of this literature is based on analyses of gaze patterns as participants view social information, such as talking faces, on a computer screen. However, recent results suggest that generalizing gaze behaviors from computer screens to live interactions may not be valid. This study examines between- and within-group differences in gaze behaviors of children with ASD and their neurotypical (NT) peers during a screen-based and a live-interaction task. Results show between-group differences in gaze only for the screen-based, but not the live-interaction task. We also find that gaze behavior of NT children during the screen-based task significantly correlates with their gaze behavior during the live interaction; individuals who direct a higher percentage of gaze to the face in one task also did so in the other task. However, there is no significant relationship between the gaze patterns of children with ASD for those two tasks. These results strongly caution against using gaze of individuals with ASD recorded during screen-based tasks as a proxy for understanding their gaze behavior during live social interactions.

Neurotypical adults often form negative first impressions of individuals with autism spectrum disorder and are less interested in engaging with them socially. In contrast, individuals with autism spectrum disorder actively seek out the company of others who share their diagnosis. It is not clear, however, whether individuals with autism spectrum disorder form more positive first impressions of autistic peers when diagnosis is not explicitly shared. We asked adolescents with and without autism spectrum disorder to watch brief video clips of adolescents with and without autism spectrum disorder and answer questions about their impressions of the individuals in the videos. Questions were related to participants' perceptions of the social skills of the individuals in the video, as well as their own willingness to interact with that person. We also measured gaze patterns to the faces, eyes, and mouths of adolescents in the video stimuli. Both participant groups spent less time gazing at videos of autistic adolescents. Regardless of diagnostic group, all participants provided more negative judgments of autistic than neurotypical adolescents in the videos. These data indicate that, without being explicitly informed of a shared diagnosis, adolescents with autism spectrum disorder form negative first impressions of autistic adolescents that are similar to, or lower than, those formed by neurotypical peers.

Research shows that neurotypical individuals struggle to interpret the emotional facial expressions of people with Autism Spectrum Disorder (ASD). The current study uses motion-capture to objectively quantify differences between the movement patterns of emotional facial expressions of individuals with and without ASD. Participants volitionally mimicked emotional expressions while wearing facial markers. Recorded marker movement was grouped by expression valence and intensity. We used Growth Curve Analysis to test whether movement patterns were predictable by expression type and participant group. Results show significant interactions between expression type and group, and little effect of emotion valence on ASD expressions. Together, results support perceptions that expressions of individuals with ASD are different from-and more ambiguous than-those of neurotypical individuals'.

Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, are outlined herein. One such highlight is the activity of (-)-2( S )-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening., Competing Interests: The authors declare no competing financial interest.

Research into emotional responsiveness in Autism Spectrum Disorder (ASD) has yielded mixed findings. Some studies report uniform, flat and emotionless expressions in ASD; others describe highly variable expressions that are as or even more intense than those of typically developing (TD) individuals. Variability in findings is likely due to differences in study design: some studies have examined posed (i.e., not spontaneous expressions) and others have examined spontaneous expressions in social contexts, during which individuals with ASD-by nature of the disorder-are likely to behave differently than their TD peers. To determine whether (and how) spontaneous facial expressions and other emotional responses are different from TD individuals, we video-recorded the spontaneous responses of children and adolescents with and without ASD (between the ages of 10 and 17 years) as they watched emotionally evocative videos in a non-social context. Researchers coded facial expressions for intensity, and noted the presence of laughter and other responsive vocalizations. Adolescents with ASD displayed more intense, frequent and varied spontaneous facial expressions than their TD peers. They also produced significantly more emotional vocalizations, including laughter. Individuals with ASD may display their emotions more frequently and more intensely than TD individuals when they are unencumbered by social pressure. Differences in the interpretation of the social setting and/or understanding of emotional display rules may also contribute to differences in emotional behaviors between groups.

