Clock gene homologs lin-42 and kin-20 regulate circadian rhythms in C. elegans.

Circadian rhythms are endogenous oscillations in nearly all organisms, from prokaryotes to humans, allowing them to adapt to cyclical environments for close to 24 h. Circadian rhythms are regulated by a central clock, based on a transcription-translation feedback loop. One important protein in the central loop in metazoan clocks is PERIOD, which is regulated in part by Casein kinase 1ε/δ (CK1ε/δ) phosphorylation. In the nematode Caenorhabditis elegans, period and casein kinase 1ε/δ are conserved as lin-42 and kin-20, respectively. Here, we studied the involvement of lin-42 and kin-20 in the circadian rhythms of the adult nematode using a bioluminescence-based circadian transcriptional reporter. We show that mutations of lin-42 and kin-20 generate a significantly longer endogenous period, suggesting a role for both genes in the nematode circadian clock, as in other organisms. These phenotypes can be partially rescued by overexpression of either gene under their native promoter. Both proteins are expressed in neurons and epidermal seam cells, as well as in other cells. Depletion of LIN-42 and KIN-20, specifically in neuronal cells after development, was sufficient to lengthen the period of oscillating sur-5 expression. Therefore, we conclude that LIN-42 and KIN-20 are critical regulators of the adult nematode circadian clock through neuronal cells.

MLT-11 is a transient apical extracellular matrix component required for cuticle patterning and function.

Apical extracellular matrices (aECMs) are associated with all epithelia and form a protective layer against biotic and abiotic threats in the environment. Despite their importance, we lack a deep understanding of their structure and dynamics in development and disease. C. elegans molting offers a powerful entry point to understanding developmentally programmed aECM remodeling. A transient matrix is formed in embryos and at the end of each larval stage, presumably to pattern the new cuticle. Focusing on targets of NHR-23, a key transcription factor which drives molting, we identified the Kunitz family protease inhibitor gene mlt-11 as an NHR-23 target. We identified NHR-23-binding sites that are necessary and sufficient for epithelial expression. mlt-11 is necessary to pattern every layer of the adult cuticle, suggesting a broad patterning role prior to the formation of the mature cuticle. MLT-11::mNeonGreen::3xFLAG transiently localized to the aECM in the cuticle and embryo. It was also detected in lining openings to the exterior (vulva, rectum, mouth). Reduction of mlt-11 function disrupted the barrier function of the cuticle. Tissue-specific RNAi suggested mlt-11 activity is primarily necessary in seam cells and we observed alae and seam cell fusion defects upon mlt-11 inactivation. Predicted mlt-11 null mutations caused fully penetrant embryonic lethality and elongation defects suggesting mlt-11 also plays an important role in patterning the embryonic sheath. Finally, we found that mlt-11 inactivation suppressed the blistered cuticle phenotype of mutants of bli-4 mutants, a subtilisin protease gene but did not affect BLI-4::sfGFP expression. These data could suggest that MLT-11 may be necessary to assure proper levels of BLI-4 activity.

A conserved chronobiological complex times C. elegans development.

The mammalian PAS-domain protein PERIOD (PER) and its C. elegans orthologue LIN-42 have been proposed to constitute an evolutionary link between two distinct, circadian and developmental, timing systems. However, while the function of PER in animal circadian rhythms is well understood molecularly and mechanistically, this is not true for the function of LIN-42 in timing rhythmic development. Here, using targeted deletions, we find that the LIN-42 PAS domains are dispensable for the protein's function in timing molts. Instead, we observe arrhythmic molts upon deletion of a distinct sequence element, conserved with PER. We show that this element mediates stable binding to KIN-20, the C. elegans CK1δ/ε orthologue. We demonstrate that CK1δ phosphorylates LIN-42 and define two conserved helical motifs, CK1δ-binding domain A (CK1BD-A) and CK1BD-B, that have distinct roles in controlling CK1δ-binding and kinase activity in vitro. KIN-20 and the LIN-42 CK1BD are required for proper molting timing in vivo. These interactions mirror the central role of a stable circadian PER-CK1 complex in setting a robust ~24-hour period. Hence, our results establish LIN-42/PER - KIN-20/CK1δ/ε as a functionally conserved signaling module of two distinct chronobiological systems.

Supplemental Patch Testing Identifies Allergens Missed by Standard Screening Series.

