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Rationale: Mechanical ventilation with high tidal volumes leads to increased permeability, generation of inflammatory mediators, and damage to alveolar epithelial cells (ATII).Objectives: To identify changes in the ATII proteome after two different ventilation strategies in rats.Methods: Rats (n = 6) were ventilated for 5 hours with high- and low tidal volumes (VTs) (high VT: 20 ml/kg; low VT: 6 ml/kg). Pooled nonventilated rats served as control animals. ATII cells were isolated and lysed, and proteins were tryptically cleaved into peptides. Cellular protein content was evaluated by peptide labeling of the ventilated groups with (18)O. Samples were fractionated by cation exchange chromatography and identified using electrospray tandem mass spectrometry. Proteins identified by 15 or more peptides were statistically compared using t tests corrected for the false discovery rate.Measurements and Main Results: High Vt resulted in a significant increase in airspace neutrophils without an increase in extravascular lung water. Compared with low-VT samples, high-VT samples showed a 32% decrease in the inositol 1,4,5-trisphosphate 3 receptor (p < 0.01), a 34% decrease in Na(+), K(+)-ATPase (p < 0.01), and a significantly decreased content in ATP synthase chains. Even low-VT samples displayed significant changes, including a 66% decrease in heat shock protein 90-beta (p < 0.01) and a 67% increase in mitochondrial pyruvate carboxylase (p < 0.01). Significant differences were found in membrane, acute phase, structural, and mitochondrial proteins.Conclusions: After short-term exposure to high-VT ventilation, significant reductions in membrane receptors, ion channel proteins, enzymes of the mitochondrial energy system, and structural proteins in ATII cells were present. The data supports the two-hit concept that an unfavorable ventilatory strategy may make the lung more vulnerable to an additional insult. [ABSTRACT FROM AUTHOR]

The effectiveness of state-of-the-art cross-linking strategies and mass spectrometry (MS) detection was explored in an important biological context, namely, the ubiquitin-proteasome system, which is responsible for most of the regulated protein degradation in eukaryotic cells. The locations of possible binding sites on the S. cerevisiae 19S proteasome regulatory particle for Lys 48 linked polyubiquitin chains were examined using cross-linking strategies and MS based detection by comparing two types of cross-linkers: a (bis)-sulfosuccinimidyl suberate (BS 3 ) and diethyl suberothioimidate (DEST). The well-established BS 3 -based strategy produced 328 cross-linked peptides; however, no ubiquitin-19S cross-links were observed. The recently developed DEST-based approach produced fewer (146) linkages overall, but these included six ubiquitin-19S cross-links. Some of these cross-links are predicted by the canonical view of ubiquitin recognition, but others suggest novel insights into how the proteasome recognizes its substrates. A discussion of these strategies and structural implications for polyubiquitin-proteasome binding is provided.

Evolutionary arms races can lead to extremely specific and effective defense mechanisms, including venoms that deter predators by targeting nociceptive (pain-sensing) pathways. The venom of velvet ants (Hymenoptera: Mutillidae) is notoriously painful. It has been described as "Explosive and long lasting, you sound insane as you scream. Hot oil from the deep fryer spilling over your entire hand." 1 The effectiveness of the velvet ant sting against potential predators has been shown across vertebrate orders, including mammals, amphibians, reptiles, and birds. 2 , 3 , 4 This leads to the hypothesis that velvet ant venom targets a conserved nociception mechanism, which we sought to uncover using Drosophila melanogaster as a model system. Drosophila larvae have peripheral sensory neurons that sense potentially damaging (noxious) stimuli such as high temperature, harsh mechanical touch, and noxious chemicals. 5 , 6 , 7 , 8 They share features with vertebrate nociceptors, including conserved sensory receptor channels. 9 , 10 We found that velvet ant venom strongly activated Drosophila nociceptors through heteromeric Pickpocket/Balboa (Ppk/Bba) ion channels, through a single venom peptide, Do6a. Drosophila Ppk/Bba is homologous to mammalian acid-sensing ion channels (ASICs). 11 However, Do6a did not produce behavioral signs of nociception in mice, which was instead triggered by other venom peptides that are non-specific and less potent on Drosophila nociceptors. This suggests that Do6a has an insect-specific function. In fact, we further demonstrated that the velvet ant's sting produced aversive behavior in a predatory praying mantis. Together, our results indicate that velvet ant venom acts through different molecular mechanisms in vertebrates and invertebrates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA repair dysregulation is a key driver of cancer development. Understanding the molecular mechanisms underlying DNA repair dysregulation in cancer cells is crucial for cancer development and therapies. Here, we report that enhancer of zeste homolog 2 (EZH2) directly methylates poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), an essential enzyme involved in DNA repair, and regulates its activity. Functionally, EZH2-catalyzed methylation represses PARP1 catalytic activity, down-regulates the recruitment of x-ray repair cross-complementing group-1 to DNA lesions and its associated DNA damage repair; on the other hand, it protects the cells from nicotinamide adenine dinucleotide overconsumption upon DNA damage formation. Meanwhile, EZH2-mediated methylation regulates PARP1 transcriptional and oncogenic activity, at least in part, through impairing PARP1-E2F1 interaction and E2F1 transcription factor activity. EZH2 and PARP1 inhibitors synergistically suppress prostate cancer growth. Collectively, our findings uncover an insight of EZH2 functions in fine-tuning PARP1 activity during DNA damage repair and cancer progression, which provides a rationale for combinational targeting EZH2 and PARP1 in cancer.

