Proteasomal control of anti-CRISPRs for the regulation of CRISPR/Cas9 activity using Cas9-ACROBAT.

Small molecule-mediated proteasomal degradation of proteins is a powerful tool for synthetic regulation of biological activity. To control Cas9 activity in cells, we engineered an anti-CRISPR protein, AcrIIA4, fused to a degradation (dTAG) or small molecule assisted shutoff (SMASh) tag. Co-expression of the tagged AcrIIA4 along with Cas9 and riboswitch-regulated sgRNAs enables precise tunable control of CRISPR activity by small molecule addition.

Skeletal Muscle Texture Assessment Using Ultrasonography: Comparison with Magnetic Resonance Imaging in Chronic Kidney Disease.

Skeletal muscle dysfunction is common in chronic kidney disease (CKD). Of interest is the concept of "muscle quality," of which measures include ultrasound-derived echo intensity (EI). Alternative parameters of muscle texture, for example, gray level of co-occurrence matrix (GCLM), are available and may circumvent limitations in EI. The validity of EI is limited in humans, particularly in chronic diseases. This study aimed to investigate the associations between ultrasound-derived parameters of muscle texture with MRI. Images of the thigh were acquired using a 3 Tesla MRI scanner. Quantification of muscle (contractile), fat (non-contractile), and miscellaneous (connective tissue, fascia) components were estimated. Anatomical rectus femoris cross-sectional area was measured using B-mode 2D ultrasonography. To assess muscle texture, first (i.e., EI)- and second (i.e., GLCM)-order statistical analyses were performed. Fourteen participants with CKD were included (age: 58.0 ± 11.9 years, 50% male, eGFR: 27.0 ± 7.4 ml/min/1.73m2, 55% Stage 4). Higher EI was associated with lower muscle % (quadriceps: ß = -.568, p = .034; hamstrings: ß = -.644, p = .010). Higher EI was associated with a higher fat % in the hamstrings (ß = -.626, p = .017). A higher angular second moment from GLCM analysis was associated with greater muscle % (ß = .570, p = .033) and lower fat % (ß = -.534, p = .049). A higher inverse difference moment was associated with greater muscle % (ß = .610, p = .021 and lower fat % (ß = -.599, p = .024). This is the first study to investigate the associations between ultrasound-derived parameters of muscle texture with MRI. Our preliminary findings suggest ultrasound-derived texture analysis provides a novel indicator of reduced skeletal muscle % and thus increased intramuscular fat.

Chromosome evolution screens recapitulate tissue-specific tumor aneuploidy patterns.

Whole chromosome and arm-level copy number alterations occur at high frequencies in tumors, but their selective advantages, if any, are poorly understood. Here, utilizing unbiased whole chromosome genetic screens combined with in vitro evolution to generate arm- and subarm-level events, we iteratively selected the fittest karyotypes from aneuploidized human renal and mammary epithelial cells. Proliferation-based karyotype selection in these epithelial lines modeled tissue-specific tumor aneuploidy patterns in patient cohorts in the absence of driver mutations. Hi-C-based translocation mapping revealed that arm-level events usually emerged in multiples of two via centromeric translocations and occurred more frequently in tetraploids than diploids, contributing to the increased diversity in evolving tetraploid populations. Isogenic clonal lineages enabled elucidation of pro-tumorigenic mechanisms associated with common copy number alterations, revealing Notch signaling potentiation as a driver of 1q gain in breast cancer. We propose that intrinsic, tissue-specific proliferative effects underlie tumor copy number patterns in cancer.

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation.

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

Healthcare Professionals' Experiences of Recovery-Oriented Collaboration Between Mental Health Centres and Municipalities: A Qualitative Study.

