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INTRODUCTION: Individuals with chronic hypoparathyroidism managed with conventional therapy (active vitamin D and calcium) have an increased risk for renal dysfunction versus age- and sex-matched controls. Treatments that replace the physiologic effects of parathyroid hormone (PTH) while reducing the need for conventional therapy may help prevent a decline in renal function in this population. This post hoc analysis examined the impact of palopegteriparatide treatment on renal function in adults with chronic hypoparathyroidism. METHODS: PaTHway is a phase 3 trial of palopegteriparatide in adults with chronic hypoparathyroidism that included a randomized, double-blind, placebo-controlled 26-week period followed by an ongoing 156-week open-label extension (OLE) period. Changes in renal function over 52 weeks (26 weeks blinded + 26 weeks OLE) were assessed using estimated glomerular filtration rate (eGFR). A subgroup analysis was performed with participants stratified by baseline eGFR < 60 or ≥ 60 mL/min/1.73 m2. RESULTS: At week 52, over 95% (78/82) of participants remained enrolled in the OLE and of those, 86% maintained normocalcemia and 95% achieved independence from conventional therapy (no active vitamin D and ≤ 600 mg/day of calcium), with none requiring active vitamin D. Treatment with palopegteriparatide over 52 weeks resulted in a mean (SD) increase in eGFR of 9.3 (11.7) mL/min/1.73 m2 from baseline (P < 0.0001) and 43% of participants had an increase ≥ 10 mL/min/1.73 m2. In participants with baseline eGFR < 60 mL/min/1.73 m2, 52 weeks of treatment with palopegteriparatide resulted in a mean (SD) increase of 11.5 (11.3) mL/min/1.73 m2 (P < 0.001). One case of nephrolithiasis was reported for a participant in the placebo group during blinded treatment; none were reported through week 52 with palopegteriparatide. CONCLUSION: In this post hoc analysis of the PaTHway trial, palopegteriparatide treatment was associated with significantly improved eGFR at week 52 in addition to previously reported maintenance and normalization of serum and urine biochemistries. Further investigation of palopegteriparatide for the preservation of renal function in hypoparathyroidism is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT04701203.

Chronic hypoparathyroidism is caused by inadequate parathyroid hormone (PTH) levels. Hypoparathyroidism is managed with conventional therapy (active vitamin D and calcium), but over time the disease itself and conventional therapy can increase the risk of medical complications including kidney problems. This study looked at how a new treatment for chronic hypoparathyroidism, palopegteriparatide (approved in the European Union under the brand name YORVIPATH^®), affects kidney function in adults in the PaTHway clinical trial. Participants were randomly assigned to receive palopegteriparatide or a placebo injection once daily along with conventional therapy. For both groups, clinicians used a protocol to eliminate conventional therapy while maintaining normal blood calcium levels. After 26 weeks, participants on placebo switched to palopegteriparatide. Ninety-five percent of participants were still enrolled in the PaTHway trial after 52 weeks. Of those, 86% had normal blood calcium levels and 95% did not need conventional therapy (not taking vitamin D and not taking therapeutic doses of calcium [> 600 mg/day]). After 52 weeks of treatment with palopegteriparatide, significant improvements were seen in a measure of kidney function called estimated glomerular filtration rate (eGFR). Improvements in eGFR from the beginning of the trial to week 52 were considered clinically meaningful for over 57% of participants. In participants with impaired kidney function at the beginning of the trial, eGFR improvements were even greater, and 74% of participants had a clinically meaningful improvement. These results suggest that palopegteriparatide treatment may be beneficial for kidney function in adults with chronic hypoparathyroidism, especially those with impaired kidney function.

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Mohs micrographic surgery (MMS) can lead to complications such as scarring and delayed wound healing, particularly in sensitive areas such as the face, neck, and chest. This study aims to assess the evidence regarding the use of lasers post-MMS for wound healing and scar revision. A comprehensive systematic review of the literature was performed using databases including MEDLINE, PubMed, EMBASE, Web of Science, Cochrane Library, and CINAHL from inception until July 25, 2022. A total of 2147 unique studies were identified, from which 17 were included in the analysis. A total of 17 studies reported applications of lasers with favourable efficacy including wound healing (n = 1), resurfacing of full-thickness skin grafts and split-thickness skin grafts (n = 4), periscar telangiectasias (n = 1), functional scar contractures (n = 2), and scar texture (n = 9). Minimal adverse effects were reported with the use of lasers post-MMS. Overall, the use of lasers post-MMS is a safe and well-tolerated option for scar revision with high patient satisfaction and is less invasive than surgical interventions.