Polycyclic polyprenylated acylphloroglucinols (PPAPs) are a class of hybrid natural products sharing the mevalonate/methylerythritol phosphate and polyketide biosynthetic pathways and showing considerable structure and bioactivity diversity. This review discusses the progress of research into the chemistry and biological activity of 421 natural PPAPs in the past 11 years as well as in-depth studies of biological activities and total synthesis of some PPAPs isolated before 2006. We created an online database of all PPAPs known to date at http://www.chem.uky.edu/research/grossman/PPAPs . Two subclasses of biosynthetically related metabolites, spirocyclic PPAPs with octahydrospiro[cyclohexan-1,5'-indene]-2,4,6-trione core and complicated PPAPs produced by intramolecular [4 + 2] cycloadditions of MPAPs, are brought into the PPAP family. Some PPAPs' relative or absolute configurations are reassigned or critically discussed, and the confusing trivial names in PPAPs investigations are clarified. Pharmacologic studies have revealed a new molecular mechanism whereby hyperforin and its derivatives regulate neurotransmitter levels by activating TRPC6 as well as the antitumor mechanism of garcinol and its analogues. The antineoplastic potential of some type B PPAPs such as oblongifolin C and guttiferone K has increased significantly. As a result of the recent appearances of innovative synthetic methods and strategies, the total syntheses of 22 natural PPAPs including hyperforin, garcinol, and plukenetione A have been accomplished.

The core of the loline family of insecticidal alkaloids is the bicyclic pyrrolizidine unit with an additional strained ether bridge between carbons 2 and 7. Previously reported genetic and in vivo biochemical analyses showed that the presumptive iron- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, LolO, is required for installation of the ether bridge upon the pathway intermediate, 1- exo-acetamidopyrrolizidine (AcAP). Here we show that LolO is, in fact, solely responsible for this biosynthetic four-electron oxidation. In sequential 2OG- and O 2 -consuming steps, LolO removes hydrogens from C2 and C7 of AcAP to form both carbon-oxygen bonds in N-acetylnorloline (NANL), the precursor to all other lolines. When supplied with substoichiometric 2OG, LolO only hydroxylates AcAP. At higher 2OG:AcAP ratios, the enzyme further processes the alcohol to the tricyclic NANL. Characterization of the alcohol intermediate by mass spectrometry and nuclear magnetic resonance spectroscopy shows that it is 2- endo-hydroxy-1- exo-acetamidopyrrolizidine (2- endo-OH-AcAP). Kinetic and spectroscopic analyses of reactions with site-specifically deuteriated AcAP substrates confirm that the C2-H bond is cleaved first and that the responsible intermediate is, as expected, an Fe IV -oxo (ferryl) complex. Analyses of the loline products from cultures fed with stereospecifically deuteriated AcAP precursors, proline and aspartic acid, establish that LolO removes the endo hydrogens from C2 and C7 and forms both new C-O bonds with retention of configuration. These findings delineate the pathway to an important class of natural insecticides and lay the foundation for mechanistic dissection of the chemically challenging oxacyclization reaction.

Several studies have established that facial expressions of children with autism are often perceived as atypical, awkward or less engaging by typical adult observers. Despite this clear deficit in the quality of facial expression production, very little is understood about its underlying mechanisms and characteristics. This paper takes a computational approach to studying details of facial expressions of children with high functioning autism (HFA). The objective is to uncover those characteristics of facial expressions, notably distinct from those in typically developing children, and which are otherwise difficult to detect by visual inspection. We use motion capture data obtained from subjects with HFA and typically developing subjects while they produced various facial expressions. This data is analyzed to investigate how the overall and local facial dynamics of children with HFA differ from their typically developing peers. Our major observations include reduced complexity in the dynamic facial behavior of the HFA group arising primarily from the eye region.

Individuals with autism spectrum disorder (ASD) often exhibit increased anxiety, even in non-stressful situations. We investigate general anxiousness (anxiety trait) and responses to stressful situations (anxiety state) in 22 adolescents with ASD and 32 typically developing controls. We measured trait anxiety with standardized self- and parent-reported questionnaires. We used a Biopac system to capture state anxiety via skin conductance responses, mean heart rate and heart rate variability during high- and low-anxiety tasks. Results reveal higher trait anxiety in adolescents with ASD (p < 0.05) and no group difference in state anxiety. Increased parent-reported trait anxiety may predict decreased state anxiety during high-stress conditions. Together, these findings suggest that higher trait anxiety may result in dampened physical responses to stress.