Collaboration within mental health centres and with municipalities in Western European healthcare has presented challenges due to structural and cultural disparities. The Danish healthcare system faces obstacles that impact mental healthcare services, particularly in cross-sectorial cooperation. Our aim was to investigate healthcare professionals' experiences of recovery-oriented collaboration within a mental healthcare setting across hospitals and municipalities to gather a deeper understanding of this issue. Twenty-four employees were purposively sampled from mental health centres in Copenhagen and focus group interviews were conducted to explore their perceptions of working together. Inductive content analysis was used to analyse the data and identify themes and categories. The participants emphasised challenges in communication and coordination to improve collaboration within across the two sectors. This study can contribute to a greater understanding of collaboration between mental health centres and municipalities. It aims to inspire improvements in communication, coordination, and the optimisation of mental health service delivery across sectors.

Corrigendum to "Advances in In Vitro and In Vivo Bioreactor-Based Bone Generation for Craniofacial Tissue Engineering".

[This corrects the article DOI: 10.34133/bmef.0004.].

Advances in In Vitro and In Vivo Bioreactor-Based Bone Generation for Craniofacial Tissue Engineering.

Craniofacial reconstruction requires robust bone of specified geometry for the repair to be both functional and aesthetic. While native bone from elsewhere in the body can be harvested, shaped, and implanted within a defect, using either an in vitro or in vivo bioreactors eliminates donor site morbidity while increasing the customizability of the generated tissue. In vitro bioreactors utilize cells harvested from the patient, a scaffold, and a device to increase mass transfer of nutrients, oxygen, and waste, allowing for generation of larger viable tissues. In vivo bioreactors utilize the patient's own body as a source of cells and of nutrient transfer and involve the implantation of a scaffold with or without growth factors adjacent to vasculature, followed by the eventual transfer of vascularized, mineralized tissue to the defect site. Several different models of in vitro bioreactors exist, and several different implantation sites have been successfully utilized for in vivo tissue generation and defect repair in humans. In this review, we discuss the specifics of each bioreactor strategy, as well as the advantages and disadvantages of each and the future directions for the engineering of bony tissues for craniofacial defect repair.

Exploiting Chemical Protein Synthesis to Study the Role of Tyrosine Sulfation on Anticoagulants from Hematophagous Organisms.

Tyrosine sulfation is a post-translational modification (PTM) that modulates function by mediating key protein-protein interactions. One of the early proteins shown to possess this PTM was hirudin, produced in the salivary glands of the medicinal leech Hirudo medicinalis, whereby tyrosine sulfation led to a ∼10-fold improvement in α-thrombin inhibitory activity. Outside of this pioneering discovery, the involvement of tyrosine sulfation in modulating the activity of salivary proteins from other hematophagous organisms was unknown. We hypothesized that the intrinsic instability of the tyrosine sulfate functionality, particularly under the acidic conditions used to isolate and analyze peptides and proteins, has led to poor detection during the isolation and/or expression of these molecules.Herein, we summarize our efforts to interrogate the functional role of tyrosine sulfation in the thrombin inhibitory and anticoagulant activity of salivary peptides and proteins from a range of different blood feeding organisms, including leeches, ticks, mosquitoes, and flies. Specifically, we have harnessed synthetic chemistry to efficiently generate homogeneously sulfated peptides and proteins for detailed structure-function studies both in vitro and in vivo.Our studies began with the leech protein hirudin P6 (from Hirudinaria manillensis), which is both sulfated on tyrosine and O-glycosylated at a nearby threonine residue. Synthetically, this was achieved through solid-phase peptide synthesis (SPPS) with a late-stage on-resin sulfation, followed by native chemical ligation and a folding step to generate six differentially modified variants of hirudin P6 to assess the functional interplay between O-glycosylation and tyrosine sulfation. A one-pot, kinetically controlled ligation of three peptide fragments was used to assemble homogeneously sulfoforms of madanin-1 and chimadanin from the tick Haemaphysalis longicornis. Dual tyrosine sulfation at two distinct sites was shown to increase the thrombin inhibitory activity by up to 3 orders of magnitude through a novel interaction with exosite II of thrombin. The diselenide-selenoester ligation developed by our lab provided us with a means to rapidly assemble a library of different sulfated tick anticoagulant proteins: the andersonins, hyalomins, madanin-like proteins, and hemeathrins, thus enabling the generation of key structure-activity data on this family of proteins. We have also confirmed the presence of tyrosine sulfation in the anticoagulant proteins of Anopheles mosquitoes (anophelins) and the Tsetse fly (TTI) via insect expression and mass spectrometric analysis. These molecules were subsequently synthesized and assessed for thrombin inhibitory and anticoagulant activity. Activity was significantly improved by the addition of tyrosine sulfate modifications and led to molecules with potent antithrombotic activity in an in vivo murine thrombosis model.The Account concludes with our most recent work on the design of trivalent hybrids that tandemly occupy the active site and both exosites (I and II) of α-thrombin, with a TTI-anophelin hybrid (Ki = 20 fM against α-thrombin) being one of the most potent protease inhibitors and anticoagulants ever generated. Taken together, this Account highlights the importance of the tyrosine sulfate post-translational modification within salivary proteins from blood feeding organisms for enhancing anticoagulant activity. This work lays the foundation for exploiting native or engineered variants as therapeutic leads for thrombotic disorders in the future.

