Molecular signatures for CCN1, p21 and p27 in progressive mantle cell lymphoma

Mantle cell lymphoma (MCL) is a comparatively rare non-Hodgkin’s lymphoma characterised by overexpression of cyclin D1.Many patients present with or progress to advanced stage disease within 3 years. MCL is considered an incurable disease withmedian survival between 3 and 4 years. We have investigated the role(s) of CCN1 (CYR61) and cell cycle regulators inprogressive MCL. We have used the human MCL cell lines REC1 G519 > JVM2 cells by RQ-PCR, depicting a decrease in CCN1expression with disease progression. Investigation of CCN1 isoform expression by western blotting showed that whilst expres-sion of full-length CCN1 was barely altered in the cell lines, expression of truncated forms (18–20 and 28–30 kDa) decreasedwith disease progression. We have then demonstrated that cyclin D1 and cyclin dependent kinase inhibitors (p21CIP1and p27KIP1)are also involved in disease progression. Cyclin D1 was highly expressed in REC1 cells (OD: 1.0), reduced to one fifth in G519cells (OD: 0.2) and not detected by western blotting in JVM2 cells. p27KIP1followed a similar profile of expression as cyclin D1.Conversely, p21CIP1was absent in the REC1 cells and showed increasing expression in G519 and JVM2 cells. Subcellularlocalization detected p21CIP1/p27KIP1primarily within the cytoplasm and absent from the nucleus, consistent with altered roles in treatment resistance. Dysregulation of the CCN1 truncated forms are associated with MCL progression. In conjunction withreduced expression of cyclin D1 and increased expression of p21, this molecular signature may depict aggressive disease andtreatment resistance

Kalibracja przenośnego spektrometru fluorescencji rentgenowskiej (XRF) na granitach zawierających Sn-W ze złoża Cínovec (Erzgebirge/Krušné Hory Mts., Republika Czeska)

This study evaluates the suitability of portable X-Ray fluorescence spectrometry (PXRF) in the environment of granites and greisens with Sn-W and secondary mineralization of other important elements (Rb,Y, La, Ce, Nb, Ta and Tl). International certificated reference materials (CRM), historical data from the sixties based on the borehole CS-1, current analyses in accredited laboratory and data measured by a portable X-ray fluorescence spectrometer (PXRF) with factory settings were evaluated in this study. Powder CRMs and samples from borehole CS-1 were analysed by PXRF. The results of PXRF analysis were then tested for reliability, credibility, accuracy and precision. The accuracy was calculated as a relative percent difference (RPD), regression equations and correlation coefficients. A detailed statistical evaluation of the results proves, that PXRF can be a very useful method for a primary determination of main, secondary and trace elements. Comparison of the conventional methods and the PXRF shows a good correlation of different analytical methods and a good possibility of using the PXRF method for a future selection of samples for subsequent more demanding and expensive conventional and special analyses. A calibration of the PXRF spectrometer to the lithological environment of Sn-W ore-bearing granites and greisens was made by the statistical comparison of the methods.Niniejsze badania sprawdzają przydatność przenośnego spektrometru fluorescencji rentgenowskiej (ang. skrót PXRF) w warunkach występowania granitów i grejzenów z Sn-W oraz wtórnej mineralizacji innych pierwiastków (Rb, Y, La, Ce, Nb, Ta i Tl). W badaniach uwzględniono międzynarodowe certyfikowane materiały odniesienia (ang. skrót CRM), dane historyczne z lat 60 oparte na odwiertach CS-1, bieżące analizy w akredytowanych laboratoriach i dane uzyskane z przenośnego spektrometru fluorescencji rentgenowskiej (PXRF) z ustawieniami fabrycznymi. CRM proszkowe oraz próbki pobrane z odwiertu CS-1 przeanalizowano w PXRF. Wyniki analizy PXRF zostały następnie poddane testom na niezawodność, wiarygodność, dokładność i precyzję. Dokładność została policzona jako względna różnica procentowa (RPD), równania regresyjne i współczynniki korelacji. Szczegółowa ocena statystyczna wyników dowodzi, że PXRF może być pomocną metodą we wstępnym określaniu zawartości pierwiastków głównych, wtórnych i śladowych. Porównanie metod konwencjonalnych z metodą PXRF pokazało pozytywną korelację różnych metod analitycznych i możliwość stosowania metody PXRF do przyszłych doborów próbek w celu dalszych trudniejszych i droższych analiz konwencjonalnych i specjalistycznych. Kalibracja spektrometru PXRF do warunków występowania litu w granitach i grejzenach zawierających rudę Sn-W została sporządzona została na podstawie statystycznego porównania wymienionych metod

