Effects of Nighttime Applications of Germicidal Ultraviolet Light Upon Powdery Mildew (Erysiphe necator), Downy Mildew (Plasmopara viticola), and Sour Rot of Grapevine.

Nighttime applications of germicidal ultraviolet were evaluated as a means to suppress three diseases of grapevine. In laboratory studies, UV-C light (peak 254 nm, FWHM 5 nm) applied during darkness strongly inhibited the germination of conidia of Erysiphe necator, and at a dose of 200 J/m2, germination was zero. Reciprocity of irradiance and duration of exposure with respect to conidial germination was confirmed for UV-C doses between 0 and 200 J/m2 applied at 4 or 400 s. When detached grapevine leaves were exposed during darkness to UV-C at 100 J/m2 up to 7 days before they were inoculated with zoospores of Plasmopara viticola, infection and subsequent sporulation was reduced by over 70% compared to untreated control leaves, indicating an indirect suppression of the pathogen exerted through the host. A hemicylindrical array of low-pressure discharge UV-C lamps configured for trellised grapevines was designed and fitted to both a tractor-drawn carriage and a fully autonomous robotic carriage for vineyard applications. In 2019, in a Chardonnay research vineyard with a history of high inoculum and severe disease, weekly nighttime applications of UV-C suppressed E. necator on leaves and fruit at doses of 100 and 200 J/m2. In the same vineyard in 2020, UV-C was applied once or twice weekly at doses of 70, 100, or 200 J/m2, and severity of E. necator on both leaves and fruit was significantly reduced compared to untreated controls; twice-weekly applications at 200 J/m2 provided suppression equivalent to a standard fungicide program. None of the foregoing UV-C treatments significantly reduced the severity of P. viticola on Chardonnay vines compared to the untreated control in 2020. However, twice-weekly applications of UV-C at 200 J/m2 to the more downy mildew-resistant Vitis interspecific hybrid cultivar Vignoles in 2021 significantly suppressed foliar disease severity. In commercial Chardonnay vineyards with histories of excellent disease control in Dresden, NY, E. necator remained at trace levels on foliage and was zero on fruit following weekly nighttime applications of UV-C at 200 J/m2 in 2020 and after weekly or twice-weekly application of UV-C at 100 or 200 J/m2 in 2021. In 2019, weekly nighttime applications of UV-C at 200 J/m2 also significantly reduced the severity of sour rot, a decay syndrome of complex etiology, on fruit of 'Vignoles' but not the severity of bunch rot caused by Botrytis cinerea. A similar level of suppression of sour rot was observed on 'Vignoles' vines treated twice-weekly with UV-C at 200 J/m2 in 2021. Nighttime UV-C applications did not produce detectable indications of metabolic abnormalities, phytotoxicity, growth reduction, or reductions of fruit yield or quality parameters, even at the highest doses and most frequent intervals employed.

Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing.

Ubiquitin, and components of the ubiquitin-proteasome system, feature extensively in the regulation of gene transcription. Although there are many examples of how ubiquitin controls the activity of transcriptional regulators and coregulators, there are few examples of core components of the transcriptional machinery that are directly controlled by ubiquitin-dependent processes. The budding yeast protein Asr1 is the prototypical member of the RPC (RING, PHD, CBD) family of ubiquitin-ligases, characterized by the presence of amino-terminal RING (really interesting new gene) and PHD (plant homeo domain) fingers and a carboxyl-terminal domain that directly binds the largest subunit of RNA polymerase II (pol II), Rpb1, in response to phosphorylation events tied to the initiation of transcription. Asr1-mediated oligo-ubiquitylation of pol II leads to ejection of two core subunits of the enzyme and is associated with inhibition of polymerase function. Here, we present evidence that Asr1-mediated ubiquitylation of pol II is required for silencing of subtelomeric gene transcription. We show that Asr1 associates with telomere-proximal chromatin and that disruption of the ubiquitin-ligase activity of Asr1--or mutation of ubiquitylation sites within Rpb1--induces transcription of silenced gene sequences. In addition, we report that Asr1 associates with the Ubp3 deubiquitylase and that Asr1 and Ubp3 play antagonistic roles in setting transcription levels from silenced genes. We suggest that control of pol II by nonproteolytic ubiquitylation provides a mechanism to enforce silencing by transient and reversible inhibition of pol II activity at subtelomeric chromatin.

Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species.

Caloric expenditure of aerobic, resistance, or combined high-intensity interval training using a hydraulic resistance system in healthy men.

