Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs.

Pleasurable and problematic receptive anal intercourse and diseases of the colon, rectum and anus.

The ability to experience pleasurable sexual activity is important for human health. Receptive anal intercourse (RAI) is a common, though frequently stigmatized, pleasurable sexual activity. Little is known about how diseases of the colon, rectum, and anus and their treatments affect RAI. Engaging in RAI with gastrointestinal disease can be difficult due to the unpredictability of symptoms and treatment-related toxic effects. Patients might experience sphincter hypertonicity, gastrointestinal symptom-specific anxiety, altered pelvic blood flow from structural disorders, decreased sensation from cancer-directed therapies or body image issues from stoma creation. These can result in problematic RAI - encompassing anodyspareunia (painful RAI), arousal dysfunction, orgasm dysfunction and decreased sexual desire. Therapeutic strategies for problematic RAI in patients living with gastrointestinal diseases and/or treatment-related dysfunction include pelvic floor muscle strengthening and stretching, psychological interventions, and restorative devices. Providing health-care professionals with a framework to discuss pleasurable RAI and diagnose problematic RAI can help improve patient outcomes. Normalizing RAI, affirming pleasure from RAI and acknowledging that the gastrointestinal system is involved in sexual pleasure, sexual function and sexual health will help transform the scientific paradigm of sexual health to one that is more just and equitable.

Artificial intelligence improves resident detection of pediatric and young adult upper extremity fractures.

PURPOSE: We wished to evaluate if an open-source artificial intelligence (AI) algorithm ( https://www.childfx.com ) could improve performance of (1) subspecialized musculoskeletal radiologists, (2) radiology residents, and (3) pediatric residents in detecting pediatric and young adult upper extremity fractures. MATERIALS AND METHODS: A set of evaluation radiographs drawn from throughout the upper extremity (elbow, hand/finger, humerus/shoulder/clavicle, wrist/forearm, and clavicle) from 240 unique patients at a single hospital was constructed (mean age 11.3 years, range 0-22 years, 37.9% female). Two fellowship-trained musculoskeletal radiologists, three radiology residents, and two pediatric residents were recruited as readers. Each reader interpreted each case initially without and then subsequently 3-4 weeks later with AI assistance and recorded if/where fracture was present. RESULTS: Access to AI significantly improved area under the receiver operator curve (AUC) of radiology residents (0.768 [0.730-0.806] without AI to 0.876 [0.845-0.908] with AI, P < 0.001) and pediatric residents (0.706 [0.659-0.753] without AI to 0.844 [0.805-0.883] with AI, P < 0.001) in identifying fracture, respectively. There was no evidence of improvement for subspecialized musculoskeletal radiology attendings in identifying fracture (AUC 0.867 [0.832-0.902] to 0.890 [0.856-0.924], P = 0.093). There was no evidence of difference between overall resident AUC with AI and subspecialist AUC without AI (resident with AI 0.863, attending without AI AUC 0.867, P = 0.856). Overall physician radiograph interpretation time was significantly lower with AI (38.9 s with AI vs. 52.1 s without AI, P = 0.030). CONCLUSION: An openly accessible AI model significantly improved radiology and pediatric resident accuracy in detecting pediatric upper extremity fractures.

Engineering Brassica Crops to Optimize Delivery of Bioactive Products Postcooking.

Glucosinolates are plant-specialized metabolites that can be hydrolyzed by glycosyl hydrolases, called myrosinases, creating a variety of hydrolysis products that benefit human health. While cruciferous vegetables are a rich source of glucosinolates, they are often cooked before consumption, limiting the conversion of glucosinolates to hydrolysis products due to the denaturation of myrosinases. Here we screen a panel of glycosyl hydrolases for high thermostability and engineer the Brassica crop, broccoli (Brassica oleracea L.), for the improved conversion of glucosinolates to chemopreventive hydrolysis products. Our transgenic broccoli lines enabled glucosinolate hydrolysis to occur at higher cooking temperatures, 20 °C higher than in wild-type broccoli. The process of cooking fundamentally transforms the bioavailability of many health-relevant bioactive compounds in our diet. Our findings demonstrate the promise of leveraging genetic engineering to tailor crops with novel traits that cannot be achieved through conventional breeding and improve the nutritional properties of the plants we consume.

Long COVID Among Wisconsin Workers in the Workers' Compensation System: Associations With Sociodemographics, Vaccination, and Predominant Variant Period From March 1, 2020 to July 31, 2022.