Individuals with autism spectrum disorder (ASD), including those who otherwise require less support, face severe difficulties in everyday social interactions. Research in this area has primarily focused on identifying the cognitive and neurological differences that contribute to these social impairments, but social interaction by definition involves more than one person and social difficulties may arise not just from people with ASD themselves, but also from the perceptions, judgments, and social decisions made by those around them. Here, across three studies, we find that first impressions of individuals with ASD made from thin slices of real-world social behavior by typically-developing observers are not only far less favorable across a range of trait judgments compared to controls, but also are associated with reduced intentions to pursue social interaction. These patterns are remarkably robust, occur within seconds, do not change with increased exposure, and persist across both child and adult age groups. However, these biases disappear when impressions are based on conversational content lacking audio-visual cues, suggesting that style, not substance, drives negative impressions of ASD. Collectively, these findings advocate for a broader perspective of social difficulties in ASD that considers both the individual's impairments and the biases of potential social partners., Competing Interests: The authors declare no competing financial interests.

We form first impressions of many traits based on very short interactions. This study examines whether typical adults judge children with high-functioning autism to be more socially awkward than their typically developing peers based on very brief exposure to still images, audio-visual, video-only, or audio-only information. We used video and audio recordings of children with and without high-functioning autism captured during a story-retelling task. Typically developing adults were presented with 1 s and 3 s clips of these children, as well as still images, and asked to judge whether the person in the clip was socially awkward. Our findings show that participants who are naïve to diagnostic differences between the children in the clips judged children with high-functioning autism to be socially awkward at a significantly higher rate than their typically developing peers. These results remain consistent for exposures as short as 1 s to visual and/or auditory information, as well as for still images. These data suggest that typical adults use subtle nonverbal and non-linguistic cues produced by children with high-functioning autism to form rapid judgments of social awkwardness with the potential for significant repercussions in social interactions., (© The Author(s) 2014.)

Conversation requires integration of information from faces and voices to fully understand the speaker's message. To detect auditory-visual asynchrony of speech, listeners must integrate visual movements of the face, particularly the mouth, with auditory speech information. Individuals with autism spectrum disorder may be less successful at such multisensory integration, despite their demonstrated preference for looking at the mouth region of a speaker. We showed participants (individuals with and without high-functioning autism (HFA) aged 8-19) a split-screen video of two identical individuals speaking side by side. Only one of the speakers was in synchrony with the corresponding audio track and synchrony switched between the two speakers every few seconds. Participants were asked to watch the video without further instructions (implicit condition) or to specifically watch the in-synch speaker (explicit condition). We recorded which part of the screen and face their eyes targeted. Both groups looked at the in-synch video significantly more with explicit instructions. However, participants with HFA looked at the in-synch video less than typically developing (TD) peers and did not increase their gaze time as much as TD participants in the explicit task. Importantly, the HFA group looked significantly less at the mouth than their TD peers, and significantly more at non-face regions of the image. There were no between-group differences for eye-directed gaze. Overall, individuals with HFA spend less time looking at the crucially important mouth region of the face during auditory-visual speech integration, which is maladaptive gaze behavior for this type of task., (© 2015 International Society for Autism Research, Wiley Periodicals, Inc.)

Children with Autism Spectrum Disorder (ASD) are known to have difficulty in producing and perceiving emotional facial expressions. Their expressions are often perceived as atypical by adult observers. This paper focuses on data driven ways to analyze and quantify atypicality in facial expressions of children with ASD. Our objective is to uncover those characteristics of facial gestures that induce the sense of perceived atypicality in observers. Using a carefully collected motion capture database, facial expressions of children with and without ASD are compared within six basic emotion categories employing methods from information theory, time-series modeling and statistical analysis. Our experiments show that children with ASD usually have less complex expression producing mechanisms; the differences in facial dynamics between children with and without ASD primarily come from the eye region. Our study also notes that children with ASD exhibit lower symmetry between left and right regions, and lower variation in motion intensity across facial regions.

