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During embryonic development, hierarchical cascades of transcription factors interact with lineage-specific chromatin structures to control the sequential steps in the differentiation of specialized cell types. While examples of transcription factor cascades have been well documented, the mechanisms underlying developmental changes in accessibility of cell type–specific enhancers remain poorly understood. Here, we show that the transcriptional “master regulator” ATOH1—which is necessary for the differentiation of two distinct mechanoreceptor cell types, hair cells in the inner ear and Merkel cells of the epidermis—is unable to access much of its target enhancer network in the progenitor populations of either cell type when it first appears, imposing a block to further differentiation. This block is overcome by a feed-forward mechanism in which ATOH1 first stimulates expression of POU4F3, which subsequently acts as a pioneer factor to provide access to closed ATOH1 enhancers, allowing hair cell and Merkel cell differentiation to proceed. Our analysis also indicates the presence of both shared and divergent ATOH1/POU4F3-dependent enhancer networks in hair cells and Merkel cells. These cells share a deep developmental lineage relationship, deriving from their common epidermal origin, and suggesting that this feed-forward mechanism preceded the evolutionary divergence of these very different mechanoreceptive cell types.

Reprogramming of the cochlea with hair-cell-specific transcription factors such as ATOH1 has been proposed as a potential therapeutic strategy for hearing loss. ATOH1 expression in the developing cochlea can efficiently induce hair cell regeneration but the efficiency of hair cell reprogramming declines rapidly as the cochlea matures. We developed Cre-inducible mice to compare hair cell reprogramming with ATOH1 alone or in combination with two other hair cell transcription factors, GFI1 and POU4F3. In newborn mice, all transcription factor combinations tested produced large numbers of cells with the morphology of hair cells and rudimentary mechanotransduction properties. However, 1 week later, only a combination of ATOH1, GFI1 and POU4F3 could reprogram non-sensory cells of the cochlea to a hair cell fate, and these new cells were less mature than cells generated by reprogramming 1 week earlier. We used scRNA-seq and combined scRNA-seq and ATAC-seq to suggest at least two impediments to hair cell reprogramming in older animals. First, hair cell gene loci become less epigenetically accessible in non-sensory cells of the cochlea with increasing age. Second, signaling from hair cells to supporting cells, including Notch signaling, can prevent reprogramming of many supporting cells to hair cells, even with three hair cell transcription factors. Our results shed light on the molecular barriers that must be overcome to promote hair cell regeneration in the adult cochlea.

Mycolactone is an exotoxin produced by Mycobacterium ulcerans that causes the neglected tropical skin disease Buruli ulcer. This toxin inhibits the Sec61 translocon in the endoplasmic reticulum (ER), preventing the host cell from producing several secretory and transmembrane proteins, resulting in cytotoxic and immunomodulatory effects. Interestingly, only one of the two dominant isoforms of mycolactone is cytotoxic. Here, we investigate the origin of this specificity by performing extensive molecular dynamics (MD) simulations with enhanced free energy sampling to query the association trends of the two isoforms with both the Sec61 translocon, using two distinct cryo-electron microscopy (cryo-EM) models as references, and the ER membrane, which serves as a toxin reservoir prior to association. Our results suggest that mycolactone B (the cytotoxic isoform) has a stronger association with the ER membrane than mycolactone A due to more favorable interactions with membrane lipids and water molecules. This could increase the reservoir of toxin proximal to the Sec61 translocon. In one model of Sec61 inhibited by mycolactone, we find that isomer B interacts more closely with residues thought to play a key role in signal peptide recognition and, thus, are essential for subsequent protein translocation. In the other model, we find that isomer B interacts more closely with the lumenal and lateral gates of the translocon, the dynamics of which are essential for protein translocation. These interactions induce a more closed conformation, which has been suggested to block signal peptide insertion and subsequent protein translocation. Collectively, these findings suggest that isomer B’s unique cytotoxicity is a consequence of both increased localization to the ER membrane and channel-locking association with the Sec61 translocon, facets that could be targeted in the development of Buruli Ulcer diagnostics and Sec61-targeted therapeutics.

Radical reactions are a powerful class of chemical transformations. However, the formation of radical species to initiate these reactions has often required the use of stoichiometric amounts of toxic reagents, such as tributyltin hydride. Recently, the use of visible-light-mediated photoredox catalysis to generate radical species has become popular, but the scope of these radical precursors has been limited. Here, we describe the identification of reaction conditions under which photocatalysts such as fac -Ir(ppy) 3 can be utilized to form radicals from unactivated alkyl, alkenyl and aryl iodides. The generated radicals undergo reduction via hydrogen atom abstraction or reductive cyclization. The reaction protocol utilizes only inexpensive reagents, occurs under mild reaction conditions, and shows exceptional functional group tolerance. Reaction efficiency is maintained upon scale-up and decreased catalyst loading, and the reaction time can be significantly shortened when the reaction is performed in a flow reactor. Visible-light-mediated photocatalytic generation of carbon-centred radicals from alkyl, alkenyl and aryl iodides, which then undergo subsequent hydrogen-atom abstraction or reductive cyclizations, is reported. The protocol is characterized by the use of inexpensive reagents, mild conditions, exceptional functional group tolerance, and good to high yields.

