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Melanopsin (Opn4) is identified as an oncogene in cutaneous melanoma, with the loss of Opn4 found to be associated with reduced proliferation and tumor growth, due to changes in immune responses and Mitf signaling in vitro and in vivo.

Skin melanocytes harbor a complex photosensitive system comprised of opsins, which were shown, in recent years, to display light- and thermo-independent functions. Based on this premise, we investigated whether melanopsin, OPN4, displays such a role in normal melanocytes. In this study, we found that murine Opn4KO melanocytes displayed a faster proliferation rate compared to Opn4WT melanocytes. Cell cycle population analysis demonstrated that OPN4KO melanocytes exhibited a faster cell cycle progression with reduced G0–G1, and highly increased S and slightly increased G2/M cell populations compared to the Opn4WT counterparts. Expression of specific cell cycle-related genes in Opn4KO melanocytes exhibited alterations that corroborate a faster cell cycle progression. We also found significant modification in gene and protein expression levels of important regulators of melanocyte physiology. PER1 protein level was higher while BMAL1 and REV-ERBα decreased in Opn4KO melanocytes compared to Opn4WT cells. Interestingly, the gene expression of microphthalmia-associated transcription factor (MITF) was upregulated in Opn4KO melanocytes, which is in line with a higher proliferative capability. Taken altogether, we demonstrated that OPN4 regulates cell proliferation, cell cycle, and affects the expression of several important factors of the melanocyte physiology; thus, arguing for a putative tumor suppression role in melanocytes.

This paper aims to report clinical, laboratory and pathological features in a case of suppurative meningoencephalitis by P. aeruginosa from the direct extension of chronic otitis in a Gir cow. The cow was recumbent during physical examination, and neurological examination revealed depression, absence of left eyelid and auricular motor reflex, and hypotonic tongue. Hematology revealed hemoconcentration, leukocytosis by neutrophilia, and hyperfibrinogenemia. Cerebrospinal fluid was slightly turbid, and presented polymorphonuclear pleocytosis, and hyperproteinorrachia. Grossly, the skull floor showed a purulent green–yellow exudate that drained from the left inner ear to the cisterna magna. There was diffuse congestion of the telencephalon, and meninges showed severe hyperemia, moderate thickening, and opacity with the deposition of fibrinosuppurative material ventrally, extending to the cerebellum and brainstem. The left cerebellar hemisphere showed an approximately 1.5 cm in diameter liquefaction area surrounded by a hemorrhagic halo. Histologically, cerebellar, mesencephalic, thalamic, and brain base meninges were intensely thickened and showed severe suppurative inflammation and fibrin deposition. Small multifocal suppurative areas were observed in the cerebellum and brainstem, characterized by a necrotic core, a number of neutrophils, and Gram-negative intralesional bacillary myriads. Pure cultures of P. aeruginosa were obtained and identified in the suppurative CNS lesions, meninges, and inner ear samples. This report highlights an uncommon clinical evolution of secondary P. aeruginosa suppurative meningoencephalitis, probably triggered by recurrent parasitic otitis in an adult Gir cow. Veterinarians, practitioners, and farmers must be aware of the risk of CNS infections after unresolved media and inner otitis, especially in cattle breeds more prone to developing parasitic otitis, such as the Gir and Indubrasil breeds.