The lolines are a class of bioprotective alkaloids that are produced by Epichloë species, fungal endophytes of grasses. These alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group: -NH2 (norloline), -NHCH3 (loline), -N(CH3)2 (N-methylloline), -N(CH3)Ac (N-acetylloline), -NHAc (N-acetylnorloline), and -N(CH3)CHO (N-formylloline). Other than the LolP cytochrome P450, which is required for conversion of N-methylloline to N-formylloline, the enzymatic steps for loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorloline is the first fully cyclized loline alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in loline alkaloid diversification. Two genes of the loline alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorloline, and complementation with the two wild-type genes restored production of N-formylloline and N-acetylloline. These results indicated that lolN and lolM are required in the steps from N-acetylnorloline to other lolines. The function of LolM as an N-methyltransferase was confirmed by its heterologous expression in yeast resulting in conversion of norloline to loline, and of loline to N-methylloline. One of the more abundant lolines, N-acetylloline, was observed in some but not all plants with symbiotic Epichloë siegelii, and when provided with exogenous loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylloline, suggesting that a plant acetyltransferase catalyzes N-acetylloline formation. We conclude that although most loline alkaloid biosynthesis reactions are catalyzed by fungal enzymes, both fungal and plant enzymes are responsible for the chemical diversification steps in symbio.

Lolines are potent insecticidal agents produced by endophytic fungi of cool-season grasses. These alkaloids are composed of a pyrrolizidine ring system and an uncommon ether bridge linking carbons 2 and 7. Previous results indicated that 1-aminopyrrolizidine was a pathway intermediate. We used RNA interference to knock down expression of lolO, resulting in the accumulation of an alkaloid identified as exo-1-acetamidopyrrolizidine based on high-resolution MS and NMR. Genomes of endophytes differing in alkaloid profiles were sequenced, revealing that those with mutated lolO accumulated exo-1-acetamidopyrrolizidine but no lolines. Heterologous expression of wild-type lolO complemented a lolO mutant, resulting in the production of N-acetylnorloline. These results indicated that the non-heme iron oxygenase, LolO, is required for ether bridge formation, probably through oxidation of exo-1-acetamidopyrrolizidine., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: People with high-functioning autism (HFA) have qualitative differences in facial expression and prosody production, which are rarely systematically quantified. The authors' goals were to qualitatively and quantitatively analyze prosody and facial expression productions in children and adolescents with HFA., Method: Participants were 22 male children and adolescents with HFA and 18 typically developing (TD) controls (17 males, 1 female). The authors used a story retelling task to elicit emotionally laden narratives, which were analyzed through the use of acoustic measures and perceptual codes. Naïve listeners coded all productions for emotion type, degree of expressiveness, and awkwardness., Results: The group with HFA was not significantly different in accuracy or expressiveness of facial productions, but was significantly more awkward than the TD group. Participants with HFA were significantly more expressive in their vocal productions, with a trend for greater awkwardness. Severity of social communication impairment, as captured by the Autism Diagnostic Observation Schedule (ADOS; Lord, Rutter, DiLavore, & Risi, 1999), was correlated with greater vocal and facial awkwardness., Conclusions: Facial and vocal expressions of participants with HFA were as recognizable as those of their TD peers but were qualitatively different, particularly when listeners coded samples with intact dynamic properties. These preliminary data show qualitative differences in nonverbal communication that may have significant negative impact on the social communication success of children and adolescents with HFA.

We focus on the analysis, quantification and visualization of atypicality in affective facial expressions of children with High Functioning Autism (HFA). We examine facial Motion Capture data from typically developing (TD) children and children with HFA, using various statistical methods, including Functional Data Analysis, in order to quantify atypical expression characteristics and uncover patterns of expression evolution in the two populations. Our results show that children with HFA display higher asynchrony of motion between facial regions, more rough facial and head motion, and a larger range of facial region motion. Overall, subjects with HFA consistently display a wider variability in the expressive facial gestures that they employ. Our analysis demonstrates the utility of computational approaches for understanding behavioral data and brings new insights into the autism domain regarding the atypicality that is often associated with facial expressions of subjects with HFA.

Data on emotion processing by individuals with ASD suggest both intact abilities and significant deficits. Signal intensity may be a contributing factor to this discrepancy. We presented low- and high-intensity emotional stimuli in a face-voice matching task to 22 adolescents with ASD and 22 typically developing (TD) peers. Participants heard semantically neutral sentences with happy, surprised, angry, and sad prosody presented at two intensity levels (low, high) and matched them to emotional faces. The facial expression choice was either across- or within-valence. Both groups were less accurate for low-intensity emotions, but the ASD participants' accuracy levels dropped off more sharply. ASD participants were significantly less accurate than their TD peers for trials involving low-intensity emotions and within-valence face contrasts.