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation.

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

A Randomized Trial of Intravenous Iron Supplementation and Exercise on Exercise Capacity in Iron-Deficient Nonanemic Patients With CKD.

Introduction: Patients with chronic kidney disease (CKD) are often iron deficient, even when not anemic. This trial evaluated whether iron supplementation enhances exercise capacity of nonanemic patients with CKD who have iron-deficiency. Methods: Prospective, multicenter double-blind randomized controlled trial of nondialysis patients with CKD and iron-deficiency but without anemia (Hemoglobin [Hb] >110 g/l). Patients were assigned 1:1 to intravenous (IV) iron therapy, or placebo. An 8-week exercise program commenced at week 4. The primary outcome was the mean between-group difference in 6-minute walk test (6MWT) at 4 weeks. Secondary outcomes included 6MWT at 12 weeks, transferrin saturation (TSAT), serum ferritin (SF), Hb, renal function, muscle strength, functional capacity, quality of life, and adverse events at baseline, 4 weeks, and at 12 weeks. Mean between-group differences were analyzed using analysis of covariance models. Results: Among 75 randomized patients, mean (SD) age for iron therapy (n = 37) versus placebo (n = 38) was 54 (16) versus 61 (12) years; estimated glomerular filtration rate (eGFR) (34 [12] vs. 35 [11] ml/min per 1.73 m2], TSAT (23 [12] vs. 21 [6])%; SF (57 [64] vs. 62 [33]) µg/l; Hb (122.4 [9.2] vs. 127 [13.2] g/l); 6MWT (384 [95] vs. 469 [142] meters) at baseline, respectively. No significant mean between-group difference was observed in 6MWT distance at 4 weeks. There were significant increases in SF and TSAT at 4 and 12 weeks (P < 0.02), and Hb at 12 weeks (P = 0.009). There were no between-group differences in other secondary outcomes and no adverse events attributable to iron therapy. Conclusion: This trial did not demonstrate beneficial effects of IV iron therapy on exercise capacity at 4 weeks. A larger study is needed to confirm if IV iron is beneficial in nondialysis patients with CKD who are iron-deficient.

Mapping Health Disparities in 11 High-Income Nations.