Abstract Background Members of the family of NEK protein kinases (NIMA-related kinases) were described to have crucial roles in regulating different aspects of the cell cycle. NEK10 was reported to take part in the maintenance of the G2/M checkpoint after exposure to ultraviolet light. NEK1, NEK5, NEK2 and NEK4 proteins on the other hand have been linked to mitochondrial functions. Methods HEK293T cells were transfected with FLAG empty vector or FLAG-NEK10 and treated or not with Zeocin. For proteomic analysis, proteins co-precipitated with the FLAG constructs were digested by trypsin, and then analyzed via LC-MS/MS. Proteomic data retrieved were next submitted to Integrated Interactome System analysis and differentially expressed proteins were attributed to Gene Ontology biological processes and assembled in protein networks by Cytoscape. For functional, cellular and molecular analyses two stable Nek10 silenced HeLa cell clones were established. Results Here, we discovered the following possible new NEK10 protein interactors, related to mitochondrial functions: SIRT3, ATAD3A, ATAD3B, and OAT. After zeocin treatment, the spectrum of mitochondrial interactors increased by the proteins: FKBP4, TXN, PFDN2, ATAD3B, MRPL12, ATP5J, DUT, YWHAE, CS, SIRT3, HSPA9, PDHB, GLUD1, DDX3X, and APEX1. We confirmed the interaction of NEK10 and GLUD1 by proximity ligation assay and confocal microscopy. Furthermore, we demonstrated that NEK10-depleted cells showed more fragmented mitochondria compared to the control cells. The knock down of NEK10 resulted further in changes in mitochondrial reactive oxygen species (ROS) levels, decreased citrate synthase activity, and culminated in inhibition of mitochondrial respiration, affecting particularly ATP-linked oxygen consumption rate and spare capacity. NEK10 depletion also decreased the ratio of mtDNA amplification, possibly due to DNA damage. However, the total mtDNA content increased, suggesting that NEK10 may be involved in the control of mtDNA content. Conclusions Taken together these data place NEK10 as a novel regulatory player in mitochondrial homeostasis and energy metabolism.

Xeroderma pigmentosum (XP) is a rare genetic condition in which exposure to sunlight leads to a high tumor incidence due to defective DNA repair machinery. Herein, we investigated seven patients clinically diagnosed with XP living in a small city, Montanhas (Rio Grande do Norte), in the Northeast region of Brazil. We performed high-throughput sequencing and, surprisingly, identified two different mutated genes. Six patients carry a novel homozygote mutation in the POLH/XPV gene, c.672_673insT (p.Leu225Serfs*33), while one patient carries a homozygote mutation in the XPC gene, c.2251-1G>C. This latter mutation was previously described in Southeastern Africa (Comoro Island and Mozambique), Pakistan, and in a high incidence in Brazil. The XP-C patient had the first symptoms before the first year of life with aggressive ophthalmologic tumor progression and a melanoma onset at 7 years of age. The XP-V patients presented a milder phenotype with later onset of the disorder (mean age of 16 years old), and one of the six XP-V patients developed melanoma at 72 years. The photoprotection is minimal among them, mainly for the XP-V patients. The differences in the disease severity between XP-C (more aggressive) and XP-V (milder) patients are obvious and point to the major role of photoprotection in the XPs. We estimate that the incidence of XP patients at Montanhas can be higher, but with no diagnosis, due to poor health assistance. Patients still suffer from the stigmatization of the condition, impairing diagnosis, education for sun protection, and medical care.

Infection with some mucosal human papillomavirus (HPV) types is the etiological cause of cervical cancer and of a significant fraction of vaginal, vulvar, anal, penile, and head and neck carcinomas. DNA repair machinery is essential for both HPV replication and tumor cells survival suggesting that cellular DNA repair machinery may play a dual role in HPV biology and pathogenesis. Here, we silenced genes involved in DNA Repair pathways to identify genes that are essential for the survival of HPV-transformed cells. We identified that inhibition of the ATM/CHK2/BRCA1 axis selectively affects the proliferation of cervical cancer-derived cell lines, without altering normal primary human keratinocytes (PHK) growth. Silencing or chemical inhibition of ATM/CHK2 reduced the clonogenic and proliferative capacity of cervical cancer-derived cells. Using PHK transduced with HPV16 oncogenes we observed that the effect of ATM/CHK2 silencing depends on the expression of the oncogene E6 and on its ability to induce p53 degradation. Our results show that inhibition of components of the ATM/CHK2 signaling axis reduces p53-deficient cells proliferation potential, suggesting the existence of a synthetic lethal association between CHK2 and p53. Altogether, we present evidence that synthetic lethality using ATM/CHK2 inhibitors can be exploited to treat cervical cancer and other HPV-associated tumors.