Deuterium Oxide (D2O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

There are two stable isotopes of hydrogen, protium (1H) and deuterium (2H; D). Cellular stress response dysregulation in cancer represents both a major pathological driving force and a promising therapeutic target, but the molecular consequences and potential therapeutic impact of deuterium (2H)-stress on cancer cells remain largely unexplored. We have examined the antiproliferative and apoptogenic effects of deuterium oxide (D2O; ‘heavy water’) together with stress response gene expression profiling in panels of malignant melanoma (A375V600E, A375NRAS, G361, LOX-IMVI), and pancreatic ductal adenocarcinoma (PANC-1, Capan-2, or MIA PaCa-2) cells with inclusion of human diploid Hs27 skin fibroblasts. Moreover, we have examined the efficacy of D2Obased pharmacological intervention in murine models of human melanoma tumor growth and metastasis. D2O-induction of apoptosis was substantiated by AV-PI flow cytometry, immunodetection of PARP-1, and pro-caspase 3 cleavage, and rescue by pan-caspase inhibition. Differential array analysis revealed early modulation of stress response gene expression in both A375 melanoma and PANC-1 adenocarcinoma cells elicited by D2O (90%; ≤6 h) (upregulated: CDKN1A, DDIT3, EGR1, GADD45A, HMOX1, NFKBIA, or SOD2 (up to 9-fold; p < 0.01)) confirmed by independent RT-qPCR analysis. Immunoblot analysis revealed rapid onset of D2O-induced stress response phospho-protein activation (p-ERK, p-JNK, p-eIF2α, or p-H2AX) or attenuation (p- AKT). Feasibility of D2O-based chemotherapeutic intervention (drinking water (30% w/w)) was demonstrated in a severe combined immunodeficiency (SCID) mouse melanoma metastasis model using luciferase-expressing A375-Luc2 cells. Lung tumor burden (visualized by bioluminescence imaging) was attenuated by D2O, and inhibition of invasiveness was also confirmed in an in vitro Matrigel transwell invasion assay. D2O supplementation also suppressed tumor growth in a murine xenograft model of human melanoma, and median survival was significantly increased without causing adverse effects. These data demonstrate for the first time that systemic D2O administration impairs growth and metastasis of malignant melanoma through the pharmacological induction of deuterium (2H)-stress. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.National Institutes of HealthOpen access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Genetic Target Modulation Employing CRISPR/Cas9 Identifies Glyoxalase 1 as a Novel Molecular Determinant of Invasion and Metastasis in A375 Human Malignant Melanoma Cells In Vitro and In Vivo

Metabolic reprogramming is a molecular hallmark of cancer. Recently, we have reported the overexpression of glyoxalase 1 (encoded by GLO1), a glutathione-dependent enzyme involved in detoxification of the reactive glycolytic byproduct methylglyoxal, in human malignant melanoma cell culture models and clinical samples. However, the specific role of GLO1 in melanomagenesis remains largely unexplored. Here, using genetic target modulation, we report the identification of GLO1 as a novel molecular determinant of invasion and metastasis in malignant melanoma. First, A375 human malignant melanoma cells with GLO1 deletion (A375-GLO1_KO) were engineered using CRISPR/Cas9, and genetic rescue clones were generated by stable transfection of KO clones employing a CMV-driven GLO1 construct (A375-GLO1_R). After confirming GLO1 target modulation at the mRNA and protein levels (RT-qPCR, immunodetection, enzymatic activity), phenotypic characterization indicated that deletion of GLO1 does not impact proliferative capacity while causing significant sensitization to methylglyoxal-, chemotherapy-, and starvation-induced cytotoxic stress. Employing differential gene expression array analysis (A375-GLO1_KO versus A375-GLO1_WT), pronounced modulation of epithelial--mesenchymal transition (EMT)-related genes [upregulated: CDH1, OCLN, IL1RN, PDGFRB, SNAI3; (downregulated): BMP1, CDH2, CTNNB1, FN1, FTH1, FZD7, MELTF, MMP2, MMP9, MYC, PTGS2, SNAI2, TFRC, TWIST1, VIM, WNT5A, ZEB1, and ZEB2 (up to tenfold; p < 0.05)] was observed-all of which are consistent with EMT suppression as a result of GLO1 deletion. Importantly, these expression changes were largely reversed upon genetic rescue employing A375-GLO1_R cells. Differential expression of MMP9 as a function of GLO1 status was further substantiated by enzymatic activity and ELISA analysis; phenotypic assessment revealed the pronounced attenuation of morphological potential, transwell migration, and matrigel 3D-invasion capacity displayed by A375-GLO1_KO cells, reversed again in genetic rescue clones. Strikingly, in a SCID mouse metastasis model, lung tumor burden imposed by A375-GLO1_KO cells was strongly attenuated as compared to A375-GLO1_WT cells. Taken together, these prototype data provide evidence in support of a novel function of GLO1 in melanoma cell invasiveness and metastasis, and ongoing investigations explore the function and therapeutic potential of GLO1 as a novel melanoma target.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Topical hypochlorous acid (HOCl) blocks inflammatory gene expression and tumorigenic progression in UV-exposed SKH-1 high risk mouse skin