Background: Supplemental patch testing is an adjunct to standard patch test screening series. Objective: To determine the demographics, characteristics, frequency, relevance, and interpretation of patch test reactions for supplemental patch testing. Methods: Retrospective study of patients tested 2017-2020 with North American Contact Dermatitis Group (NACDG) and supplemental screening series (Supplemental Series A [SSA], Supplemental Series B [SSB]). Demographics, characteristics, reaction strengths, relevance, and final interpretation were recorded. Results: Cohort included 791 patients; 73.5% female, 68.6% age >40 years. 74.1% were White, 15.2% Black, 5.7% Asian, and 1.5% Hispanic. The most common dermatitis sites were scattered/generalized (27.2%), face (24.0%), and hands (23.5%). For 2017-2018 and 2019-2020, respectively, 82% (318/388) and 78.4% (316/403) had ≥1 "allergic" reaction. In addition, 13.5% (52/385) and 11.7% (47/403) had SSA reactions, and 38.1% (115/302) and 31.5% (101/321) had SSB reactions. In the 87 (2017-2018) and 99 (2019-2020) patients with negative NACDG testing, 17 (19.5%) and 12 (12.1%) had supplemental reactions. Of the 34 supplemental allergens with reaction frequency ≥1%, 58.8% (20/34) are not part of the American Contact Dermatitis Society 90 (2020) or NACDG 2021-2022 screening series. The highest frequency allergens from this group were dodecyl and octyl gallate, cinnamic alcohol, phenyl salicylate, hexahydro-1,3,5-tris-(2-hydroxyethyl) triazine, and abitol. Conclusions: Supplemental patch testing identifies additional relevant allergens in patients with suspected allergic contact dermatitis.

Telemedicine and Its Perceptions in a Border Community: A Review of How Health Care Technology Has Helped Increase Access.

Introduction: Telemedicine is a practical way of offering medical services to remote and underserved areas. During the COVID-19 pandemic, telemedicine has provided convenient access to health care and has overcome barriers such as distance that prevent patients from receiving care. Border populations are impacted by this change in health care delivery. The goal of this study was to investigate how a border patient population perceives their experiences with telemedicine. Methods: We utilized telephone surveys of patients who had a recent telehealth visit at the Texas Tech University Health Science Center (TTUHSC) Family Medicine Center clinic in El Paso, Texas. Survey measures included patients' demographics, a quality assessment of the patients' most recent telehealth visit and their experience, a comparison of the patients' telehealth visit to past in-person visits, and a rating of their telehealth visit. Result: Over 2,000 individuals (n = 2,040), primarily Hispanic females, older than the age of 44 years were identified for potential inclusion in the study. Of these, 928 had a contact attempt, of which 1,378 could not be contacted, 592 were invited, 70 declined leading to a response rate of 67.6% (number invited/completed the survey). Most patients agreed that during their most recent telehealth visit their clinician listened well (98.7%), spent adequate time with them (98.2%), was prompt (94.5%), explained things well (98.0%), and was someone they would recommend to others (97.2%). When comparing telehealth to in-person visits, patients reported the following: less wait time, easier convenience, and similar quality between virtual and in-person visits. Patients rated both their likelihood of using telehealth again and their likelihood of recommending telehealth to others as an 8.68 out of 10, on average. Patients 65 years old or older had 3.17 times greater likelihood of satisfaction with virtual visits when compared with patients younger than 45 years old (confidence interval [95% CI], 1.24-11.11). Patients also had less satisfaction with virtual visits if they had lower educational attainment (odds ratio = 0.10; 95% CI, 0.01-0.81). Conclusions: We found that individuals in a border community had a positive experience with telehealth primary care visits. This approach may improve access to health care.

Split-GFP lamin as a tool for studying C. elegans LMN-1 dynamics in vivo.

We engineered a fluorescent fusion protein of C. elegans lamin, by fusing the eleventh beta strand of GFP to the N-terminus of LMN-1 at the endogenous lmn-1 locus. When co-expressed with GFP1-10, GFP11::LMN-1 was observed at the nuclear periphery of a wide variety of somatic cells. Homozygous gfp11::lmn-1 animals had normal numbers of viable embryos. However, the gfp11::lmn-1 animals had a mild swimming defect. While not completely functional, the GFP11::LMN-1 strain is more healthy than other published fluorescent LMN-1 lines, making it a valuable reagent for studying lamins.

Regulation of the circadian clock in C. elegans by clock gene homologs kin-20 and lin-42.