Air pollution represents a considerable threat to health worldwide. The São Paulo Metropolitan area, in Brazil, has a unique composition of atmospheric pollutants with a population of nearly 20 million people and 9 million passenger cars. It is long known that exposure to particulate matter less than 2.5 µm (PM2.5) can cause various health effects such as DNA damage. One of the most versatile defense mechanisms against the accumulation of DNA damage is the nucleotide excision repair (NER), which includes XPC protein. However, the mechanisms by which NER protects against adverse health effects related to air pollution are largely unknown. We hypothesized that reduction of XPC activity may contribute to inflammation response, oxidative stress and DNA damage after PM2.5 exposure. To address these important questions, XPC knockout and wild type mice were exposed to PM2.5 using the Harvard Ambient Particle concentrator. Results from one-single exposure have shown a significant increase in the levels of anti-ICAM, IL-1β, and TNF-α in the polluted group when compared to the filtered air group. Continued chronic PM2.5 exposure increased levels of carbonylated proteins, especially in the lung of XPC mice, probably as a consequence of oxidative stress. As a response to DNA damage, XPC mice lungs exhibit increased γ-H2AX, followed by severe atypical hyperplasia. Emissions from vehicles are composed of hazardous substances, with polycyclic aromatic hydrocarbons (PAHs) and metals being most frequently cited as the major contributors to negative health impacts. This analysis showed that benzo[b]fluoranthene, 2-nitrofluorene and 9,10-anthraquinone were the most abundant PAHs and derivatives. Taken together, these findings demonstrate the participation of XPC protein, and NER pathway, in the protection of mice against the carcinogenic potential of air pollution. This implicates that DNA is damaged directly (forming adducts) or indirectly (Reactive Oxygen Species) by the various compounds detected in urban PM2.5.

Objetivo: Evaluar el efecto protector del extracto acuoso de Cymbopogon citratus (DC) Stapf, ante el daño inducido por las radiaciones UVC. Material y Métodos: Para evaluar si el extracto acuoso de C. citratus era capaz de inducir roturas de cadenas en el ADN, moléculas de plásmido pBluescript SK II fueron tratadas con diferentes concentraciones del extracto (0,01 - 4,0 mg/mL), en los tiempos de exposición: 30, 60 y 90 min. El efecto fotoprotector fue evaluado aplicando el extracto vegetal antes, durante, y después de la irradiación del ADN plasmídico con 200 J/m² de UVC. La actividad enzimática de T4 endonucleasa V fue empleada para detectar formación de CPDs. Las formas superenrollada y relajada de las moléculas de plásmido fueron separadas electroforéticamente en gel de agarosa. Adicionalmente, se midió la transmitancia del extracto acuoso a la DO de 254 nm. Resultados: Ninguna de las concentraciones evaluadas resultó genotóxica con 30 min de tratamiento. Las concentraciones ≥ 2 mg/mL indujeron roturas de cadenas a los 90 min de incubación. El extracto de C. citratus a concentraciones ≥ 0,5 mg/mL protegió al ADN frente a las radiaciones UVC. Conclusiones: En nuestras condiciones experimentales, el extracto acuoso de C. citratus protege al ADN frente a la genotoxicidad inducida por la luz UVC, previniendo la generación de CPDs, pero no es capaz de eliminarlas una vez formadas.

Glioblastoma is a severe type of brain tumor with a poor prognosis and few therapy options. Temozolomide (TMZ) is one of these options, however, with limited success, and failure is mainly due to tumor resistance. In this work, genome-wide CRISPR-Cas9 lentiviral screen libraries for gene knockout or activation were transduced in the human glioblastoma cell line, aiming to identify genes that modulate TMZ resistance. The sgRNAs enriched in both libraries in surviving cells after TMZ treatment were identified by next-generation sequencing (NGS). Pathway analyses of gene candidates on knockout screening revealed several enriched pathways, including the mismatch repair and the Sonic Hedgehog pathways. Silencing three genes ranked on the top 10 list (MSH2, PTCH2, and CLCA2) confirm cell protection from TMZ-induced death. In addition, a CRISPR activation library revealed that NRF2 and Wnt pathways are involved in TMZ resistance. Consistently, overexpression of FZD6, CTNNB1, or NRF2 genes significantly increased cell survival upon TMZ treatment. Moreover, NRF2 and related genes detected in this screen presented a robust negative correlation with glioblastoma patient survival rates. Finally, several gene candidates from knockout or activation screening are targetable by inhibitors or small molecules, and some of them have already been used in the clinic.