Tissue nanotransfection (TNT)-based fluorescent labeling of cell-specific exosomes has shown that exosomes play a central role in physiological keratinocyte-macrophage (mϕ) crosstalk at the wound-site. Here, we report that during the early phase of wound reepithelialization, macrophage-derived exosomes (Exo mϕ ), enriched with the outer mitochondrial membrane protein TOMM70, are localized in leading-edge keratinocytes. TOMM70 is a 70 kDa adaptor protein anchored in the mitochondrial outer membrane and plays a critical role in maintaining mitochondrial function and quality. TOMM70 selectively recognizes cytosolic chaperones by its tetratricopeptide repeat (TPR) domain and facilitates the import of preproteins lacking a positively charged mitochondrial targeted sequence. Exosomal packaging of TOMM70 in mϕ was independent of mitochondrial fission. TOMM70-enriched Exo mϕ compensated for the hypoxia-induced depletion of epidermal TOMM70, thereby rescuing mitochondrial metabolism in leading-edge keratinocytes. Thus, macrophage-derived TOMM70 is responsible for the glycolytic ATP supply to power keratinocyte migration. Blockade of exosomal uptake from keratinocytes impaired wound closure with the persistence of proinflammatory mϕ in the wound microenvironment, pointing toward a bidirectional crosstalk between these two cell types. The significance of such bidirectional crosstalk was established by the observation that in patients with nonhealing diabetic foot ulcers, TOMM70 is deficient in keratinocytes of wound-edge tissues.

Evolutionary arms races between predator and prey can lead to extremely specific and effective defense mechanisms. Such defenses include venoms that deter predators by targeting nociceptive (pain-sensing) pathways. Through co-evolution, venom toxins can become extremely efficient modulators of their molecular targets. The venom of velvet ants (Hymenoptera: Mutillidae) is notoriously painful. The intensity of a velvet ant sting has been described as "Explosive and long lasting, you sound insane as you scream. Hot oil from the deep fryer spilling over your entire hand." [1] The effectiveness of the velvet ant sting as a deterrent against potential predators has been shown across vertebrate orders, including mammals, amphibians, reptiles, and birds [2-4]. The venom's low toxicity suggests it has a targeted effect on nociceptive sensory mechanisms [5]. This leads to the hypothesis that velvet ant venom targets a conserved nociception mechanism, which we sought to uncover using Drosophila melanogaster as a model system. Drosophila larvae have peripheral sensory neurons that sense potentially damaging (noxious) stimuli such as high temperature, harsh mechanical touch, and noxious chemicals [6-9]. These polymodal nociceptors are called class IV multidendritic dendritic arborizing (cIV da) neurons, and they share many features with vertebrate nociceptors, including conserved sensory receptor channels [10,11]. We found that velvet ant venom strongly activated Drosophila nociceptors through heteromeric Pickpocket/Balboa (Ppk/Bba) ion channels. Furthermore, we found a single venom peptide (Do6a) that activated larval nociceptors at nanomolar concentrations through Ppk/Bba. Drosophila Ppk/Bba is homologous to mammalian Acid Sensing Ion Channels (ASICs) [12]. However, the Do6a peptide did not produce behavioral signs of nociception in mice, which was instead triggered by other non-specific, less potent, peptides within the venom. This suggests that Do6a is an insect-specific venom component that potently activates insect nociceptors. Consistent with this, we showed that the velvet ant's defensive sting produced aversive behavior in a predatory praying mantis. Together, our results indicate that velvet ant venom evolved to target nociceptive systems of both vertebrates and invertebrates, but through different molecular mechanisms.

