Apoptotic cells represent a dynamic stem cell niche governing proliferation and tissue regeneration.

Stem cells (SCs) play a key role in homeostasis and repair. While many studies have focused on SC self-renewal and differentiation, little is known regarding the molecular mechanism regulating SC elimination and compensation upon loss. Here, we report that Caspase-9 deletion in hair follicle SCs (HFSCs) attenuates the apoptotic cascade, resulting in significant temporal delays. Surprisingly, Casp9-deficient HFSCs accumulate high levels of cleaved caspase-3 and are improperly cleared due to an essential caspase-3/caspase-9 feedforward loop. These SCs are retained in an apoptotic-engaged state, serving as mitogenic signaling centers by continuously releasing Wnt3 and instructing proliferation. Investigating the underlying mechanism, we reveal a caspase-3/Dusp8/p38 module responsible for Wnt3 induction, which operates in both normal and Casp9-deleted HFSCs. Notably, Casp9-deleted mice display accelerated wound repair and de novo hair follicle regeneration. Taken together, we demonstrate that apoptotic cells represent a dynamic SC niche, from which emanating signals drive SC proliferation and tissue regeneration.

Site-specific ubiquitylation acts as a regulator of linker histone H1.

Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.

Formulations for Bacteriophage Therapy and the Potential Uses of Immobilization.

The emergence of antibiotic-resistant pathogens is becoming increasingly problematic in the treatment of bacterial diseases. This has led to bacteriophages receiving increased attention as an alternative form of treatment. Phages are effective at targeting and killing bacterial strains of interest and have yielded encouraging results when administered as part of a tailored treatment to severely ill patients as a last resort. Despite this, success in clinical trials has not always been as forthcoming, with several high-profile trials failing to demonstrate the efficacy of phage preparations in curing diseases of interest. Whilst this may be in part due to reasons surrounding poor phage selection and a lack of understanding of the underlying disease, there is growing consensus that future success in clinical trials will depend on effective delivery of phage therapeutics to the area of infection. This can be achieved using bacteriophage formulations instead of purely liquid preparations. Several encapsulation-based strategies can be applied to produce phage formulations and encouraging results have been observed with respect to efficacy as well as long term phage stability. Immobilization-based approaches have generally been neglected for the production of phage therapeutics but could also offer a viable alternative.

Webcams and Social Interaction During Online Classes: Identity Work, Presentation of Self, and Well-Being.

The well-being of children and young people has been affected by the COVID-19 pandemic. The shift to online education disrupted daily rhythms, transformed learning opportunities, and redefined social connections with peers and teachers. We here present a qualitative content analysis of responses to open-ended questions in a large-scale survey of teachers and students in Romania. We explore how their well-being has been impacted by online education through (1) overflow effects of the sudden move to online classes; (2) identity work at the individual and group levels; and (3) Students' and teachers' presentations of self in the online environment, with a focus on problematic aspects of webcam use. The results indicate that both students and teachers experienced ambivalence and diverse changes in well-being, generated by the flexibility, burdens, and disruptions of school-from-home. The identities associated with the roles of teacher and student have been challenged and opened for re-negotiation. Novel patterns have emerged in teachers' and Students' identity work. Failure or success at the presentation of self in online situations is relevant for the emotional valence of learning encounters, impacting well-being. Online classes have brought about new ways to control one's presentation of self while also eliminating previous tactics and resources. The controversy regarding webcams has captured this duality: for some, the home remained a backstage that could not be safely exposed; for others, the home became a convenient front stage for school. Well-being was affected by the success of individual and collective performances, and by student-teacher asymmetries. Overall, our study of online learning indicates powerful yet variable influences on subjective well-being, which are related to overflow effects, identity work, and presentation of self.

A Model for the Remote Deployment, Update, and Safe Recovery for Commercial Sensor-Based IoT Systems.