The main goal of chemotherapeutic drugs is to induce massive cell death in tumors. Cisplatin is an antitumor drug widely used to treat several types of cancer. Despite its remarkable efficiency, most tumors show intrinsic or acquired drug resistance. The primary biological target of cisplatin is genomic DNA, and it causes a plethora of DNA lesions that block transcription and replication. These cisplatin-induced DNA lesions strongly induce cell death if they are not properly repaired or processed. To counteract cisplatin-induced DNA damage, cells use an intricate network of mechanisms, including DNA damage repair and translesion synthesis. In this review, we describe how cisplatin-induced DNA lesions are repaired or tolerated by cells and focus on the pivotal role of DNA repair and tolerance mechanisms in tumor resistance to cisplatin. In fact, several recent clinical findings have correlated the tumor cell status of DNA repair/translesion synthesis with patient response to cisplatin treatment. Furthermore, these mechanisms provide interesting targets for pharmacological modulation that can increase the efficiency of cisplatin chemotherapy.

The objective of this study was to compare the efficiency of bovine semen cryopreservation using the controlled-rate freezing machine versus the conventional method (uncontrolled curve) by the parameters of sperm quality and viability in post-thaw period. Semen from four adult crossbreed bulls was cryopreserved in Tris-yolk-glycerol medium. The computer assisted analysis of thawed semen detected the following results: PM 56.50±22.25%; VAP 34.77±4.25μm/s; VSL 28.17±4.25μm/s; VCL 58.45±6.85μm/s; STR 82.00±2.31%; LIN 49.50±3.32%, to automated system and PM 57.00±13.11%; VAP 25.75±1.66μm/s; VSL 23.32±1.99μm/s; VCL 63.32±1.79μm/s; STR 82.25±3.59μm/s; LIN 50.00±4.97μm/s to conventional cryopreservation system. The results of plasma membrane and acrosome integrity evaluation were 54.7±12.55% and 36.13±22.20% for the automated system and 53.22±13.22% and 47.26±5.74% for the conventional system, respectively. The parameters evaluated demonstrated that there was no statistical difference between the cryopreservation systems. Thus, the choice of the bovine semen cryopreservation method to be used on a farm is a responsibility of the technician, and should be based on the reality of each farm. Therefore, it is always necessary to consider that the conventional system of bovine semen cryopreservation can vary more than the automated system, which, despite the cost of the equipment, can ensure repeatability of the results and consequent quality of cryopreserved bovine semen.