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DNA i-motifs (iMs) are non-canonical C-rich secondary structures implicated in numerous cellular processes. Though iMs exist throughout the genome, our understanding of iM recognition by proteins or small molecules is limited to a few examples. We designed a DNA microarray containing 10976 genomic iM sequences to examine the binding profiles of four iM-binding proteins, mitoxantrone and the iMab antibody. iMab microarray screens demonstrated that pH 6.5, 5% BSA buffer was optimal, and fluorescence was correlated with iM C-tract length. hnRNP K broadly recognizes diverse iM sequences, favoring 3-5 cytosine repeats flanked by thymine-rich loops of 1-3 nucleotides. Array binding mirrored public ChIP-Seq datasets, in which 35% of well-bound array iMs are enriched in hnRNP K peaks. In contrast, other reported iM-binding proteins had weaker binding or preferred G-quadruplex (G4) sequences instead. Mitoxantrone broadly binds both shorter iMs and G4s, consistent with an intercalation mechanism. These results suggest that hnRNP K may play a role in iM-mediated regulation of gene expression in vivo, whereas hnRNP A1 and ASF/SF2 are possibly more selective in their binding preferences. This powerful approach represents the most comprehensive investigation of how biomolecules selectively recognize genomic iMs to date.

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. ALS is on a pathogenetic disease spectrum with frontotemporal dementia, referred to as ALS-frontotemporal spectrum disorder (ALS-FTSD). For mutations associated with ALS-FTSD, such as the C9orf72 hexanucleotide repeat expansion, the molecular factors associated with heterogeneity along this spectrum require further characterization. Here, using a targeted NanoString molecular barcoding approach, we interrogate neuroinflammatory dysregulation and heterogeneity at the level of gene expression in post-mortem motor cortex tissue from a cohort of clinically heterogeneous C9-ALS-FTSD cases. We identified 20 dysregulated genes in C9-ALS-FTSD, with enrichment of microglial and inflammatory response gene sets. Two genes with significant correlations to available clinical metrics were selected for validation: FKBP5, a correlate of cognitive function, and brain-derived neurotrophic factor (BDNF), a correlate of disease duration. FKBP5 and its signalling partner, NF-κB, appeared to have a cell type-specific staining distribution, with activated (i.e. nuclear) NF-κB immunoreactivity in C9-ALS-FTSD. Expression of BDNF, a correlate of disease duration, was confirmed to be higher in individuals with long compared to short disease duration using BaseScope™ in situ hybridization. Our analyses also revealed two distinct neuroinflammatory panel signatures (NPS), NPS1 and NPS2, delineated by the direction of expression of proinflammatory, axonal transport and synaptic signalling pathways. We compared NPS between C9-ALS-FTSD cases and those from sporadic ALS and SOD1-ALS cohorts and identified NPS1 and NPS2 across all cohorts. Moreover, a subset of NPS was also able to separate publicly available RNA sequencing data from independent C9-ALS and sporadic ALS cohorts into two inflammatory subgroups. Importantly, NPS subgroups did not clearly segregate with available demographic, genetic, clinical or pathological features, highlighting the value of molecular stratification in clinical trials for inflammatory subgroup identification. Our findings thus underscore the importance of tailoring therapeutic approaches based on distinct molecular signatures that exist between and within ALS-FTSD cohorts.