Internet of Things (IoT) systems deployments are becoming both ubiquitous and business critical in numerous business verticals, both for process automation and data-driven decision-making based on distributed sensors networks. Beneath the simplicity offered by these solutions, we usually find complex, multi-layer architectures-from hardware sensors up to data analytics systems. These rely heavily on software running on the on-location gateway devices designed to bridge the communication between the sensors and the cloud. This will generally require updates and improvements-raising deployment and maintenance challenges. Especially for large scale commercial solutions, a secure and fail-safe updating system becomes crucial for a successful IoT deployment. This paper explores the specific challenges for infrastructures dedicated to remote application deployment and management, addresses the management challenges related to IoT sensors systems, and proposes a mathematical model and a methodology for tackling this. To test the model's efficiency, we implemented it as a software infrastructure system for complete commercial IoT products. As proof, we present the deployment of 100 smart soda dispensing machines in three locations. Each machine relies on sensors monitoring its status and on gateways controlling its behaviour, each receiving 133 different remote software updates through our solution. In addition, 80% of the machines ran non-interrupted for 250 days, with 20% failing due to external factors; out of the 80%, 30% experienced temporary update failures due to reduced hardware capabilities and the system successfully performed automatic rollback of the system, thus recovering in 100% of the temporary failures.

Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation.

Herein we describe a simple protocol for the efficient generation of site-specific ubiquitin-protein conjugates using click chemistry. By using two different methods to expand the genetic code, the two bio-orthogonal functionalities that are necessary for Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), an alkyne and an azide, are co-translationally incorporated into the proteins of interest with unnatural amino acids. Protein ubiquitylation is subsequently carried out with the purified proteins in vitro by CuAAC. In addition, we provide a protocol for the incorporation of two unnatural amino acids into a single ubiquitin, resulting in a 'bifunctional' protein that contains both an alkyne and an azide functionality, thereby enabling assembly of free ubiquitin chains as well as ubiquitin chains conjugated to a target protein. Our procedure enables the synthesis of nonhydrolyzable ubiquitin-protein conjugates within 1 week (given that the relevant cDNAs are at hand), and it yields conjugates in milligram quantities from 1-liter expression cultures. The approach described herein is faster and less laborious than other methods, and it requires only standard molecular biology equipment. Moreover, the protocol can be readily adapted to achieve conjugation at any site of any target protein, which facilitates the generation of custom-tailored ubiquitin-protein conjugates.

Dissecting ubiquitin signaling with linkage-defined and protease resistant ubiquitin chains.

Ubiquitylation is a complex posttranslational protein modification and deregulation of this pathway has been associated with different human disorders. Ubiquitylation comes in different flavors: Besides mono-ubiquitylation, ubiquitin chains of various topologies are formed on substrate proteins. The fate of ubiquitylated proteins is determined by the linkage-type of the attached ubiquitin chains, however, the underlying mechanism is poorly characterized. Herein, we describe a new method based on codon expansion and click-chemistry-based polymerization to generate linkage-defined ubiquitin chains that are resistant to ubiquitin-specific proteases and adopt native-like functions. The potential of these artificial chains for analyzing ubiquitin signaling is demonstrated by linkage-specific effects on cell-cycle progression.

Chain-terminating and clickable NAD⁺ analogues for labeling the target proteins of ADP-ribosyltransferases.

ADP-ribosyltransferases (ARTs) use NAD(+) as a substrate and play important roles in numerous biological processes, such as the DNA damage response and cell cycle regulation, by transferring multiple ADP-ribose units onto target proteins to form poly(ADP-ribose) (PAR) chains of variable sizes. Efforts to identify direct targets of PARylation, as well as the specific ADP-ribose acceptor sites, must all tackle the complexity of PAR. Herein, we report new NAD(+) analogues that are efficiently processed by wild-type ARTs and lead to chain termination owing to a lack of the required hydroxy group, thereby significantly reducing the complexity of the protein modification. Due to the presence of an alkyne group, these NAD(+) analogues allow subsequent manipulations by click chemistry for labeling with dyes or affinity markers. This study provides insight into the substrate scope of ARTs and might pave the way for the further developments of chemical tools for investigating PAR metabolism.

Improving bioorthogonal protein ubiquitylation by click reaction.