Background: In the absence of a gold-standard diagnostic modality for cellulitis, sterile inflammatory disorders may be misdiagnosed as cellulitis., Objective: To determine the utility of skin biopsy and tissue culture for the diagnosis and management of patients admitted with a diagnosis of presumed cellulitis., Design: Pilot single-blind parallel group randomized controlled clinical trial in 56 patients with a primary diagnosis of presumed cellulitis. In the intervention group only, skin biopsy and tissue culture results were made available to the primary care team to guide diagnosis and management. Length of hospital stay and antibiotic use were evaluated as outcome measures., Results: Length of stay showed the greatest opportunity for further study as a primary outcome (intervention: 4, IQR (2-6) vs. control: 5 IQR (3-8) days; p = 0.124)., Limitations: The COVID-19 pandemic placed limitations on participant enrollment and study duration; in addition, data was collected from a single medical center., Conclusion: This study demonstrates that length of stay and anti-pseudomonal antibiotic de-escalation are endpoints that may be influenced by biopsy and tissue culture results in presumed cellulitis patients; these outcomes warrant further study., (© 2024. The Author(s).)

In the work presented herein, a simple serial-pelleting purification strategy combined with a mass spectrometry-based proteomics analysis was developed as a means of discerning differences in extracellular vesicle (EV) populations found in bovine milk samples. A sequence of ultracentrifugation speeds was used to generate changes in the abundances of EV populations, allowing for the identification of associated proteins. A metric was developed to determine the relative abundances of proteins in large EVs (>200 nm) and small EVs (<200 nm). Of the 476 proteins consistently found in this study, 340 are associated with vesicular components. Of these, 156 were heavily enriched in large EVs, 155 shared between large and small EVs, and 29 heavily enriched in small EVs. Additionally, out of 68 proteins annotated as exosome proteins, 32 were enriched in large EVs, 27 shared between large and small EVs, 5 enriched in small EVs, and 7 were found to be nonvesicular contaminant proteins. The top correlated proteins in the small EV group were predominantly membrane-bound proteins, whereas the top correlated proteins in the large EV group were mostly cytosolic enzymes for molecular processing. This method provides a means of assessing the origins of vesicle components and provides new potential marker proteins within discrete vesicle populations.

There are certain demographic characteristics that may serve as risk factors for exposure to a contact allergen. Volatile alkyl nitrites, colloquially known as "poppers," are commonly inhaled by men who have sex with men (MSM) for their psychoactive and muscle-relaxing effects. They have been reported to cause either allergic contact Dermatitis® (ACD) or irritant contact Dermatitis® (ICD), termed "poppers Dermatitis®." We searched PubMed, Embase, and Web of Science to understand the patient population, clinical presentation, diagnosis, and clinical course of poppers Dermatitis®. Our search returned 13 articles detailing 18 patients total. Most patient cases were male ( n  = 13/15; 87%), of whom 5 were MSM. The reports on the remaining 8 men did not disclose their sexual behaviors. The chemicals deemed responsible for adverse cutaneous side effects included amyl nitrite (6/18; 33%), butyl nitrite (3/18; 167%), isobutyl nitrite (2/18; 11%), and an unspecified alkyl nitrite (7/18, 39%). The most common diagnosis was ICD (10/18; 56%). Only 3 patients underwent alkyl nitrite patch testing, with 2 testing positive for ACD. The most commonly involved anatomic sites were perinasal (13/18; 72%) and perioral regions (10/18; 56%). Rash morphology has been described as yellow crusting, impetigo-like, eczematous, vesicular, edematous, and erythematous. Ulceration can occur with direct skin contact to concentrated nitrite solution. Poppers Dermatitis® was often self-limited with complete resolution. Dermatologists should consider poppers Dermatitis® in those presenting with a characteristic midfacial rash, especially in MSM.

Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk in vivo . However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes ( hExo κ ) from human chronic wound fluid. Furthermore, we studied the significance of hExo κ in diabetic wounds. LC-MS-MS detection of KRT14 in hExo κ and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of hExo κ . dSTORM nanoimaging identified KRT14 + extracellular vesicles ( EV κ ) in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated hExo κ from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated hExo κ (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed hExo κ Ø as 80-145nm. The abundance of hExo κ was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The hExo κ isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic hExo κ Uptake of hExo κ by monocyte-derived macrophages (MDM) was low for diabetics versus non-diabetics. Unlike hExo κ from non-diabetics, the addition of diabetic hExo κ to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of hExo κ from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity., Competing Interests: Financial Interest Statement The authors declare no competing financial interest. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Use of inpatient teledermatology increased during the COVID-19 pandemic. We surveyed the Society for Dermatology Hospitalists to better characterize the impact of COVID-19 on teledermatology use by inpatient dermatology providers, particularly on provider perceptions of teledermatology. Prior to the COVID-19 pandemic, 40% (8/20) of surveyed providers had used telehealth at their institution to help perform inpatient consults, while 90% (18/20) adapted use of teledermatology during the pandemic. 80% (16/20) reported that their opinion of teledermatology changed as a result of the COVID-19 pandemic, with the vast majority (87.5%, 14/16) reporting having a more positive opinion. Benefits of teledermatology included efficiency, ability to increase access safely, and ability for clinicians to focus on complex cases. Some providers expressed concerns over the potential implications regarding the perception of dermatology within medicine, limitations of inadequate photos, and breakdowns in communication with consulting teams and patients. Robust algorithms and or utilization criteria of teledermatology may help to mitigate risk, while increasing access to inpatient dermatologic evaluation., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A one-step protocol for the automated flow synthesis of protected glycosylated amino acids is described using pumps with open-source controls in overall yields of 21-50%. The resulting glycosylated amino acids could be used directly in solid-phase peptide synthesis (SPPS) protocols to quickly produce glycopeptide standards. Access to a variety of stereoisomers of the sugar enabled the development of an LC-MS/MS protocol that can distinguish between peptides modified with carbohydrates having the same exact mass. This method could definitively identify fucose in an O -glycosylation site on the transmembrane protein, Notch1.