OBJECTIVE: This analysis aimed to determine the likelihood of developing long COVID among Wisconsin workers while adjusting for sociodemographics, COVID-19 vaccination, industry, and occupation. METHODS: This retrospective analysis determined the odds ratios of developing long COVID among Wisconsin workers who were compensated for COVID-19 lost time during March 1, 2020 to July 31, 2022. RESULTS: A total of 234 workers (11.7%) were determined to have long COVID. Factors associated with long COVID were age ≥40 years, non-White race, infection occurrence during the initial and Omicron variant dominant periods, and the absence of COVID-19 vaccination. Workers in manufacturing and public administration were more likely to develop long COVID compared with those in health care and social assistance. CONCLUSIONS: Long COVID disproportionately affects some worker groups. This calls for more worker protection and preventative care to mitigate its impact.

Age-related changes of auditory sensitivity across the life span of CBA/CaJ mice.

The inbred mouse strain CBA/CaJ is a frequently used animal model of age-related hearing loss in humans. These mice display significant hearing loss at a relatively advanced age, similar to most humans, with progressive loss of hearing as the mouse continues to age. While important descriptions of hearing loss in this mouse strain at multiple ages have previously been published, shortcomings persist in the data for hearing over the lifespan of the mouse. Therefore, we analyzed auditory brainstem response threshold data from records maintained by our research group to yield an extensive database of thresholds over nearly the entire life span of the CBA/CaJ mouse (from 79 to 1085 days). Data was collected from in-house bred mice of CBA/CaJ stock, initially from The Jackson Laboratory. Data was collected using BiosigRZ software and TDT System III hardware. Thresholds were routinely measured in conjunction with behavioral and electrophysiological experiments; only responses from baseline or experimentally naïve animals were analyzed. The resulting data set comprised 376 female mice and 441 males. At the lowest and highest frequencies (8 & 32 kHz), initial thresholds were just under 30 dB SPL and increased slowly until they were significantly different at 16-18 months compared to 1-3 months age, with the difference increasing over subsequent ages. At the middle frequencies (12 & 16 kHz), initial thresholds were just under 20 dB SPL and increased until they became different from initial at 16-18 months. At 24 kHz, initial thresholds were just above 20 dB and became different from initial at 13-16 months of age. The rate of change of thresholds with age were similar for all frequencies until about 30 months of age, when 32 kHz threshold changes lagged behind other frequencies. Generally, CBA/CaJ mice in our colony display relatively low thresholds until approximately 16 months of age, depending on frequency. After 16-18 months, thresholds become significantly worse. After approximately 20-22 months thresholds increase linearly with age.

Harnessing Plant Sugar Metabolism for Glycoengineering.

Plants possess an innate ability to generate vast amounts of sugar and produce a range of sugar-derived compounds that can be utilized for applications in industry, health, and agriculture. Nucleotide sugars lie at the unique intersection of primary and specialized metabolism, enabling the biosynthesis of numerous molecules ranging from small glycosides to complex polysaccharides. Plants are tolerant to perturbations to their balance of nucleotide sugars, allowing for the overproduction of endogenous nucleotide sugars to push flux towards a particular product without necessitating the re-engineering of upstream pathways. Pathways to produce even non-native nucleotide sugars may be introduced to synthesize entirely novel products. Heterologously expressed glycosyltransferases capable of unique sugar chemistries can further widen the synthetic repertoire of a plant, and transporters can increase the amount of nucleotide sugars available to glycosyltransferases. In this opinion piece, we examine recent successes and potential future uses of engineered nucleotide sugar biosynthetic, transport, and utilization pathways to improve the production of target compounds. Additionally, we highlight current efforts to engineer glycosyltransferases. Ultimately, the robust nature of plant sugar biochemistry renders plants a powerful chassis for the production of target glycoconjugates and glycans.

Plant-based production of diverse human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates that aid in the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the ∼130 structurally diverse HMOs at scale has proven difficult. Here, we produce a vast diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high value HMOs, such as lacto-N-fucopentaose I, that have not yet been commercially produced using state-of-the-art microbial fermentative processes. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the cheap and sustainable production of HMOs.

Sexual health and treatment-related sexual dysfunction in sexual and gender minorities with prostate cancer.