Importance: Health care delivery faces a myriad of challenges globally with well-documented health inequities based on geographic location. Yet, researchers and policy makers have a limited understanding of the frequency of geographic health disparities. Objective: To describe geographic health disparities in 11 high-income countries. Design, Setting, and Participants: In this survey study, we analyzed results from the 2020 Commonwealth Fund International Health Policy (IHP) Survey-a nationally representative, self-reported, and cross-sectional survey of adults from Australia, Canada, France, Germany, the Netherlands, New Zealand, Norway, Sweden, Switzerland, the UK, and the US. Eligible adults older than age 18 years were included by random sampling. Survey data were compared for the association of area type (rural or urban) with 10 health indicators across 3 domains: health status and socioeconomic risk factors, affordability of care, and access to care. Logistic regression was used to determine the associations between countries with area type for each factor, controlling for individuals' age and sex. Main Outcomes and Measures: The main outcomes were geographic health disparities as measured by differences in respondents living in urban and rural settings in 10 health indicators across 3 domains. Results: There were 22 402 survey respondents (12 804 female [57.2%]), with a 14% to 49% response rate depending on the country. Across the 11 countries and 10 health indicators and 3 domains (health status and socioeconomic risk factors, affordability of care, access to care), there were 21 occurrences of geographic health disparities; 13 of those in which rural residence was a protective factor and 8 of those where rural residence was a risk factor. The mean (SD) number of geographic health disparities in the countries was 1.9 (1.7). The US had statistically significant geographic health disparities in 5 of 10 indicators, the most of any country, while Canada, Norway, and the Netherlands had no statistically significant geographic health disparities. The indicators with the most occurrences of geographic health disparities were in the access to care domain. Conclusions and Relevance: In this survey study of 11 high-income nations, health disparities across 10 indicators were identified. Differences in number of disparities reported by country suggest that health policy and decision makers in the US should look to Canada, Norway, and the Netherlands to improve geographic-based health equity.

ERα-associated translocations underlie oncogene amplifications in breast cancer.

Focal copy-number amplification is an oncogenic event. Although recent studies have revealed the complex structure1-3 and the evolutionary trajectories4 of oncogene amplicons, their origin remains poorly understood. Here we show that focal amplifications in breast cancer frequently derive from a mechanism-which we term translocation-bridge amplification-involving inter-chromosomal translocations that lead to dicentric chromosome bridge formation and breakage. In 780 breast cancer genomes, we observe that focal amplifications are frequently connected to each other by inter-chromosomal translocations at their boundaries. Subsequent analysis indicates the following model: the oncogene neighbourhood is translocated in G1 creating a dicentric chromosome, the dicentric chromosome is replicated, and as dicentric sister chromosomes segregate during mitosis, a chromosome bridge is formed and then broken, with fragments often being circularized in extrachromosomal DNAs. This model explains the amplifications of key oncogenes, including ERBB2 and CCND1. Recurrent amplification boundaries and rearrangement hotspots correlate with oestrogen receptor binding in breast cancer cells. Experimentally, oestrogen treatment induces DNA double-strand breaks in the oestrogen receptor target regions that are repaired by translocations, suggesting a role of oestrogen in generating the initial translocations. A pan-cancer analysis reveals tissue-specific biases in mechanisms initiating focal amplifications, with the breakage-fusion-bridge cycle prevalent in some and the translocation-bridge amplification in others, probably owing to the different timing of DNA break repair. Our results identify a common mode of oncogene amplification and propose oestrogen as its mechanistic origin in breast cancer.

Repair of complex ovine segmental mandibulectomy utilizing customized tissue engineered bony flaps.

Craniofacial defects require a treatment approach that provides both robust tissues to withstand the forces of mastication and high geometric fidelity that allows restoration of facial architecture. When the surrounding soft tissue is compromised either through lack of quantity (insufficient soft tissue to enclose a graft) or quality (insufficient vascularity or inducible cells), a vascularized construct is needed for reconstruction. Tissue engineering using customized 3D printed bioreactors enables the generation of mechanically robust, vascularized bony tissues of the desired geometry. While this approach has been shown to be effective when utilized for reconstruction of non-load bearing ovine angular defects and partial segmental defects, the two-stage approach to mandibular reconstruction requires testing in a large, load-bearing defect. In this study, 5 sheep underwent bioreactor implantation and the creation of a load-bearing mandibular defect. Two bioreactor geometries were tested: a larger complex bioreactor with a central groove, and a smaller rectangular bioreactor that were filled with a mix of xenograft and autograft (initial bone volume/total volume BV/TV of 31.8 ± 1.6%). At transfer, the tissues generated within large and small bioreactors were composed of a mix of lamellar and woven bone and had BV/TV of 55.3 ± 2.6% and 59.2 ± 6.3%, respectively. After transfer of the large bioreactors to the mandibular defect, the bioreactor tissues continued to remodel, reaching a final BV/TV of 64.5 ± 6.2%. Despite recalcitrant infections, viable osteoblasts were seen within the transferred tissues to the mandibular site at the end of the study, suggesting that a vascularized customized bony flap is a potentially effective reconstructive strategy when combined with an optimal stabilization strategy and local antibiotic delivery prior to development of a deep-seated infection.

Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons.

Neurotransmitter-filled synaptic vesicles (SVs) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading model posits that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH (retention using selective hooks) system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct transmembrane SV proteins, synaptotagmin 1 and synaptobrevin 2, from the soma of mature cultured rat and mouse neurons. For these studies, the SV reporter constructs were expressed at carefully controlled, very low levels. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons with minimal entry into dendrites. However, even moderate overexpression resulted in the spillover of SV proteins into dendrites, potentially explaining the origin of previous non-polarized transport models, revealing the limited, saturable nature of the direct axonal trafficking pathway. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway, for SV proteins, in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis.

Exercise and chronic kidney disease: potential mechanisms underlying the physiological benefits.

Increasing evidence indicates that exercise has beneficial effects on chronic inflammation, cardiorespiratory function, muscle and bone strength and metabolic markers in adults with chronic kidney disease (CKD), kidney failure or kidney transplants. However, the mechanisms that underlie these benefits have received little attention, and the available clinical evidence is mainly from small, short-duration (<12 weeks) exercise intervention studies. The available data, mainly from patients with CKD or on dialysis, suggest that exercise-mediated shifts towards a less inflammatory immune cell profile, enhanced activity of the NRF2 pathway and reduced monocyte infiltration into adipose tissue may underlie improvements in inflammatory biomarkers. Exercise-mediated increases in nitric oxide release and bioavailability, reduced angiotensin II accumulation in the heart, left ventricular remodelling and reductions in myocardial fibrosis may contribute to improvements in left ventricular hypertrophy. Exercise stimulates an anabolic response in skeletal muscle in CKD, but increases in mitochondrial mass and satellite cell activation seem to be impaired in this population. Exercise-mediated activation of the canonical wnt pathway may lead to bone formation and improvements in the levels of the bone-derived hormones klotho and fibroblast growth factor 23 (FGF23). Longer duration studies with larger sample sizes are needed to confirm these mechanisms in CKD, kidney failure and kidney transplant populations and provide evidence for targeted exercise interventions.

Factors that influence the feasibility and implementation of a complex intervention to improve the treatment of peripheral arterial disease in primary and secondary care: a qualitative exploration of patient and provider perspectives.

OBJECTIVES: Our aim was to examine the feasibility and implementation of a complex intervention to improve the care of patients with peripheral arterial disease (the LEGS intervention) from the perspective of patients, general practitioners and secondary care clinicians. DESIGN: A qualitative study involving semistructured individual interviews with patients and providers to gain an understanding of the feasibility of the LEGS intervention as well the barriers and facilitators to implementation in secondary and primary care. SETTING: Primary and secondary care settings across two National Health Service Trusts. PARTICIPANTS: Twenty-five semistructured telephone interviews were conducted with (1) patients who had received the intervention (n=11), (2) secondary care clinicians responsible for delivering the intervention (n=8) and (3) general practitioners (n=6). ANALYSIS: Data were initially analysed using inductive descriptive thematic analysis. The consolidated framework for implementation research was then used as a matrix to explore patterns in the data and to map connections between the three participant groups. Lastly, interpretive analysis allowed for refining, and a final coding frame was developed. RESULTS: Four overarching themes were identified: (1) the potential to make a difference, (2) a solution to address the gap in no man's land, (3), prioritising and making it happen and (4) personalised information and supportive conversations for taking on the advice. The impetus for prioritising and delivering the intervention was further driven by its flexibility and adaptability to be tailored to the individual and to the environment. CONCLUSIONS: The LEGS intervention can be tailored for use at early and late stages of peripheral arterial disease, provides an opportunity to meet patient needs and can be used to promote shared working across the primary-secondary care interface.