We analyzed several studies of non-verbal communication (prosody and facial expressions) completed in our lab and conducted a secondary analysis to compare performance on receptive vs. expressive tasks by adolescents with ASD and their typically developing peers. Results show a significant between-group difference for the aggregate score of expressive tasks, but not for the aggregate score of receptive tasks. There was also a significant within-group difference among individuals with ASD for expressive vs. receptive performance. Our data indicate that adolescents with ASD can achieve receptive accuracy in non-verbal communication, but show significant qualitative deficits in expressive skills across a range of tasks, which may have a significant negative impact on their success as social communicators.

Purpose: To investigate the perception and production of lexical stress and processing of affective prosody in adolescents with high-functioning autism (HFA). We hypothesized preserved processing of lexical and affective prosody but atypical lexical prosody production., Method: Sixteen children with HFA and 15 typically developing (TD) peers participated in 3 experiments that examined the following: (a) perception of affective prosody (Experiment 1), (b) lexical stress perception (Experiment 2), and (c) lexical stress production (Experiment 3). In Experiment 1, participants labeled sad, happy, and neutral spoken sentences that were low-pass filtered, to eliminate verbal content. In Experiment 2, participants disambiguated word meanings based on lexical stress (HOTdog vs. hot DOG). In Experiment 3, participants produced these words in a sentence completion task. Productions were analyzed with acoustic measures., Results: Accuracy levels showed no group differences. Participants with HFA could determine affect from filtered sentences and disambiguate words on the basis of lexical stress. They produced appropriately differentiated lexical stress patterns but demonstrated atypically long productions, indicating reduced ability in natural prosody production., Conclusions: Children with HFA were as capable as their TD peers in receptive tasks of lexical stress and affective prosody. Prosody productions were atypically long, despite accurate differentiation of lexical stress patterns. Future research should use larger samples and spontaneous versus elicited productions.

Background: It has frequently been suggested that individuals with autism spectrum disorder (ASD) have deficits in auditory-visual (AV) sensory integration. Studies of language integration have mostly used non-word syllables presented in congruent and incongruent AV combinations and demonstrated reduced influence of visual speech in individuals with ASD. The aim of our study was to test whether adolescents with high-functioning autism are able to integrate AV information of meaningful, phrase-length language in a task of onset asynchrony detection., Methods: Participants were 25 adolescents with ASD and 25 typically developing (TD) controls. The stimuli were video clips of complete phrases using simple, commonly occurring words. The clips were digitally manipulated to have the video precede the corresponding audio by 0, 4, 6, 8, 10, 12, or 14 video frames, a range of 0-500ms. Participants were shown the video clips in random order and asked to indicate whether each clip was in-synch or not., Results: There were no differences between adolescents with ASD and their TD peers in accuracy of onset asynchrony detection at any slip rate., Conclusion: These data indicate that adolescents with ASD are able to integrate auditory and visual components in a task of onset asynchrony detection using natural, phrase-length language stimuli. We propose that the meaningful nature of the language stimuli in combination with presentation in a non-distracting environment allowed adolescents with autism spectrum disorder to demonstrate preserved accuracy for bi-modal AV integration.

Studies of explicit processing of facial expressions by individuals with autism spectrum disorder (ASD) have found a variety of deficits and preserved abilities compared to their typically developing (TD) peers. However, little attention has been paid to their implicit processing abilities for emotional facial expressions. The question has also been raised whether preferential attention to the mouth region of a speaker's face by ASD individuals has resulted in a relative lipreading expertise. We present data on implicit processing of pseudo-dynamic facial emotions and visual speech in adolescents with autism. We compared 25 ASD and 25 TD participants on their ability to recreate the sequences of four dynamic emotional facial expressions (happy, sad, disgust, fear) as well as four spoken words (with, bath, thumb, watch) using six still images taken from a video sequence. Typical adolescents were significantly better at recreating the dynamic properties of emotional expressions than those of facial speech, while the autism group showed the reverse accuracy pattern. For Experiment 2 we obscured the eye region of the stimuli and found no significant difference between the 22 adolescents with ASD and 22 TD controls. Fearful faces achieved the highest accuracy results among the emotions in both groups.

The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, DeltalolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of DeltalolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.