Hypochlorous acid (HOCl) is the active oxidizing principle underlying drinking water disinfection, also delivered by numerous skin disinfectants and released by standard swimming pool chemicals used on a global scale, a topic of particular relevance in the context of the ongoing COVID-19 pandemic. However, the cutaneous consequences of human exposure to HOCl remain largely unknown, posing a major public health concern. Here, for the first time, we have profiled the HOCl-induced stress response in reconstructed human epidermis and SKH-1 hairless mouse skin. In addition, we have investigated the molecular consequences of solar simulated ultraviolet (UV) radiation and HOCl combinations, a procedure mimicking co-exposure experienced for example by recreational swimmers exposed to both HOCl (pool disinfectant) and UV (solar radiation). First, gene expression elicited by acute topical HOCl exposure was profiled in organotypic human reconstructed epidermis. Next, co-exposure studies (combining topical HOCl and UV) performed in SKH-1 hairless mouse skin revealed that the HOCl-induced cutaneous stress response blocks redox and inflammatory gene expression elicited by subsequent acute UV exposure (Nos2, Ptgs2, Hmox1, Srxn1), a finding consistent with emerging clinical evidence in support of a therapeutic role of topical HOCl formulations for the suppression of inflammatory skin conditions (e.g. atopic dermatitis, psoriasis). Likewise, in AP-1 transgenic SKH-1 luciferase-reporter mice, topical HOCl suppressed UV-induced inflammatory signaling assessed by bioluminescent imaging and gene expression analysis. In the SKH-1 high-risk mouse model of UV-induced human keratinocytic skin cancer, topical HOCl blocked tumorigenic progression and inflammatory gene expression (Ptgs2, Il19, Tlr4), confirmed by immunohistochemical analysis including 3-chloro-tyrosine-epitopes. These data illuminate the molecular consequences of HOCl-exposure in cutaneous organotypic and murine models assessing inflammatory gene expression and modulation of UV-induced carcinogenesis. If translatable to human skin these observations provide novel insights on molecular consequences of chlorination stress relevant to environmental exposure and therapeutic intervention

Muscle hypertrophy and architectural changes in response to eight-week neuromuscular electrical stimulation training in healthy older people

Loss of muscle mass of the lower limbs and of the spine extensors markedly impairs locomotor ability and spine stability in old age. In this study, we investigated whether 8 w of neuromuscular electrical stimulation (NMES) improves size and architecture of the lumbar multifidus (LM) and vastus lateralis (VL) along with locomotor ability in healthy older individuals. Eight volunteers (aged 65 65 years) performed NMES 3 times/week. Eight sex-and age-matched individuals served as controls. Functional tests (Timed Up and Go test (TUG) and Five Times Sit-to-Stand Test (FTSST)), VL muscle architecture (muscle thickness (MT), pennation angle (PA), and fiber length (FL)), along with VL cross-sectional area (CSA) and both sides of LM were measured before and after by ultrasound. By the end of the training period, MT and CSA of VL increased by 8.6% and 11.4%, respectively. No significant increases were observed in FL and PA. LM CSA increased by 5.6% (left) and 7.1% (right). Interestingly, all VL architectural parameters significantly decreased in the control group. The combined NMES had a large significant effect on TUG (r = 0.50, p = 0.046). These results extend previous findings on the hypertrophic effects of NMES training, suggesting to be a useful mean for combating age-related sarcopenia