Although exercise regimens vary in content and duration, few studies have compared the caloric expenditure of multiple exercise modalities with the same duration. The purpose of this study was to compare the energy expenditure of single sessions of resistance, aerobic, and combined exercise with the same duration. Nine recreationally active men (age: 25 ± 7 years; height: 181.6 ± 7.6 cm; weight: 86.6 ± 7.5 kg) performed the following 4 exercises for 30 minutes: a resistance training session using 75% of their 1-repetition maximum (1RM), an endurance cycling session at 70% maximum heart rate (HRmax), an endurance treadmill session at 70% HRmax, and a high-intensity interval training (HIIT) session on a hydraulic resistance system (HRS) that included repeating intervals of 20 seconds at maximum effort followed by 40 seconds of rest. Total caloric expenditure, substrate use, heart rate (HR), and rating of perceived exertion (RPE) were recorded. Caloric expenditure was significantly (p ≤ 0.05) greater when exercising with the HRS (12.62 ± 2.36 kcal·min), compared with when exercising with weights (8.83 ± 1.55 kcal·min), treadmill (9.48 ± 1.30 kcal·min), and cycling (9.23 ± 1.25 kcal·min). The average HR was significantly (p ≤ 0.05) greater with the HRS (156 ± 9 b·min), compared with that using weights (138 ± 16 b·min), treadmill (137 ± 5 b·min), and cycle (138 ± 6 b·min). Similarly, the average RPE was significantly (p ≤ 0.05) higher with the HRS (16 ± 2), compared with that using weights (13 ± 2), treadmill (10 ± 2), and cycle (11 ± 1). These data suggest that individuals can burn more calories performing an HIIT session with an HRS than spending the same amount of time performing a steady-state exercise session. This form of exercise intervention may be beneficial to individuals who want to gain the benefits of both resistance and cardiovascular training but have limited time to dedicate to exercise.

Multiple interactions of the oncoprotein transcription factor MYC with the SWI/SNF chromatin remodeler.

The SNF5 subunit of the SWI/SNF chromatin remodeling complex has been shown to act as a tumor suppressor through multiple mechanisms, including impairing the ability of the oncoprotein transcription factor MYC to bind chromatin. Beyond SNF5, however, it is unknown to what extent MYC can access additional SWI/SNF subunits or how these interactions affect the ability of MYC to drive transcription, particularly in SNF5-null cancers. Here, we report that MYC interacts with multiple SWI/SNF components independent of SNF5. We show that MYC binds the pan-SWI/SNF subunit BAF155 through the BAF155 SWIRM domain, an interaction that is inhibited by the presence of SNF5. In SNF5-null cells, MYC binds with remaining SWI/SNF components to essential genes, although for a purpose that is distinct from chromatin remodeling. Analysis of MYC-SWI/SNF target genes in SNF5-null cells reveals that they are associated with core biological functions of MYC linked to protein synthesis. These data reveal that MYC can bind SWI/SNF in an SNF5-independent manner and that SNF5 modulates access of MYC to core SWI/SNF complexes. This work provides a framework in which to interrogate the influence of SWI/SNF on MYC function in cancers in which SWI/SNF or MYC are altered.

KDM3A/Ets1/MCAM axis promotes growth and metastatic properties in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy of childhood. RMS exists as two major disease subtypes, with oncofusion-positive RMS (FP-RMS) typically carrying a worse prognosis than oncofusion-negative RMS (FN-RMS), in part due to higher propensity for metastasis. Epigenetic mechanisms have recently emerged as critical players in the pathogenesis of pediatric cancers, as well as potential new therapeutic vulnerabilities. Herein, we show that the epigenetic regulator KDM3A, a member of the Jumonji-domain histone demethylase (JHDM) family, is overexpressed, potently promotes colony formation and transendothelial invasion, and activates the expression of genes involved in cell growth, migration and metastasis, in both FN-RMS and FP-RMS. In mechanistic studies, we demonstrate that both RMS subtypes utilize a KDM3A/Ets1/MCAM disease-promoting axis recently discovered in Ewing Sarcoma, another aggressive pediatric cancer of distinct cellular and molecular origin. We further show that KDM3A depletion in FP-RMS cells inhibits both tumor growth and metastasis in vivo, and that RMS cells are highly sensitive to colony growth inhibition by the pan-JHDM inhibitor JIB-04. Together, our studies reveal an important role for the KDM3A/Ets1/MCAM axis in pediatric sarcomas of distinct cellular and molecular ontogeny, and identify new targetable vulnerabilities in RMS.