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Conventional therapy for hypoparathyroidism consisting of active vitamin D and calcium aims to alleviate hypocalcemia but fails to restore normal parathyroid hormone (PTH) physiology. PTH replacement therapy is the ideal physiologic treatment for hypoparathyroidism. The double-blind, placebo-controlled, 26-week, phase 3 PaTHway trial assessed the efficacy and safety of PTH replacement therapy for hypoparathyroidism individuals with the investigational drug TransCon PTH (palopegteriparatide). Participants (n = 84) were randomized 3:1 to once-daily TransCon PTH (initially 18 µg/d) or placebo, both co-administered with conventional therapy. The study drug and conventional therapy were titrated according to a dosing algorithm guided by serum calcium. The composite primary efficacy endpoint was the proportion of participants at week 26 who achieved normal albumin-adjusted serum calcium levels (8.3-10.6 mg/dL), independence from conventional therapy (requiring no active vitamin D and ≤600 mg/d of calcium), and no increase in study drug over 4 weeks before week 26. Other outcomes of interest included health-related quality of life measured by the 36-Item Short Form Survey (SF-36), hypoparathyroidism-related symptoms, functioning, and well-being measured by the Hypoparathyroidism Patient Experience Scale (HPES), and urinary calcium excretion. At week 26, 79% (48/61) of participants treated with TransCon PTH versus 5% (1/21) wiplacebo met the composite primary efficacy endpoint (p < 0.0001). TransCon PTH treatment demonstrated a significant improvement in all key secondary endpoint HPES domain scores (all p < 0.01) and the SF-36 Physical Functioning subscale score (p = 0.0347) compared with placebo. Additionally, 93% (57/61) of participants treated with TransCon PTH achieved independence from conventional therapy. TransCon PTH treatment normalized mean 24-hour urine calcium. Overall, 82% (50/61) treated with TransCon PTH and 100% (21/21) wiplacebo experienced adverse events; most were mild (46%) or moderate (46%). No study drug-related withdrawals occurred. In conclusion, TransCon PTH maintained normocalcemia while permitting independence from conventional therapy and was well-tolerated in individuals with hypoparathyroidism. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

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RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases.

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Clinical heterogeneity observed across patients with amyotrophic lateral sclerosis (ALS) is a known complicating factor in identifying potential therapeutics, even within cohorts with the same mutation, such as C9orf72 hexanucleotide repeat expansions (HREs). Thus, further understanding of pathways underlying this heterogeneity is essential for appropriate ALS trial stratification and the meaningful assessment of clinical outcomes. It has been shown that both inflammation and protein misfolding can influence ALS pathogenesis, such as the manifestation or severity of motor or cognitive symptoms. However, there has yet to be a systematic and quantitative assessment of immunohistochemical markers to interrogate the potential relevance of these pathways in an unbiased manner. To investigate this, we extensively characterised features of commonly used glial activation and protein misfolding stains in thousands of images of post-mortem tissue from a heterogeneous cohort of deeply clinically profiled patients with a C9orf72 HRE. Using a random forest model, we show that microglial staining features are the most accurate classifiers of disease status in our panel and that clinicopathological relationships exist between microglial activation status, TDP-43 pathology, and language dysfunction. Furthermore, we detected spatially resolved changes in fused in sarcoma (FUS) staining, suggesting that liquid-liquid phase shift of this aggregation-prone RNA-binding protein may be important in ALS caused by a C9orf72 HRE. Interestingly, no one feature alone significantly impacted the predictiveness of the model, indicating that the collective examination of all features, or a combination of several features, is what allows the model to be predictive. Our findings provide further support to the hypothesis of dysfunctional immune regulation and proteostasis in the pathogenesis of C9-ALS and provide a framework for digital analysis of commonly used neuropathological stains as a tool to enrich our understanding of clinicopathological relationships within and between cohorts. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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Recently, Chen and colleagues reported the development of phosphatase-targeting chimeric molecules (PhosTACs), heterobifunctional small molecules that promote targeted, proximity-induced protein dephosphorylation. This strategy represents an innovative approach to selectively manipulate phosphoprotein function and provides proof-of-concept for a new class of bifunctional small molecules in the chemical biologist's toolbox.

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Amyotrophic lateral sclerosis is a rapidly progressive and fatal disease. Although astrocytes are increasingly recognized contributors to the underlying pathogenesis, the cellular autonomy and uniformity of astrocyte reactive transformation in different genetic forms of amyotrophic lateral sclerosis remain unresolved. Here we systematically examine these issues by using highly enriched and human induced pluripotent stem cell-derived astrocytes from patients with VCP and SOD1 mutations. We show that VCP mutant astrocytes undergo cell-autonomous reactive transformation characterized by increased expression of complement component 3 (C3) in addition to several characteristic gene expression changes. We then demonstrate that isochronic SOD1 mutant astrocytes also undergo a cell-autonomous reactive transformation, but that this is molecularly distinct from VCP mutant astrocytes. This is shown through transcriptome-wide analyses, identifying divergent gene expression profiles and activation of different key transcription factors in SOD1 and VCP mutant human induced pluripotent stem cell-derived astrocytes. Finally, we show functional differences in the basal cytokine secretome between VCP and SOD1 mutant human induced pluripotent stem cell-derived astrocytes. Our data therefore reveal that reactive transformation can occur cell autonomously in human amyotrophic lateral sclerosis astrocytes and with a striking degree of early molecular and functional heterogeneity when comparing different disease-causing mutations. These insights may be important when considering astrocyte reactivity as a putative therapeutic target in familial amyotrophic lateral sclerosis.