Circadian rhythms are endogenous oscillations present in nearly all organisms from prokaryotes to humans, allowing them to adapt to cyclical environments close to 24 hours. Circadian rhythms are regulated by a central clock, which is based on a transcription-translation feedback loop. One important protein in the central loop in metazoan clocks is PERIOD, which is regulated in part by Casein kinase 1 ε/δ (CK1 ε/δ ) phosphorylation. In the nematode Caenorhabditis elegans , period and casein kinase 1ε/δ are conserved as lin-42 and kin-20 , respectively. Here we studied the involvement of lin-42 and kin-20 in circadian rhythms of the adult nematode using a bioluminescence-based circadian transcriptional reporter. We show that mutations of lin-42 and kin-20 generate a significantly longer endogenous period, suggesting a role for both genes in the nematode circadian clock, as in other organisms. These phenotypes can be partially rescued by overexpression of either gene under their native promoter. Both proteins are expressed in neurons and seam cells, a population of epidermal stem cells in C. elegans that undergo multiple divisions during development. Depletion of LIN-42 and KIN-20 specifically in neuronal cells after development was sufficient to lengthen the period of oscillating sur-5 expression. Therefore, we conclude that LIN-42 and KIN-20 are critical regulators of the adult nematode circadian clock through neuronal cells.

An nhr-85::GFP::AID*::3xFLAG knock-in allele for investigation of molting and oscillatory gene regulation.

C. elegans NHR-85 is a poorly characterized nuclear hormone receptor transcription factor with an emerging role in regulating microRNA expression to control developmental timing. We generated the first NHR-85 translational fusion by knocking a GFP::AID*::3xFLAG cassette into the endogenous locus to tag all known isoforms. nhr-85 ::GFP::AID*::3xFLAG animals have wild-type broodsizes and NHR-85 ::GFP peaks in expression at the start of the L4 stage in epithelial cells. NHR-85 is not expressed in the germline, suggesting that while it might cooperate with the NHR-23 transcription factor to control microRNA expression, NHR-23 promotes spermatogenesis independent of NHR-85 . This nhr-85 ::GFP::AID*::3xFLAG strain will be a valuable resource for studying when and where NHR-85 acts to promote developmental timing.

A spontaneous TIR1 loss-of-function allele in C. elegans.

The auxin-inducible degron (AID) system is a widely-used system for conditional protein depletion. During the course of an experiment, we depleted the nuclear hormone receptor transcription factor NHR-23 to study molting, and we recovered a spontaneous suppressor allele that bypassed the L1 larval arrest caused by NHR-23 depletion. These mutants also failed to deplete a BFP::AID reporter in the strain background, suggesting a broader defect in the AID system. These animals carried an in-frame 18 base pair insertion that produced a 6 amino acid repeat in TIR1. The larval arrest in these animals could be restored by expressing a wild-type TIR1 transgene from an extrachromosomal array. Sister siblings that lost this array developed normally on auxin. Together, these experiments indicate that the TIR1 mutation was causing the loss of developmental arrest in the nhr-23::AID strain. This result highlights the importance of setting up a robust secondary screen to detect such mutants if performing forward genetic screens in conjunction with the AID system.

An nhr-23::mScarlet::3xMyc knock-in allele for studying spermatogenesis and molting.

C. elegans NHR-23 is a nuclear hormone receptor transcription factor involved in molting, apical extracellular matrix structure, and spermatogenesis. To determine NHR-23 expression dynamics, we previously tagged the endogenous nhr-23 locus with a GFP::AID*::3xFLAG tag. To allow co-localization of NHR-23 with green fluorescent protein-tagged factors of interest, we generated an equivalent strain carrying an mScarlet::3xMyc tag to produce a C-terminal fusion. Similar to the GFP::AID*::3xFLAG knock-in, NHR-23 ::mScarlet::3xMyc was expressed in seam and hypodermal cells, vulval precursor cells, and the spermatogenic germline. We also observed a diffuse NHR-23::mScarlet expression pattern in spermatids and residual bodies after NHR-23 ceased to localize on chromatin. Further examination of this novel localization may provide insight into NHR-23 regulation of spermatogenesis.

NHR-23 activity is necessary for C. elegans developmental progression and apical extracellular matrix structure and function.

Nematode molting is a remarkable process where animals must repeatedly build a new apical extracellular matrix (aECM) beneath a previously built aECM that is subsequently shed. The nuclear hormone receptor NHR-23 (also known as NR1F1) is an important regulator of C. elegans molting. NHR-23 expression oscillates in the epidermal epithelium, and soma-specific NHR-23 depletion causes severe developmental delay and death. Tissue-specific RNAi suggests that nhr-23 acts primarily in seam and hypodermal cells. NHR-23 coordinates the expression of factors involved in molting, lipid transport/metabolism and remodeling of the aECM. NHR-23 depletion causes dampened expression of a nas-37 promoter reporter and a loss of reporter oscillation. The cuticle collagen ROL-6 and zona pellucida protein NOAH-1 display aberrant annular localization and severe disorganization over the seam cells after NHR-23 depletion, while the expression of the adult-specific cuticle collagen BLI-1 is diminished and frequently found in patches. Consistent with these localization defects, the cuticle barrier is severely compromised when NHR-23 is depleted. Together, this work provides insight into how NHR-23 acts in the seam and hypodermal cells to coordinate aECM regeneration during development.