KDM3A/Ets1 epigenetic axis contributes to PAX3/FOXO1-driven and independent disease-promoting gene expression in fusion-positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young adults. RMS exists as two major disease subtypes, oncofusion-negative RMS (FN-RMS) and oncofusion-positive RMS (FP-RMS). FP-RMS is characterized by recurrent PAX3/7-FOXO1 driver oncofusions and is a biologically and clinically aggressive disease. Recent studies have revealed FP-RMS to have a strong epigenetic basis. Epigenetic mechanisms represent potential new therapeutic vulnerabilities in FP-RMS, but their complex details remain to be defined. We previously identified a new disease-promoting epigenetic axis in RMS, involving the chromatin factor KDM3A and the Ets1 transcription factor. In the present study, we define the KDM3A and Ets1 FP-RMS transcriptomes and show that these interface with the recently characterized PAX3/FOXO1-driven gene expression program. KDM3A and Ets1 positively control numerous known and candidate novel PAX3/FOXO1-induced RMS-promoting genes, including subsets under control of PAX3/FOXO1-associated superenhancers (SE), such as MEST. Interestingly, KDM3A and Ets1 also positively control a number of known and candidate novel FP-RMS-promoting, but not PAX3/FOXO1-dependent, genes. Epistatically, Ets1 is downstream of, and exerts disease-promoting effects similar to, both KDM3A and PAX3/FOXO1. MEST also manifests disease-promoting properties in FP-RMS, and KDM3A and Ets1 each impacts activation of the PAX3/FOXO1-associated MEST SE. Taken together, our studies show that the KDM3A/Ets1 epigenetic axis plays an important role in disease promotion in FP-RMS, and provide insight into potential new ways to target aggressive phenotypes in this disease.

KDM5A and PHF2 positively control expression of pro-metastatic genes repressed by EWS/Fli1, and promote growth and metastatic properties in Ewing sarcoma.

Ewing sarcoma is an aggressive malignant neoplasm with high propensity for metastasis and poor clinical outcomes. The EWS/Fli1 oncofusion is the disease driver in > 90% of cases, but presents a difficult therapeutic target. Moreover, EWS/Fli1 plays a complex role in disease progression, with inhibitory effects on critical steps of metastasis. Like many other pediatric cancers, Ewing sarcoma is a disease marked by epigenetic dysregulation. Epigenetic mechanisms present alternative targeting opportunities, but their contributions to Ewing sarcoma metastasis and disease progression remain poorly understood. Here, we show that the epigenetic regulators KDM5A and PHF2 promote growth and metastatic properties in Ewing sarcoma, and, strikingly, activate expression many pro-metastatic genes repressed by EWS/Fli1. These genes include L1CAM, which is associated with adverse outcomes in Ewing sarcoma, and promotes migratory and invasive properties. KDM5A and PHF2 retain their growth promoting effects in more metastatically potent EWS/Fli1low cells, and PHF2 promotes both invasion and L1CAM expression in this cell population. Furthermore, KDM5A and PHF2 each contribute to the increased metastatic potency of EWS/Fli1low cells in vivo. Together, these studies identify KDM5A and PHF2 as novel disease-promoting factors, and potential new targets, in Ewing sarcoma, including the more metastatically potent EWS/Fli1low cell population.

Biology and targeting of the Jumonji-domain histone demethylase family in childhood neoplasia: a preclinical overview.

INTRODUCTION: Epigenetic mechanisms of gene regulatory control play fundamental roles in developmental morphogenesis, and, as more recently appreciated, are heavily implicated in the onset and progression of neoplastic disease, including cancer. Many epigenetic mechanisms are therapeutically targetable, providing additional incentive for understanding of their contribution to cancer and other types of neoplasia. Areas covered: The Jumonji-domain histone demethylase (JHDM) family exemplifies many of the above traits. This review summarizes the current state of knowledge of the functions and pharmacologic targeting of JHDMs in cancer and other neoplastic processes, with an emphasis on diseases affecting the pediatric population. Expert opinion: To date, the JHDM family has largely been studied in the context of normal development and adult cancers. In contrast, comparatively few studies have addressed JHDM biology in cancer and other neoplastic diseases of childhood, especially solid (non-hematopoietic) neoplasms. Encouragingly, the few available examples support important roles for JHDMs in pediatric neoplasia, as well as potential roles for JHDM pharmacologic inhibition in disease management. Further investigations of JHDMs in cancer and other types of neoplasia of childhood can be expected to both enlighten disease biology and inform new approaches to improve disease outcomes.

The Jumonji-domain histone demethylase inhibitor JIB-04 deregulates oncogenic programs and increases DNA damage in Ewing Sarcoma, resulting in impaired cell proliferation and survival, and reduced tumor growth.