Prostate cancer treatment has substantial effects on sexual health and function. Sexual function is a vital aspect of human health and a critical component of cancer survivorship, and understanding the potential effects of different treatment modalities on sexual health is crucial. Existing research has extensively described the effects of treatment on male erectile tissues necessary for heterosexual intercourse; however, evidence regarding their effects on sexual health and function in sexual and gender minority populations is minimal. These groups include sexual minority - gay and bisexual - men, and transgender women or trans feminine people in general. Such unique effects in these groups might include altered sexual function in relation to receptive anal and neovaginal intercourse and changes to patients' role-in-sex. Sexual dysfunctions following prostate cancer treatment affecting quality of life in sexual minority men include climacturia, anejaculation, decreased penile length, erectile dysfunction, and problematic receptive anal intercourse, including anodyspareunia and altered pleasurable sensation. Notably, clinical trials investigating sexual outcomes after prostate cancer treatment do not collect sexual orientation and gender identity demographic data or outcomes specific to members of these populations, which perpetuates the uncertainty regarding optimal management. Providing clinicians with a solid evidence base is essential to communicate recommendations and tailor interventions for sexual and gender minority patients with prostate cancer.

Identifying ex vivo acute ischemic stroke thrombus composition using electrochemical impedance spectroscopy.

BACKGROUND: Intra-procedural characterization of stroke thromboemboli might guide mechanical thrombectomy (MT) device choice to improve recanalization rates. Electrochemical impedance spectroscopy (EIS) has been used to characterize various biological tissues in real time but has not been used in thrombus. OBJECTIVE: To perform a feasibility study of EIS analysis of thrombi retrieved by MT to evaluate: (1) the ability of EIS and machine learning to predict red blood cell (RBC) percentage content of thrombi and (2) to classify the thrombi as "RBC-rich" or "RBC-poor" based on a range of cutoff values of RBC. METHODS: ClotbasePilot was a multicentric, international, prospective feasibility study. Retrieved thrombi underwent histological analysis to identify proportions of RBC and other components. EIS results were analyzed with machine learning. Linear regression was used to evaluate the correlation between the histology and EIS. Sensitivity and specificity of the model to classify the thrombus as RBC-rich or RBC-poor were also evaluated. RESULTS: Among 514 MT,179 thrombi were included for EIS and histological analysis. The mean composition in RBC of the thrombi was 36% ± 24. Good correlation between the impedance-based prediction and histology was achieved (slope of 0.9, R2 = 0.53, Pearson coefficient = 0.72). Depending on the chosen cutoff, ranging from 20 to 60% of RBC, the calculated sensitivity for classification of thrombi ranged from 77 to 85% and the specificity from 72 to 88%. CONCLUSION: Combination of EIS and machine learning can reliably predict the RBC composition of retrieved ex vivo AIS thrombi and then classify them into groups according to their RBC composition with good sensitivity and specificity.

A Suite of Constitutive Promoters for Tuning Gene Expression in Plants.

The need for convenient tools to express transgenes over a large dynamic range is pervasive throughout plant synthetic biology; however, current efforts are largely limited by the heavy reliance on a small set of strong promoters, precluding more nuanced and refined engineering endeavors in planta. To address this technical gap, we characterize a suite of constitutive promoters that span a wide range of transcriptional levels and develop a GoldenGate-based plasmid toolkit named PCONS, optimized for versatile cloning and rapid testing of transgene expression at varying strengths. We demonstrate how easy access to a stepwise gradient of expression levels can be used for optimizing synthetic transcriptional systems and the production of small molecules in planta. We also systematically investigate the potential of using PCONS as an internal standard in plant biology experimental design, establishing the best practices for signal normalization in experiments. Although our library has primarily been developed for optimizing expression in N. benthamiana, we demonstrate the translatability of our promoters across distantly related species using a multiplexed reporter assay with barcoded transcripts. Our findings showcase the advantages of the PCONS library as an invaluable toolkit for plant synthetic biology.

Interpreting the Evolutionary Echoes of a Protein Complex Essential for Inner-Ear Mechanosensation.

The sensory epithelium of the inner ear, found in all extant lineages of vertebrates, has been subjected to over 500 million years of evolution, resulting in the complex inner ear of modern vertebrates. Inner-ear adaptations are as diverse as the species in which they are found, and such unique anatomical variations have been well studied. However, the evolutionary details of the molecular machinery that is required for hearing are less well known. Two molecules that are essential for hearing in vertebrates are cadherin-23 and protocadherin-15, proteins whose interaction with one another acts as the focal point of force transmission when converting sound waves into electrical signals that the brain can interpret. This "tip-link" interaction exists in every lineage of vertebrates, but little is known about the structure or mechanical properties of these proteins in most non-mammalian lineages. Here, we use various techniques to characterize the evolution of this protein interaction. Results show how evolutionary sequence changes in this complex affect its biophysical properties both in simulations and experiments, with variations in interaction strength and dynamics among extant vertebrate lineages. Evolutionary simulations also characterize how the biophysical properties of the complex in turn constrain its evolution and provide a possible explanation for the increase in deafness-causing mutants observed in cadherin-23 relative to protocadherin-15. Together, these results suggest a general picture of tip-link evolution in which selection acted to modify the tip-link interface, although subsequent neutral evolution combined with varying degrees of purifying selection drove additional diversification in modern tetrapods.