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Single cell transcriptome profiling has emerged as a breakthrough technology for the high-resolution understanding of complex cellular systems. Here we report a flexible, cost-effective and user-friendly droplet-based microfluidics system, called the Nadia Instrument, that can allow 3' mRNA capture of ~ 50,000 single cells or individual nuclei in a single run. The precise pressure-based system demonstrates highly reproducible droplet size, low doublet rates and high mRNA capture efficiencies that compare favorably in the field. Moreover, when combined with the Nadia Innovate, the system can be transformed into an adaptable setup that enables use of different buffers and barcoded bead configurations to facilitate diverse applications. Finally, by 3' mRNA profiling asynchronous human and mouse cells at different phases of the cell cycle, we demonstrate the system's ability to readily distinguish distinct cell populations and infer underlying transcriptional regulatory networks. Notably this provided supportive evidence for multiple transcription factors that had little or no known link to the cell cycle (e.g. DRAP1, ZKSCAN1 and CEBPZ). In summary, the Nadia platform represents a promising and flexible technology for future transcriptomic studies, and other related applications, at cell resolution.

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The sphingosine 1-phosphate (S1P) signaling pathway is an attractive target for pharmacological manipulation due to its involvement in cancer progression and immune cell chemotaxis. The synthesis of S1P is catalyzed by the action of sphingosine kinase 1 or 2 (SphK1 or SphK2) on sphingosine and ATP. While potent and selective inhibitors of SphK1 or SphK2 have been reported, development of potent dual SphK1/SphK2 inhibitors are still needed. Towards this end, we report the structure-activity relationship profiling of 2-(hydroxymethyl)pyrrolidine-based inhibitors with 22d being the most potent dual SphK1/SphK2 inhibitor (SphK1 Ki = 0.679 µM, SphK2 Ki = 0.951 µM) reported in this series. 22d inhibited the growth of engineered Saccharomyces cerevisiae and decreased S1P levels in histiocytic lymphoma myeloid cell line (U937 cells), demonstrating inhibition of SphK1 and 2 in vitro. Molecular modeling studies of 22d docked inside the Sph binding pocket of both SphK1 and SphK2 indicate essential hydrogen bond between the 2-(hydroxymethyl)pyrrolidine head to interact with aspartic acid and serine residues near the ATP binding pocket, which provide the basis for dual inhibition. In addition, the dodecyl tail adopts a "J-shape" conformation found in crystal structure of sphingosine bound to SphK1. Collectively, these studies provide insight into the intermolecular interactions in the SphK1 and 2 active sites to achieve maximal dual inhibitory activity.

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Single-cell RNA-sequencing (scRNA-seq) has emerged in recent years as a breakthrough technology to understand RNA metabolism at cellular resolution. In addition to allowing new cell types and states to be identified, scRNA-seq can permit cell-type specific differential gene expression changes, pre-mRNA processing events, gene regulatory networks and single-cell developmental trajectories to be uncovered. More recently, a new wave of multi-omic adaptations and complementary spatial transcriptomics workflows have been developed that facilitate the collection of even more holistic information from individual cells. These developments have unprecedented potential to provide penetrating new insights into the basic neural cell dynamics and molecular mechanisms relevant to the nervous system in both health and disease. In this review we discuss this maturation of single-cell RNA-sequencing over the past decade, and review the different adaptations of the technology that can now be applied both at different scales and for different purposes. We conclude by highlighting how these methods have already led to many exciting discoveries across neuroscience that have furthered our cellular understanding of the neurological disease.

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Tattoo pigment can precipitate numerous inflammatory states, and granulomatous tattoo reactions are a diagnostically challenging form. The skin is the most common site of inflammation, but systemic inflammation can occur. Reactions to black tattoo ink have a broad differential of cutaneous and systemic conditions. Sarcoidosis is an important consideration because it is unclear whether it is a separate entity. Here we present a 31-year-old male who developed an inflammatory eruption where he had black tattoos. He also developed circular patches of scalp alopecia, monocular uveitis, and an enlarged axillary lymph node, initially thought to represent lymphoma. Tissue biopsy of the skin and lymph node revealed findings consistent with granulomatous tattoo reaction. Investigations for other diagnoses, including sarcoidosis, were negative. He was treated with systemic corticosteroids and then with topical corticosteroids and oral hydroxychloroquine. This case report demonstrates the diagnostic challenge associated with granulomatous tattoo ink reactions. Further studies are needed to improve characterization and management of this condition.