Ewing Sarcoma is an aggressive malignant neoplasm affecting children and young adults. Ewing Sarcoma is driven by transcription factor fusion oncoproteins, most commonly EWS/Fli1. While some patients can be cured with high-dose, multi-agent, chemotherapy, those that cannot currently have few options. Targeting of the driver oncofusion remains a logical therapeutic approach, but has proven difficult. Recent work has pointed to epigenetic mechanisms as key players, and potential new therapeutic targets, in Ewing Sarcoma. In this study we examined the activity of the pan-JHDM pharmacologic inhibitor JIB-04 in this disease. We show that JIB-04 potently inhibits the growth and viability of Ewing Sarcoma cells, and also impairs tumor xenograft growth. Effects on histone methylation at growth-inhibitory doses vary among cell lines, with most cell lines exhibiting increased total H3K27me3 levels, and some increased H3K4me3 and H3K9me3. JIB-04 treatment widely alters expression of oncogenic and tumor suppressive pathways, including downregulation of known oncogenic members of the Homeobox B and D clusters. JIB-04 also disrupts the EWS/Fli1 expression signature, including downregulation of pro-proliferative pathways normally under positive oncofusion control. Interestingly, these changes are accompanied by increased levels of the EWS/Fli1 oncofusion, suggesting that the drug could be uncoupling EWS/Fli1 from its oncogenic program. All Ewing Sarcoma cell lines examined also manifest increased DNA damage upon JIB-04 treatment. Together, the findings suggest that JIB-04 acts via multiple mechanisms to compromise Ewing Sarcoma cell growth and viability.

Subcutaneous and segmental fat loss with and without supportive supplements in conjunction with a low-calorie high protein diet in healthy women.

BACKGROUND: Weight loss benefits of multi-ingredient supplements in conjunction with a low-calorie, high-protein diet in young women are unknown. Therefore, the purpose of this study was to investigate the effects of a three-week low-calorie diet with and without supplementation on body composition. METHODS: Thirty-seven recreationally-trained women (n = 37; age = 27.1 ± 4.2; height = 165.1 ± 6.4; weight = 68.5 ± 10.1; BMI = 25.1 ± 3.4) completed one of the following three-week interventions: no change in diet (CON); a high-protein, low-calorie diet supplemented with a thermogenic, conjugated linoleic acid (CLA), a protein gel, and a multi-vitamin (SUP); or the high-protein diet with isocaloric placebo supplements (PLA). Before and after the three-week intervention, body weight, %Fat via dual X-ray absorptiometry (DXA), segmental fat mass via DXA, %Fat via skinfolds, and skinfold thicknesses at seven sites were measured. RESULTS: SUP and PLA significantly decreased body weight (SUP: PRE, 70.47 ± 8.01 kg to POST, 67.51 ± 8.10 kg; PLA: PRE, 67.88 ± 12.28 kg vs. POST, 66.38 ± 11.94 kg; p ≤ 0.05) with a greater (p ≤ 0.05) decrease in SUP than PLA or CON. SUP and PLA significantly decreased %Fat according to DXA (SUP: PRE, 34.98 ± 7.05% to POST, 32.99 ± 6.89%; PLA: PRE, 34.22 ± 6.36% vs. POST, 32.69 ± 5.84%; p ≤ 0.05), whereas only SUP significantly decreased %Fat according to skinfolds (SUP: PRE, 27.40 ± 4.09% to POST, 24.08 ± 4.31%; p ≤ 0.05). SUP significantly (p ≤ 0.05) decreased thicknesses at five skinfolds (chest, waist, hip, subscapular, and tricep) compared to PLA, but not at two skinfolds (axilla and thigh). CONCLUSIONS: The addition of a thermogenic, CLA, protein, and a multi-vitamin to a three-week low-calorie diet improved weight loss, total fat loss and subcutaneous fat loss, compared to diet alone.

Functions of the proteasome on chromatin.

The proteasome is a large self-compartmentalized protease complex that recognizes, unfolds, and destroys ubiquitylated substrates. Proteasome activities are required for a host of cellular functions, and it has become clear in recent years that one set of critical actions of the proteasome occur on chromatin. In this review, we discuss some of the ways in which proteasomes directly regulate the structure and function of chromatin and chromatin regulatory proteins, and how this influences gene transcription. We discuss lingering controversies in the field, the relative importance of proteolytic versus non-proteolytic proteasome activities in this process, and highlight areas that require further investigation. Our intention is to show that proteasomes are involved in major steps controlling the expression of the genetic information, that proteasomes use both proteolytic mechanisms and ATP-dependent protein remodeling to accomplish this task, and that much is yet to be learned about the full spectrum of ways that proteasomes influence the genome.