Nonpurulent cellulitis lacks a gold standard to distinguish noninfectious sources of inflammation. Two models have been created that evaluate cellulitis. The ALT-70 model was created to reduce the overdiagnosis of cellulitis and provide clinical direction. The Dundee classification was developed to grade the severity of previously diagnosed cases of cellulitis and enhance treatment and clinical outcomes. We analyzed a dataset of 56 patients who were admitted to the OSU Wexner Medical Center with a primary admission diagnosis of cellulitis. Each patient underwent extensive tissue culture sampling to identify potential pathogens. Patients were scored using both models, then evaluated based on the positive tissue culture and skin and soft tissue infection. In both models, we found low sensitivity and specificity to predict patients with positive tissue culture cellulitis. Determination of a gold standard for classification of cellulites is important to improve future diagnosis and risk models. We recommend further study to develop a scalable consensus standard in the diagnosis of nonpurulent cellulitis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Factitial Dermatitis (FD) is a notoriously difficult disease to diagnose, as patients produce self-induced cutaneous lesions and provide an inadequate or inaccurate history. We performed a cross-sectional study, querying an inpatient consultation database of all patients admitted to the Ohio State University Wexner Medical Center from 2012 to 2017 with a dermatologic ICD as a discharge diagnosis. Our exhaustive keyword search produced 189 candidates. Consult notes were thoroughly examined, and 32 patients were found to meet case definition of FD. Our analysis of this cohort revealed a significantly greater proportion of cases in the female population. Lesions were more often found to involve the skin on the upper extremities. Isolated secondary skin changes such as erosions, ulcers and excoriations in the absence primary morphologies were also significant in our cohort. As FD is difficult to identify, further understanding of its presentation pattern will decrease time to diagnosis and improve both hospital resource allocation and patient care., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Asynchronous electronic consultations (e-consults) can be a useful tool for the screening of cutaneous lesions, but may offer a malpractice risk. We characterized factors affecting initial eConsult office follow-up in a cohort of patients with documented neoplasm of uncertain behavior. Patients with an ICD 10 code of neoplasm with uncertain behavior (D48.5) at The Ohio State University that received an E-consult order from May 2017 to May 2021 were queried. Information collected included patient demographics, status of follow-up in-office appointment, referral status, and health care utilization. In-office follow-up appointments were defined as completed, cancelled/no-show or no-contact. 667 patients with a diagnosis of D48.5 were identified as having completed an eConsult. 427 (64%) patients had a documented phone/electronic message notifying the patient of the results of the eConsult. Year of encounter (0.88 [0.79-0.97]) and number of previously completed ambulatory visits (0.86 [0.77-0.96]) were significantly associated with documentation of phone/electronic message in the univariate and multivariate model. 429 (84%) patients had a dermatology office follow-up encounter while 82 (16%) had no appointment scheduled. Language spoken, referral status and race were significant in the univariate model, though race was the only significant variable in the multivariate model (P < 0.003). Asynchronous electronic consults to assess possible cutaneous neoplasms is an important tool for population screening of skin cancer. Dermatologists and health systems implementing an eConsult model for screening purposes should be aware of risk factors for loss of follow-up. Additional systems need to be implemented to ensure minorities and non-native English speakers are obtaining adequate dermatologic care., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Current understanding of the etiology, natural history, and outcomes of acute generalized exanthematous pustulosis (AGEP) has been limited, with most available studies consisting of small or heterogenous cohorts., Objectives: The aim of this study was to further characterize associated factors and disease outcomes of AGEP., Methods: A cross-sectional study design was employed with formal inclusion and causality criteria. Patients were identified from an inpatient database at an academic medical center, including 65 patients with AGEP and a control group of 61 patients with non-severe cutaneous adverse reactions., Results: Increased age and body mass index (BMI) were associated with higher risk of AGEP (p < 0.001). Length of stay was longer for both the overall AGEP cohort (13.1 days) and a subcohort with a primary discharge diagnosis of AGEP (9.7 days) compared with the control group (3.6 days) [p < 0.001]. Patients with AGEP were more likely to be discharged to a long-term care facility compared with control patients (p < 0.001)., Conclusions: AGEP was associated with longer length of hospitalization, higher rates of discharge to long-term care facilities, and higher mortality compared with non-severe cutaneous adverse drug reaction (SCAR) medication reactions. Future research should examine the association between morbid obesity and this particular drug reaction, and the possibility of decreasing hospitalization length given the relatively low risk of mortality among patients with AGEP., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 ( Dmp1 )-8kb- Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.

In response to bacterial infection, the vertebrate host employs the metal-sequestering protein calprotectin (CP) to withhold essential transition metals, notably Zn(II), to inhibit bacterial growth. Previous studies of the impact of CP-imposed transition-metal starvation in A. baumannii identified two enzymes in the de novo biosynthesis pathway of queuosine-transfer ribonucleic acid (Q-tRNA) that become cellularly abundant, one of which is QueD2, a 6-carboxy-5,6,7,8-tetrahydropterin (6-CPH 4 ) synthase that catalyzes the initial, committed step of the pathway. Here, we show that CP strongly disrupts Q incorporation into tRNA. As such, we compare the Ab QueD2 "low-zinc" paralog with a housekeeping, obligatory Zn(II)-dependent enzyme QueD. The crystallographic structure of Zn(II)-bound Ab QueD2 reveals a distinct catalytic site coordination sphere and assembly state relative to QueD and possesses a dynamic loop, immediately adjacent to the catalytic site that coordinates a second Zn(II) in the structure. One of these loop-coordinating residues is an invariant Cys18, that protects QueD2 from dissociation of the catalytic Zn(II) while maintaining flux through the Q-tRNA biosynthesis pathway in cells. We propose a "metal retention" model where Cys18 introduces coordinative plasticity into the catalytic site which slows metal release, while also enhancing the metal promiscuity such that Fe(II) becomes an active cofactor. These studies reveal a complex, multipronged evolutionary adaptation to cellular Zn(II) limitation in a key Zn(II) metalloenzyme in an important human pathogen.