Plant glycosyltransferases for expanding bioactive glycoside diversity.

Glycosylation is a successful strategy to alter the pharmacological properties of small molecules, and it has emerged as a unique approach to expand the chemical space of natural products that can be explored in drug discovery. Traditionally, most glycosylation events have been carried out chemically, often requiring many protection and deprotection steps to achieve a target molecule. Enzymatic glycosylation by glycosyltransferases could provide an alternative strategy for producing new glycosides. In particular, the glycosyltransferase family has greatly expanded in plants, representing a rich enzymatic resource to mine and expand the diversity of glycosides with novel bioactive properties. This article highlights previous and prospective uses for plant glycosyltransferases in generating bioactive glycosides and altering their pharmacological properties.

Characterization of natural killer cells in the blood and airways of cynomolgus macaques during Mycobacterium tuberculosis infection.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and kills more than 1.5 million people each year. METHODS: We examine the frequency and function of NK cells in the blood and airways over the course of Mtb infection in a TB macaque model and demonstrate differences in NK marker expression between the two compartments. Flow cytometry and intracellular cytokine staining were utilized to identify NK cell subsets (expressing NKG2A, CD56, or CD16) and function (IL-10, TNF, IL-2, IFN-g, IL-17, and CD107a). RESULTS: Blood and airway NK cell frequencies were similar during infection though there were differences in subset populations between blood and airway. Increased functional (cytokine/CD107a) parameters were observed in airway NK cells during the course of infection while none were seen in the blood. CONCLUSIONS: This study suggests that NK cells in the airway may play an important role in TB host response.

Work and Life in the Balance: COVID-19 Mortality by Usual Occupation and Industry in Wisconsin.

INTRODUCTION: Work is central to the discourse surrounding the pandemic. Going to work during the COVID-19 pandemic put individuals at risk for both disease and death. This study assesses COVID-19 mortality by industry and occupation for working-age adults in Wisconsin and applies a health equity lens to understand COVID-19, demographics, work, and mortality in the state. METHODS: We used vital records data to evaluate COVID-19 mortality in Wisconsin. We assessed the demographics of working-age decedents using chi-square tests and logistic regression. We also classified decedents by usual occupation with Standard Occupational Classification (2018) and North American Industry Classification System (2017) codes to calculate mortality rates. We then calculated proportional mortality ratios to evaluate if mortality rates from COVID-19 in industry or occupation groups were significantly higher than the overall (ie, average) mortality rate from COVID-19 among all working-age Wisconsin adults. RESULTS: Both Asian/Pacific Islander and Hispanic individuals in Wisconsin had elevated likelihoods of dying from COVID-19. Lower levels of education also were associated with a higher likelihood of COVID-19-attributable death. Additionally, we found several occupations and industries that had elevated mortality rates from COVID-19. Proportional mortality ratios showed higher than expected mortality for several occupations including Protective Service; Office and Administrative Support; Farming, Fishing, and Forestry; and Installation, Maintenance, and Repair. Moreover, several industries had elevated proportional mortality ratios, including Agriculture, Forestry, Fishing, and Hunting; Finance and Insurance; Transportation and Warehousing; and Public Administration. DISCUSSION: The lessons of the pandemic are important for public health and worker safety. Understanding who bears disparate risks allows us to prepare, communicate, and mitigate risk.

Lost time: COVID-19 indemnity claim reporting and results in the Wisconsin workers' compensation system from March 12 to December 31, 2020.

BACKGROUND: The COVID-19 pandemic introduced a new compensable infectious disease to workplaces. METHODS: This was a descriptive analysis of Wisconsin COVID workers' compensation (WC) claims between March 12 and December 31, 2020. The impact of the presumption law (March 12 to June 10, 2020) was also evaluated. RESULTS: Less than 1% of working-age residents with COVID-19 filed a claim. COVID-19 WC claim rates (per 100,000 FTE) were notably low for frontline industry sectors such as Retail Trade (n = 115), Manufacturing (n = 88), and Wholesale Trade (n = 31). Healthcare workers (764 claims per 100,000 FTE) comprised 73.2% of COVID-19 claims. Most claims (52.8%) were denied and the proportion of denied claims increased significantly after the presumption period for both first responders and other occupations. CONCLUSION: The presumption law made benefits accessible primarily to first responders. Further changes to WC systems are needed to offset the individual and collective costs of infectious diseases.