Experimental considerations for study of C. elegans lysosomal proteins.

Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and metabolic diseases. The acidic and protease-rich environment of lysosomes poses experimental challenges. Many fluorescent proteins are quenched or degraded, while specific red fluorescent proteins can be cleaved from translational fusion partners and accumulate. While studying MLT-11, a Caenorhabditis elegans molting factor that localizes to lysosomes and the cuticle, we sought to optimize several experimental parameters. We found that, in contrast to mNeonGreen fusions, mScarlet fusions to MLT-11 missed cuticular and rectal epithelial localization. Rapid sample lysis and denaturation were critical for preventing MLT-11 fragmentation while preparing lysates for western blots. Using a model lysosomal substrate (NUC-1), we found that rigid polyproline linkers and truncated mCherry constructs do not prevent cleavage of mCherry from NUC-1. We provide evidence that extended localization in lysosomal environments prevents the detection of FLAG epitopes in western blots. Finally, we optimize an acid-tolerant green fluorescent protein (Gamillus) for use in C. elegans. These experiments provide important experimental considerations and new reagents for the study of C. elegans lysosomal proteins.

Emojis and Emoticons in Health Care and Dermatology Communication: Narrative Review.

Background: Emoticons and emojis have become staple additions to modern-day communication. These graphical icons are now embedded in daily society through the various forms of popular social media and through users' personal electronic conversations. With ever-increasing use and inclusivity, exploration of the possible health care and dermatology applications of these tools is imperative. Objective: The goal of this narrative review was to provide and evaluate an up-to-date literature survey examining the utility of emoticons and emojis in medicine. Special attention was paid to their existing and potential uses in the field of dermatology, especially during the COVID-19 pandemic. Methods: A PubMed search of peer-reviewed publications was performed in mid-2021 to collect articles with emoticon or emoji keywords in combination with other health care-relevant or dermatology-relevant keywords. Screening of publications and described studies was performed by the authors with education and research experience in health care, dermatology, social media, and electronic communication trends. Selected articles were grouped based on common subjects for qualitative analysis and presentation for in-depth discussion. Results: From this extensive search, researchers were able to identify a wide variety of publications detailing the use of emoticons and emojis in general health care, pediatric health care, public health, and dermatology. Key subject areas that emerged from the investigation included the ability of emoticons and emojis to improve communication within pediatric health care, enhance mood and psychological assessment or mental health screening in adults, develop interventions to improve patient medication adherence, complement novel means of public health and COVID-19 surveillance, and bolster dermatology-specific applications. Conclusions: This review illuminated the repurposing of emojis and emoticons for a myriad of advantageous functions in health care and public health, with applications studied in many populations and situations. Dermatology-specific uses were relatively sparse in the literature, highlighting potential opportunities for growth in future studies and practices. The importance of diversity and inclusivity has extended to emojis, with the recent introduction of skin color customization and new emojis better representing the comprehensive spectrum of users' experiences. A continuously evolving and technology-driven population creates a unique niche for emoticons and emojis to ease worldwide communication and understanding, transcending the barriers of age, language, and background. We encourage future studies and innovations to better understand and expand their utility.

NHR-23 and SPE-44 regulate distinct sets of genes during Caenorhabditis elegans spermatogenesis.

Spermatogenesis is the process through which mature male gametes are formed and is necessary for the transmission of genetic information. While much work has established how sperm fate is promoted and maintained, less is known about how the sperm morphogenesis program is executed. We previously identified a novel role for the nuclear hormone receptor transcription factor, NHR-23, in promoting Caenorhabditis elegans spermatogenesis. The depletion of NHR-23 along with SPE-44, another transcription factor that promotes spermatogenesis, caused additive phenotypes. Through RNA-seq, we determined that NHR-23 and SPE-44 regulate distinct sets of genes. The depletion of both NHR-23 and SPE-44 produced yet another set of differentially regulated genes. NHR-23-regulated genes are enriched in phosphatases, consistent with the switch from genome quiescence to post-translational regulation in spermatids. In the parasitic nematode Ascaris suum, MFP1 and MFP2 control the polymerization of Major Sperm Protein, the molecule that drives sperm motility and serves as a signal to promote ovulation. NHR-23 and SPE-44 regulate several MFP2 paralogs, and NHR-23 depletion from the male germline caused defective localization of MSD/MFP1 and NSPH-2/MFP2. Although NHR-23 and SPE-44 do not transcriptionally regulate the casein kinase gene spe-6, a key regulator of sperm development, SPE-6 protein is lost following NHR-23+SPE-44 depletion. Together, these experiments provide the first mechanistic insight into how NHR-23 promotes spermatogenesis and an entry point to understanding the synthetic genetic interaction between nhr-23 and spe-44.