O objetivo deste estudo foi avaliar as características morfológicas e funcionais dos espermatozóides bovinos recuperados de epidídimos resfriados por longos períodos e posteriormente criopreservados. Testículos bovinos foram coletados no abatedouro, transportados ao laboratório e armazenados a 5°C por 0, 24, 48 e 72 horas (n=10 para cada tratamento). Os espermatozóides foram extraídos de cada epidídimo, avaliados e diluídos em meio tris-gema-glicerol a 7% e criopreservados em nitrogênio líquido. As características morfológicas e funcionais dos espermatozóides foram avaliadas in vitro por análise microscópica e in vivo, por meio de inseminação artificial. Foram observadas alterações morfológicas características da imaturidade dos espermatozóides e redução da motilidade após 72 horas de refrigeração dos epidídimos. Esses parâmetros também foram alterados após o descongelamento, em todos os tratamentos. A manutenção dos espermatozoides a 5°C por 72h reduziu a motilidade espermática. Em todos os tratamentos foram observadas alterações morfológicas características da imaturidade dos espermatozoides e redução da motilidade após o descongelamento. A integridade de membrana plasmática e acrossoma somente foram afetadas pós criopreservação nos grupos mantidos a 5°C durante 48 ou 72h antes da criopreservação. Contudo, a capacidade de fecundação dos espermatozóides mantidos a 5°C durante 24 ou 72h antes da criopreservação foi suficiente para promover duas gestações e nascimento de bezerros saudáveis. Esses resultados indicam que a recuperação e a criopreservação de espermatozóides obtidos de epidídimos mantidos a 5°C, até 72h, provenientes de animais mortos é uma opção viável para preservar gametas masculinos para compor um banco de germoplasma.The objective of this study was to evaluate the morphological and functional characteristics of bovine spermatozoa retrieved from chilled epidydimides for long periods and cryopreserved. Bovine testicles were collected in abattoir, transported to the laboratory and stored at 5°C for 0, 24, 48h e 72 hours (n=10 for each storage time treatment group). The spermatozoa were retrieved from each epidydimides, evaluated and diluted in tris-egg yolk-glycerol 7% medium and cryopreserved in liquid nitrogen. The morphological and functional characteristics of the spermatozoa were analyzed in vitro, by microscopic evaluation and in vivo, using artificial insemination. Morphological alterations as sperm immaturity and motility reduction decreased after 72h of epididymides refrigeration and after thaw sperm were observed. The membrane and acrosome integrity were only affected in G48 and G72 groups after cryopreservation. However, the sperm capacity of fertilization post-cryopreservation was sufficient to promote two pregnancies and birth of healthy calves from G24 h and G72h groups. These results indicated that recovery and cryopreservation of chilled epididymal sperm until 72h from dead animals is a viable option to preserve male gametes to compose a germplasm bank.

To investigate oxidative lesions and strand breaks induction by singlet molecular oxygen (¹O2), supercoiled-DNA plasmid was treated with thermo-dissociated DHPNO2 and photoactivated-methylene blue. DNA lesions were detected by Fpg that cleaves DNA at certain oxidized bases, and T4-endoV, which cleaves DNA at cyclobutane pyrimidine dimers and apurinic/apyrimidinic (AP) sites. These cleavages form open relaxed-DNA structures, which are discriminated from supercoiled-DNA. DHPNO2 or photoactivated-MB treatments result in similar plasmid damage profile: low number of single-strand breaks or AP-sites and high frequency of Fpg-sensitive sites; confirming that base oxidation is the main product for both reactions and that ¹O2 might be the most likely intermediate that reacts with DNA.

The objective of this study was to identify and to correlate parameters that characterize puberty and estimate the age and weight of Suffolk ewe lambs in Mato Grosso do Sul. This study was carried out with twenty-two Suffolk ewe lambs for 256 days. Blood samples were collected from the jugular vein and centrifugated and the plasma were stored at -20°C until analysis of progesterone (P4) concentration by radioimmunoassay (RIA). The morphometric measurements included: head length and width, body length, elbow height, chest perimeter, back-sternal diameter, croup length, width and height, cannon bone perimeter and shoulder and hip width, using a tape. The animals became pubertal when the plasma progesterone concentration was >1.0 ng/mL, at 34.1 ± 5.2 kg and 39.5 ± 8.5 weeks of age. Meanwhile, the lambs that did not reach puberty (37.7 ± 11.4 weeks of age) were lighter. The morphometric measurements has medium to low correlations with age and weight at puberty.Os objetivos neste trabalho foram identificar e correlacionar parâmetros que caracterizem a puberdade e estimar a idade e o peso à puberdade de cordeiras mestiças Suffolk. Amostras de sangue de 22 borregas foram coletadas e centrifugadas e o plasma armazenado a -20 ºC para posterior análise das concentrações de progesterona (P4) por meio de radioimunoensaio. As medidas morfométricas aferidas foram: comprimento e largura da cabeça; comprimento do corpo; altura de cernelha; perímetro torácico; diâmetro dorso-esternal; comprimento, altura e largura de garupa; perímetro de canela; e largura entre ísquios e ílios, utilizando-se fita métrica. Definiu-se como início da puberdade quando os animais apresentaram concentração plasmática de P4 >1,0 ng/mL, o que ocorreu aos 34,1 ± 5,2 kg de peso vivo e 39,5 ± 8,5 semanas de idade. Entretanto, as cordeiras que não atingiram a puberdade (37,7 ± 11,4 semanas de idade) apresentaram peso menor. As medidas morfométricas apresentam médias e baixas correlações com idade e peso à puberdade.