Size Doesn't Matter: Integrative Taxonomy Shows Crepidula adunca and Crepidula norrisiarum Have Overlapping Shell Sizes and Broadly Concordant Distributions.

AbstractThe calyptraeids Crepidula adunca and Crepidula norrisiarum, both direct developers, are abundant in the shallow waters of the northeastern Pacific. They have long been considered as two allopatric species that live on different hosts and differ in body size. In this study, we rigorously test this historical hypothesis by assessing molecular taxonomy, museum records, new morphological and host observations, and population genetic structure along the northeast Pacific coast. Results show that, contrary to previous understanding, the distributions of the two species largely overlap and that size does not effectively distinguish them, especially in the northern part of the range where the nominal "C. adunca" has been studied. Newly recognized northern occurrences of C. norrisiarum demonstrate that both species have similar, disrupted distributions that range from British Colombia through southern California. Neither species is reported to occur on the outer shores of southern Washington or Oregon, the exception being records of C. adunca at Cape Arago, Oregon. Despite this apparent geographic gap, neither species shows appreciable genetic differentiation between the northern and southern parts of its ranges. Despite body size having been used to distinguish these species, our observations do not support body size as a species-specific trait; rather, they support a new hypothesis that body size variation reflects regional differences in host use and host availability.

Optimization of Heterologous Glucoraphanin Production In Planta.

Glucoraphanin is a plant specialized metabolite found in cruciferous vegetables that has long been a target for production in a heterologous host because it can subsequently be hydrolyzed to form the chemopreventive compound sulforaphane before and during consumption. However, previous studies have only been able to produce small amounts of glucoraphanin in heterologous plant and microbial systems compared to the levels found in glucoraphanin-producing plants, suggesting that there may be missing auxiliary genes that play a role in improving production in planta. In an effort to identify auxiliary genes required for high glucoraphanin production, we leveraged transient expression in Nicotiana benthamiana to screen a combination of previously uncharacterized coexpressed genes and rationally selected genes alongside the glucoraphanin biosynthetic pathway. This strategy alleviated metabolic bottlenecks, which improved glucoraphanin production by 4.74-fold. Our optimized glucoraphanin biosynthetic pathway provides a pathway amenable for high glucoraphanin production.

Collective mechanical responses of cadherin-based adhesive junctions as predicted by simulations.

Cadherin-based adherens junctions and desmosomes help stabilize cell-cell contacts with additional function in mechano-signaling, while clustered protocadherin junctions are responsible for directing neuronal circuits assembly. Structural models for adherens junctions formed by epithelial cadherin (CDH1) proteins indicate that their long, curved ectodomains arrange to form a periodic, two-dimensional lattice stabilized by tip-to-tip trans interactions (across junction) and lateral cis contacts. Less is known about the exact architecture of desmosomes, but desmoglein (DSG) and desmocollin (DSC) cadherin proteins are also thought to form ordered junctions. In contrast, clustered protocadherin (PCDH)-based cell-cell contacts in neuronal tissues are thought to be responsible for self-recognition and avoidance, and structural models for clustered PCDH junctions show a linear arrangement in which their long and straight ectodomains form antiparallel overlapped trans complexes. Here, we report all-atom molecular dynamics simulations testing the mechanics of minimalistic adhesive junctions formed by CDH1, DSG2 coupled to DSC1, and PCDHγB4, with systems encompassing up to 3.7 million atoms. Simulations generally predict a favored shearing pathway for the adherens junction model and a two-phased elastic response to tensile forces for the adhesive adherens junction and the desmosome models. Complexes within these junctions first unbend at low tensile force and then become stiff to unbind without unfolding. However, cis interactions in both the CDH1 and DSG2-DSC1 systems dictate varied mechanical responses of individual dimers within the junctions. Conversely, the clustered protocadherin PCDHγB4 junction lacks a distinct two-phased elastic response. Instead, applied tensile force strains trans interactions directly, as there is little unbending of monomers within the junction. Transient intermediates, influenced by new cis interactions, are observed after the main rupture event. We suggest that these collective, complex mechanical responses mediated by cis contacts facilitate distinct functions in robust cell-cell adhesion for classical cadherins and in self-avoidance signaling for clustered PCDHs.