The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 A (1 A=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr(178) as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr(2) in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr(178) to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr(178) can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

We present a case of upper arm compartment syndrome following a biceps tendon rupture in a 77-year old man on warfarin sodium. Compartment syndrome is common in the forearm and leg, but rare in the upper arm with only a handful of cases reported in the literature. Our patient's anticoagulated state predisposed him to the development of compartment syndrome. To the best of our knowledge there has been only one other case reported in the literature of upper arm compartment syndrome following biceps tendon rupture in a patient on warfarin sodium. Compartment syndrome of the upper arm is a rare occurrence. Previous cases have occurred due to malposition of blood pressure cuffs, injections, venepuncture, trauma, tourniquets, shoulder dislocation, surgical complication, subatmospheric pressure induced, biceps rupture, and triceps rupture. The fascia of the upper arm is relatively thinner and more distensible than the fascia of the leg or forearm. This creates more room for the compartment to swell before pressures builds up to a significant level. Thus, a significantly increased amount of pressure needs to build up before compartment syndrome will occur in the upper arm. Once the diagnosis was formed, the patient was treated with emergent fasciotomy and evacuation of hematoma. After a prolonged hospital stay, the patient was released with minor neurological deficits. At final follow-up, the patient was neurovascularly intact with no complaints of numbness or tingling, and he had regained full motor function throughout.

Several species of Lolium and other cool-season grasses (Poaceae subfamily Pooideae) tend to harbor symbiotic, seed-transmitted, fungi that enhance their fitness by various means. These fungal endophytes--species of Neotyphodium or Epichloë (Clavicipitaceae)--are known for production of antiherbivore metabolites such as the bioprotective loline alkaloids. Lolines are saturated pyrrolizidines with an exo-1-amine and an ether bridge between C-2 and C-7. The ether bridge is an unusual feature for a biogenic compound in that it links two bridgehead carbon atoms. Much of the loline-biosynthetic pathway has been elucidated by administering isotopically labeled precursors to fungal cultures and by comparisons of loline biosynthesis genes to known gene families. The first step appears to be an unusual gamma-substitution reaction involving an enzyme related to O-acetylhomoserine (thiol) lyase, but which uses the secondary amine of L-proline rather than a sulfhydryl group as the nucleophile. The strained ether bridge is added after formation of the pyrrolizidine rings. Lolines with dimethylated or acylated 1-amines have insect antifeedant and insecticidal activities comparable to nicotine, but little or no toxicity to mammals. Considering the surprising abundance of lolines in some grass-endophyte symbiota, possible additional effects on plant stress tolerance and physiology are worth future consideration. In this review, we discuss the history of loline discovery, methods of analysis, biological activities and distribution in nature, as well as progress on the genetics and biochemistry of their biosynthesis, and on the chemical synthesis of these alkaloids.

Loline alkaloids are saturated pyrrolizidines with an oxygen bridge between carbon atoms C-2 and C-7 and an amino group on C-1. They are bioprotective alkaloids produced by Epichloë and Neotyphodium species, mutualistic fungal endophytes that are symbiotic with cool-season grasses. The sequence of bond formation in loline alkaloid biosynthesis was determined by synthesizing deuterated forms of potential intermediates and feeding them to cultures of the endophyte Neotyphodium uncinatum. These cultures incorporated deuterium from labeled N-(3-amino-3-carboxypropyl)proline and exo-1-aminopyrrolizidine into N-formylloline. The first result suggests that N-(3-amino-3-carboxypropyl)proline is the first committed intermediate in loline biosynthesis, and the second result demonstrates that the pyrrolizidine rings form before the ether bridge. The incorporation of these two compounds into lolines and the lack of incorporation of several related compounds clarify the order of bond formation in loline alkaloid biosynthesis.

Loline alkaloids are saturated pyrrolizidines with a substituted 1-amino group and an oxygen bridge between C2 and C7, and are insecticidal metabolites of plant-symbiotic fungi (endophytes). Cultures of the endophyte, Neotyphodium uncinatum, incorporated labeled L-proline and L-homoserine into the 1-aminopyrrolizidine, N-formylloline. The A-ring carbons C1-C3 and the N1 were derived from L-homoserine; the B-ring carbons C5-C8 and the ring nitrogen were derived from L-proline. Incorporation of both deuterium atoms from L-[4,4-(2H2)]homoserine and feeding tests with labeled L-methionine indicated that L-homoserine incorporation was not achieved via aspartyl semialdehyde or S-adenosylmethionine, but probably involved a highly novel N--C bond-forming gamma-substitution reaction.