A central role for regulated protein stability in the control of TFE3 and MITF by nutrients.

The TFE3 and MITF master transcription factors maintain metabolic homeostasis by regulating lysosomal, melanocytic, and autophagy genes. Previous studies posited that their cytosolic retention by 14-3-3, mediated by the Rag GTPases-mTORC1, was key for suppressing transcriptional activity in the presence of nutrients. Here, we demonstrate using mammalian cells that regulated protein stability plays a fundamental role in their control. Amino acids promote the recruitment of TFE3 and MITF to the lysosomal surface via the Rag GTPases, activating an evolutionarily conserved phospho-degron and leading to ubiquitination by CUL1ß-TrCP and degradation. Elucidation of the minimal functional degron revealed a conserved alpha-helix required for interaction with RagA, illuminating the molecular basis for a severe neurodevelopmental syndrome caused by missense mutations in TFE3 within the RagA-TFE3 interface. Additionally, the phospho-degron is recurrently lost in TFE3 genomic translocations that cause kidney cancer. Therefore, two divergent pathologies converge on the loss of protein stability regulation by nutrients.

Substance Exposure and Adverse Neonatal Outcomes: A Population-Based Cohort Study.

OBJECTIVE: To estimate the independent and combined effects of in utero exposures on birth outcomes in a rural population. STUDY DESIGN: The study used population-level data (2020-2022) from a state-wide surveillance tool (Working in Appalachia to identify at-risk infants, Critical congenital heart disease, and Hearing loss) in West Virginia. Outcomes included low birth weight, preterm birth, small for gestational age, and birth weight in grams. Exposure included a composite variable with 8 levels of 3 exposure (opioids, stimulants, and cannabis) categories. Analyses were adjusted for sociodemographic covariates using multiple logistic and linear regression analyses. RESULTS: Of the 34 412 singleton live births, 1 in 8 newborns (12.2%) had in utero exposure(s) to opioids, stimulants, and/or cannabis, 11.5% were preterm, 7.9% had low birthweight, 9.6% were small for gestational age, and mean birth weight was 3249 ± 563.6 g. Preterm birth was associated with stimulant alone exposure (aOR, 1.40; 95% CI, 1.03-1.89) and stimulant and cannabis concurrent exposure (aOR, 1.69; 95% CI, 1.16, 2.47). Low birthweight was associated with opioids alone (aOR, 1.34; 95% CI, 1.10, 1.63), cannabis alone (aOR, 1.31; 95% CI, 1.13 to -1.52), opioid and cannabis (aOR, 1.61; 95% CI, 1.12 to -2.31), and opioids, stimulants, and cannabis concurrent exposures (aOR, 2.27; 95% CI, 1.43-3.61). Five exposure categories were associated with lower birth weights (adjusted mean difference range. -72 to -211 g). Small for gestational age was associated with opioids alone (aOR, 1.48; 95% CI, 1.24-1.78), cannabis alone (aOR, 1.49; 95% CI, 1.31-1.69), and opioids and cannabis concurrent exposures (aOR, 1.91; 95% CI, 1.36-2.67). CONCLUSIONS: We showed complex associations between in utero substance exposures, preterm birth, birth weight, and sociodemographic factors in a rural population. The results may inform policy efforts to improve maternal and child health in socioeconomically disadvantaged and underserved rural populations.

Site-selective photocatalytic functionalization of peptides and proteins at selenocysteine.

The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide.

Synthesis of Protein-Oligonucleotide Conjugates.

Nucleic acids and proteins form two of the key classes of functional biomolecules. Through the ability to access specific protein-oligonucleotide conjugates, a broader range of functional molecules becomes accessible which leverages both the programmability and recognition potential of nucleic acids and the structural, chemical and functional diversity of proteins. Herein, we summarize the available conjugation strategies to access such chimeric molecules and highlight some key case study examples within the field to showcase the power and utility of such technology.