The sunless tanning agent dihydroxyacetone induces stress response gene expression and signaling in cultured human keratinocytes and reconstructed epidermis

Sunless (chemical) tanning is widely regarded as a safe alternative to solar UV-induced skin tanning known to be associated with epidermal genotoxic stress, but the cutaneous biology impacted by chemical tanning remains largely unexplored. Chemical tanning is based on the formation of melanin-mimetic cutaneous pigments ('melanoidins') from spontaneous amino-carbonyl ('glycation') reactions between epidermal amino acid/protein components and reactive sugars including the glycolytic ketose dihydroxyacetone (DHA). Here, we have examined the cutaneous effects of acute DHA-exposure on cultured human HaCaT keratinocytes and epidermal reconstructs, profiled by gene expression array analysis and immunodetection. In keratinocytes, DHA-exposure performed at low millimolar concentrations did not impair viability while causing a pronounced cellular stress response as obvious from rapid activation of phospho-protein signal transduction [p-p38, p-Hsp27(S15/S78), p-elF2a] and gene expression changes (HSPA6, HMOX1, CRYAB, CCL3), not observable upon exposure to the nonketose, tanning-inactive DHA-control glycerol. Formation of advanced glycation end products (AGEs) from posttranslational protein-adduction was confirmed by quantitative mass spectrometric detection of N-E-(carboxyethyl)-L-lysine (CEL) and N-7 -carboxyethyl-L-arginine, and skin cells with CRISPR-Cas9-based elimination of the carbonyl stress response gene GLO1 (encoding glyoxalase 1) displayed hypersensitivity to DHA-cytotoxicity. In human epidermal reconstructs a topical use-relevant DHA-dose regimen elicited a comparable stress response as revealed by gene expression array (HSPAIA, HSPA6, HSPD1, 1L6, DDIT3, EGR1) and immunohistochemical analysis (CEL, HO-1, p-Hsp27-S78). In DHA-treated SKH-1 hairless mouse skin IHC-detection revealed epidermal occurrence of CEL- and p-Hsp27-epitopes. For comparison, stress response gene expression array analysis was performed in epidermis exposed to a supra-erythemal dose of solar simulated UV (2 MEDs), identifying genes equally or differentially sensitive to either one of these cutaneous stimuli [DHA ('sunless tanning') versus solar UV ('sun-induced tanning')]. Given the worldwide use of chemical tanners in consumer products, these prototype data documenting a DHA-induced specific cutaneous stress response deserve further molecular exploration in living human skin.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Use of a hand-assisted laparoscopic surgical technique for closure of an extensive mesojejunal rent in a horse

CASE DESCRIPTION A 7-year-old 573-kg (1,261 -lb) Swiss Warmblood gelding was evaluated because of signs of acute abdominal pain. CLINICAL FINDINGS Physical examination revealed a markedly distended abdomen with subjectively reduced borborygmi in all abdominal quadrants. A large, gas-distended viscus was present at the pelvic brim preventing complete palpation of the abdomen per rectum. Ultrasonographic evaluation could not be safely performed in the initial evaluation because of severe signs of abdominal pain. TREATMENT AND OUTCOME Ventral midline celiotomy was performed, and right dorsal displacement of the ascending colon was corrected. Progressive signs of abdominal pain after surgery prompted repeat ventral midline celiotomy, and small intestinal incarceration in a large, radial mesojejunal rent was detected. The incarceration was reduced, but the defect was not fully accessible for repair via the celiotomy. Repair of the mesenteric defect was not attempted, and conservative management was planned after surgery; however, signs of colic returned. A standard laparoscopic approach was attempted from both flanks in the standing patient, but the small intestine could not be adequately mobilized for full evaluation of the rent. Hand-assisted laparoscopic surgery (HALS) allowed identification and reduction of jejunal incarceration and repair of the mesenteric rent. Although minor ventral midline incisional complications were encountered, the horse recovered fully. CLINICAL RELEVANCE HALS techniques should be considered for repair of mesenteric rents in horses. In the horse of this report, HALS facilitated identification, evaluation, and repair of a large radial mesenteric rent that was not accessible from a ventral median celiotomy