Although liposarcoma is the second most common soft-tissue sarcoma in adults, it is exceedingly rare in the head and neck. According to our MEDLINE search, only 11 cases of liposarcoma of the parotid gland have been reported since 1968. We report a new case of primary atypical lipomatous tumor/well-differentiated liposarcoma of the parotid gland in a 77-year-old man. Because only a very limited number of case reports and small series have been published on liposarcoma in the head and neck, we also provide a review of the literature on this uncommon disease entity.

[...] correct analyses with susceptibility weighted imaging, such as GRE, facilitate the differentiation and support the correct diagnosis, encouraging appropriate surgical treatment. [...] it is important to be able to characterize these lesions on radiological imaging.

Mycolactone is an exotoxin produced by that causes the neglected tropical skin disease Buruli ulcer. This toxin inhibits the Sec61 translocon in the endoplasmic reticulum (ER), preventing the host cell from producing many secretory and transmembrane proteins, resulting in cytotoxic and immunomodulatory effects. Interestingly, only one of the two dominant isoforms of mycolactone is cytotoxic. Here, we investigate the origin of this specificity by performing extensive molecular dynamics (MD) simulations with enhanced free energy sampling to query the association trends of the two isoforms with both the Sec61 translocon and the ER membrane, which serves as a toxin reservoir prior to association. Our results suggest that mycolactone B (the cytotoxic isoform) has a stronger association with the ER membrane than mycolactone A due to more favorable interactions with membrane lipids and water molecules. This could increase the reservoir of toxin proximal to the Sec61 translocon. Isomer B also interacts more closely with the lumenal and lateral gates of the translocon, the dynamics of which are essential for protein translocation. These interactions induce a more closed conformation, which has been suggested to block signal peptide insertion and subsequent protein translocation. Collectively, these findings suggest that isomer B's unique cytotoxicity is a consequence of both increased localization to the ER membrane and channel-locking association with the Sec61 translocon, facets that could be targeted in the development of Buruli Ulcer diagnostics and Sec61-targeted therapeutics.

Lasso peptides are a unique class of natural products with distinctively threaded structures, conferring exceptional stability against thermal and proteolytic degradation. Despite their promising biotechnological and pharmaceutical applications, reported attempts to prepare them by chemical synthesis result in forming the nonthreaded branched-cyclic isomer, rather than the desired lassoed structure. This is likely due to the entropic challenge of folding a short, threaded motif prior to chemically mediated cyclization. Accordingly, this study aims to better understand and enhance the relative stability of pre-lasso conformations-the essential precursor to lasso peptide formation-through sequence optimization, chemical modification, and disulfide incorporation. Using Rosetta fixed backbone design, optimal sequences for several class II lasso peptides are identified. Enhanced sampling with well-tempered metadynamics confirmed that designed sequences derived from the lasso structures of rubrivinodin and microcin J25 exhibit a notable improvement in pre-lasso stability relative to the competing nonthreaded conformations. Chemical modifications to the isopeptide bond-forming residues of microcin J25 further increase the probability of pre-lasso formation, highlighting the beneficial role of non-canonical amino acid residues. Counterintuitively, the introduction of a disulfide cross-link decreased pre-lasso stability. Although cross-linking inherently constrains the peptide structure, decreasing the entropic dominance of unfolded phase space, it hinders the requisite wrapping of the N-terminal end around the tail to adopt the pre-lasso conformation. However, combining chemical modifications with the disulfide cross-link results in further pre-lasso stabilization, indicating that the ring modifications counteract the constraints and provide a cooperative benefit with cross-linking. These findings lay the groundwork for further design efforts to enable synthetic access to the lasso peptide scaffold. Lasso peptides are a unique class of ribosomally synthesized and post-translationally modified natural products with diverse biological activities and potential for therapeutic applications. Although direct synthesis would facilitate therapeutic design, it has not yet been possible to fold these short sequences to their threaded architecture without the help of biosynthetic enzyme stabilization. Our work explores strategies to enhance the stability of the pre-lasso structure, the essential precursor to lasso peptide formation. We find that sequence design, incorporating non-canonical amino acid residues, and design-guided cross-linking can augment stability to increase the likelihood of lasso motif accessibility. This work presents several strategies for the continued design of foldable lasso peptides.

Circulating tumor cells (CTCs) can be isolated via a minimally invasive blood draw and are considered a liquid biopsy of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs, and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small molecule drug screening to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetylcysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single cell expansion. RNA-seq analysis of growing clones and non-growing clones confirmed the effect by NAC, but also indicate that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with heterogeneous quiescent and senescent features. Despite the challenge in expanding all CTCs, NAC treatment lead to establishment of single CTC clones that have similar tumorigenic features, which will facilitate future functional analyses. Competing Interest Statement Min Yu is the founder and president for CanTraCer Biosciences Inc. and a consultant for Microsensor labs.