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An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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PURPOSE: To determine the relationship between the American College of Cardiology/American Heart Association (ACC/AHA) risk score and plaque phenotype of the coronary and carotid arteries assessed directly using CT angiography and MRI. MATERIALS AND METHODS: Asymptomatic subjects eligible for statin therapy by risk score were enrolled in a prospective study of disease burden using coronary artery calcium (CAC) scoring, coronary CT angiography, and MRI of the carotid arteries. Quartiles were calculated for noncalcified plaque, CAC, and average carotid wall volume and were compared with ACC/AHA risk quartiles. RESULTS: Two hundred three subjects were studied (60% men; mean age, 65 years). There were weak correlations between risk and carotid wall volume (Kendall tau = 0.29), noncalcified plaque (tau = 0.16), and CAC (tau = 0.33). ACC/AHA risk alone misclassified plaque extent compared with measurement by carotid wall volume, CAC, and noncalcified plaque in 22.1%, 24.1%, and 29.6% of subjects, respectively. On average, 13% of the subjects were underclassified, and 12.5% were overclassified. CONCLUSION: Approximately 25% of subjects had large discrepancies between ACC/AHA risk and plaque burden at imaging. These results suggest that clinical risk score models alone do not fully reflect the amount of atherosclerotic disease present.© RSNA, 2020See also the commentary by Truong and Villines in this issue.

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The sphingosine-1-phosphate (S1P) signaling pathway is an attractive drug target due to its involvement in immune cell chemotaxis and vascular integrity. The formation of S1P is catalyzed by sphingosine kinase 1 or 2 (SphK1 or SphK2) from sphingosine (Sph) and ATP. Inhibition of SphK1 and SphK2 to attenuate levels of S1P has been reported to be efficacious in animal models of diseases such as cancer, sickle cell disease, and renal fibrosis. While inhibitors of both SphKs have been reported, improvements in potency and selectivity are still needed. Toward that end, we performed structure-activity relationship profiling of 8 (SLM6031434) and discovered a heretofore unrecognized side cavity that increased inhibitor potency toward SphK2. Interrogating this region revealed that relatively small hydrophobic moieties are preferred, with 10 being the most potent SphK2-selective inhibitor (Ki = 89 nM, 73-fold SphK2-selective) with validated in vivo activity.

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The cutaneous microbiome has potential for therapeutic intervention in inflammatory-driven skin disease. Research into atopic dermatitis and acne vulgaris has highlighted the importance of the skin microbiota in disease pathogenesis, prognostication, and targets for therapeutic intervention. Current management of these conditions aims to control the inflammatory response thought to be associated with specific pathogens using both topical and systemic antimicrobials. However, commensal microbiota found naturally on the skin have been shown to play an important role in the resolution of disease flares. Although often efficacious, the mainstay treatments are not without adverse effects and raise concerns regarding the development of antimicrobial resistance. Augmentation of microbial communities with targeted biotherapy could revolutionize the way inflammatory conditions of the skin are treated. Herein, we review evidence for the role of the cutaneous microbiome in atopic dermatitis and acne vulgaris and suggest that these conditions highlight the potential for microbiome-directed therapeutics.

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Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites. We identified RNA binding proteins that crosslink to each peak, including known and candidate splicing factors. Moreover, we detected the use of over 40,000 BPs with strong sequence consensus and structural accessibility, which align well to nearby crosslinking peaks. We show how the position and strength of BPs affect the crosslinking patterns of spliceosomal factors, which bind more efficiently upstream of strong or proximally located BPs and downstream of weak or distally located BPs. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

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A method for the preparation of air stable difluoroboryl acrylamides is reported. In contrast to the ubiquitous organotrifluoroborate salts, difluoroboryl acrylamides are relatively nonpolar and are readily purified by silica chromatography. Difluoroboryl acrylamides serve as efficient substrates in cross-coupling reactions to afford the corresponding trisubstituted acrylamides in good to excellent yields. The utility of the difluoroboryl group in various chemical transformations is presented.