Posttranslational modification of proteins with ubiquitin (ubiquitylation) regulates numerous cellular processes. Besides functioning as a signal for proteasomal degradation, ubiquitylation has also non-proteolytic functions by altering the biochemical properties of the modified protein. To investigate the effect(s) of ubiquitylation on the properties of a protein, sufficient amounts of homogenously and well-defined ubiquitylated proteins are required. Here, we report on the elaboration of a method for the generation of high amounts of site-specifically mono-ubiquitylated proteins. Firstly, a one-step affinity purification scheme was developed for ubiquitin containing the unnatural amino acid azidohomoalanine at the C-terminal position. This ubiquitin was conjugated in a click reaction to recombinant DNA polymerase ß, equipped with an alkyne function at a distinct position. Secondly, addition of defined amounts of SDS to the reaction significantly improved product formation. With these two technical improvements, we have developed a straight forward procedure for the efficient generation of site-specifically ubiquitylated proteins that can be used to study the effect(s) of ubiquitylation on the activities/properties of a protein.

Thermophoretically modified aerosol brownian coagulation.

A theory of aerosol coagulation rates resulting from continuum-regime brownian coagulation in the presence of size-dependent particle thermophoresis is developed and explored here. We are motivated by a wide variety of applications in which particle brownian coagulation occurs in a nonisothermal gas where differential thermophoretic drift contributes to, but does not dominate, the encounter frequency between suspended spherical particles (e.g., mist droplets) of different sizes. We employ a Smoluchowski-like population-balance to demonstrate the relative roles of brownian diffusion and thermophoresis in shaping the short and long time (asymptotic or "coagulation-aged") mist-droplet size distribution (DSD) function. To carry out these combined-mechanism DSD-evolution calculations we developed a rational "coupled" coagulation rate constant (allowing for simultaneous brownian diffusion and relative thermophoretic drift) rather than simply adding the relevant individual coagulation "kernels." Dimensionless criteria are provided to facilitate precluding other coagulation mechanisms not considered here (such as simultaneous sedimentation or Marangoni-flow-induced mist-droplet phoresis) and potential complications not included in the present model [as finite-rate coalescence, initial departures from the continuum (Stokes drag-) limit, and even dense (nonideal) vapor effects].

Thermophoretically dominated aerosol coagulation.

A theory of aerosol coagulation due to size-dependent thermophoresis is presented. This previously overlooked effect is important when local temperature gradients are large, the sol population is composed of particles of much greater thermal conductivity than the carrier gas, with mean diameters much greater than the prevailing gas mean free path, and an adequate "spread" in sizes (as in metallurgical mists or fumes). We illustrate this via a population-balance analysis of the evolution of an initially log-normal distribution when this mechanism dominates ordinary Brownian diffusion.

Bivariate Extension of the Quadrature Method of Moments for Modeling Simultaneous Coagulation and Sintering of Particle Populations.

We extendthe application of moment methods to multivariate suspended particle population problems-those for which size alone is insufficient to specify the state of a particle in the population. Specifically, a bivariate extension of the quadrature method of moments (QMOM) (R. McGraw, Aerosol Sci. Technol. 27, 255 (1997)) is presented for efficiently modeling the dynamics of a population of inorganic nanoparticles undergoing simultaneous coagulation and particle sintering. Continuum regime calculations are presented for the Koch-Friedlander-Tandon-Rosner model, which includes coagulation by Brownian diffusion (evaluated for particle fractal dimensions, D(f), in the range 1.8-3) and simultaneous sintering of the resulting aggregates (P. Tandon and D. E. Rosner, J. Colloid Interface Sci. 213, 273 (1999)). For evaluation purposes, and to demonstrate the computational efficiency of the bivariate QMOM, benchmark calculations are carried out using a high-resolution discrete method to evolve the particle distribution function n(nu, a) for short to intermediate times (where nu and a are particle volume and surface area, respectively). Time evolution of a selected set of 36 low-order mixed moments is obtained by integration of the full bivariate distribution and compared with the corresponding moments obtained directly using two different extensions of the QMOM. With the more extensive treatment, errors of less than 1% are obtained over substantial aerosol evolution, while requiring only a few minutes (rather than days) of CPU time. Longer time QMOM simulations lend support to the earlier finding of a self-preserving limit for the dimensionless joint (nu, a) particle distribution function under simultaneous coagulation and sintering (Tandon and Rosner, 1999; D. E. Rosner and S. Yu, AIChE J., 47 (2001)). We demonstrate that, even in the bivariate case, it is possible to use the QMOM to rapidly model the approach to asymptotic behavior, allowing an immediate assessment of when previously established asymptotic results can be applied to dynamical situations of current/future interest. Copyright 2001 Academic Press.