The mIAA7 degron improves auxin-mediated degradation in Caenorhabditiselegans.

Auxin-inducible degradation is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the auxin-inducible degradation system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in Caenorhabditiselegans, as previously reported in human cells. We tested the depletion of a series of proteins with various subcellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for 5 out of 6 proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for 2 of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system, which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that the degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the auxin-inducible degradation system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

Efficient generation of a single-copy eft-3p::TIR1::F2A:: BFP::AID*::NLS allele in the C. elegans ttTi5605 insertion site through recombination-mediated cassette exchange.

The auxin-inducible degron (AID) system is a widely used system to conditionally deplete proteins. Using CRISPR/Cas9-based genome editing in C. elegans, we recently generated a set of single-copy, tissue-specific and pan-somatic TIR1-expressing strains carrying a BFP reporter inserted in single-copy into two commonly used, well-characterized genetic loci. However, we were unable to obtain a strain carrying a pan-somatic eft-3p::TIR1::F2A::BFP::AID*::NLS transgene inserted into the chromosome II ttTi5605 insertion site. Using recombination-mediated cassette exchange (RMCE) we were able to efficiently obtain this knock-in. The resulting strain displayed equivalent depletion of an AID*::GFP reporter compared to our previously generated eft-3p::TIR1::F2A::BFP::AID*::NLS transgene knocked into the chromosome I ttTi4348 insertion site. This work highlights the power of RMCE for generating new reagents for the AID system and provides an eft-3p::TIR1::F2A::BFP::AID*::NLS allele on chromosome II which will simplify genetic crossing schemes when using the AID system.

Contact Allergy in Canada Versus United States: Analysis of the North American Contact Dermatitis Group Data 2005-2016.

OBJECTIVE: The aim of the study was to describe the differences in contact allergy between the United States (US) and Canada. METHODS: This is a retrospective cross-sectional analysis of the North American Contact Dermatitis Group data from 2005 to 2016. Frequencies of demographics, clinical characteristics, positive reactions, trends, and occupations were calculated. RESULTS: A total of 28,640 patients underwent patch testing. At least 1 positive patch test was observed in 18,599 patients (US, 11,641 [66.5%]; Canada, 6958 [62.5%]). When comparing the 2 groups, US positive reactions were more likely to occur in male patients (odds ratio [OR] = 1.40, 95% confidence interval [CI] = 1.31-1.49), older than 40 years (OR = 1.30, 95% CI = 1.22-1.38), Black (OR = 2.67, 95% CI = 2.24-3.19) or Hispanic race (OR = 3.53, 95% CI = 2.61-4.78), and/or patients with scattered generalized dermatitis (OR = 1.96, 95% CI = 1.80-2.13). They were less likely to occur in patients with eczema (OR = 0.61, 95% CI = 0.57-0.65) and Asian race (OR = 0.50, 95% CI = 0.44-0.56). Nickel (US, 16.0%; Canada, 22.4%) and methylisothiazolinone (US, 13.4%; Canada, 11.0%) were the top allergens. The third most frequent was neomycin (US, 11.7%) and fragrance mix I (Canada, 10.2%). CONCLUSIONS: National differences in allergen prevalence and trends exist in North America.

MFP1/MSD-1 and MFP2/NSPH-2 co-localize with MSP during C. elegans spermatogenesis.

Until recently, the only verified component of Fibrous Bodies (FBs) within Caenorhabditis elegans spermatocytes was the Major Sperm Protein (MSP), a nematode-specific cytoskeletal element. Earlier studies in the pig parasite Ascaris suum had identified accessory proteins that facilitate MSP polymerization and depolymerization within the pseudopod of crawling spermatozoa. In this study, we show that C. elegans homologs of the two Ascaris accessory proteins MFP1 and MFP2 co-localize with MSP in both the pseudopods of C. elegans sperm and the FBs of C. elegans spermatocytes.