A descoberta de que nossas células dispõem de um mecanismo de silenciamento gênico empregando RNA interferência ainda é muito recente. Apesar disso, em menos de uma década a investigação científica já alcançou progresso, suficiente para muito brevemente nos apropriarmos desse conhecimento com fins terapêuticos. Duplexes de RNA são potenciais fármacos e há investimentos altos nessa nova abordagem. Aparentemente, a promessa de terapia gênica parece finalmente atingir sua maturidade com essas novas ferramentas.The discovery of gene silencing mechanisms in our own cells using RNA interference is very recent. However, in less than a decade, the scientific investigation have progressed enough to make us see that, very soon, we will use this knowledge for therapeutic purposes. RNA duplexes are potential pharmaceutical drugs and there are high investments in this new strategy. The promising gene therapy seems to finally reach maturity with these new tools.

BackgroundDue to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter.Methodology/principal findingsThe Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations.Conclusions/significanceThe application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.

ABSTRACT: Wharton's jelly is a source of mesenchymal stem cells (MSCs) that had not yet been tested for bovine embryo production by nuclear transfer (NT). Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton's jelly for embryo and pregnancy production by NT in cattle. The umbilical cord was collected during calving and cells derived from Wharton's jelly (WJCs) were isolated by explant and cultured in Dulbecco's Modified Eagle Medium. Skin Fibroblasts (FB) were isolated after 6 months of life. Morphological analysis was performed by bright field and scanning electron microscopy (SEM) during cell culture. Phenotypic and genotypic characterization by flow cytometry, immunocytochemistry, RT-PCR and differentiation induction in cell lineages were performed for WJC. In the NT procedure, oocytes at the arrested metaphase II stage were enucleated using micromanipulators, fused with WJCs or FB and later activated artificially. SEM micrographs revealed that WJCs have variable shape under culture. Mesenchymal markers of MSCs (CD29+, CD73+, CD90+ and CD105+) were expressed in bovine-derived WJC cultures, as evidenced by flow cytometry, immunocytochemistry and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes and adipocytes. After classification, the WJCs were used in NT. Blastocyst formation rate by NT with WJCs at day 7 was 25.80±0.03%, similar to blatocyst rate with NT using skin fibroblasts (19.00±0.07%). Pregnancies were obtained and showed that WJCs constitute a new cell type for use in animal cloning.

Xeroderma pigmentosum (XP) variant cells are deficient in the translesion synthesis (TLS) DNA polymerase Polη (eta). This protein contributes to DNA damage tolerance, bypassing unrepaired UV photoproducts and allowing S‐phase progression with minimal delay. In the absence of Polη, backup polymerases perform TLS of UV lesions. However, which polymerase plays this role in human cells remains an open question. Here, we investigated the potential role of Polι (iota) in bypassing ultraviolet (UV) induced photoproducts in the absence of Polη, using NER‐deficient (XP‐C) cells knocked down for Polι and/or Polη genes. Our results indicate that cells lacking either Polι or Polη have increased sensitivity to UVC radiation. The lack of both TLS polymerases led to increased cell death and defects in proliferation and migration. Loss of both polymerases induces a significant replication fork arrest and G1/S‐phase blockage, compared to the lack of Polη alone. In conclusion, we propose that Polι acts as a bona fide backup for Polη in the TLS of UV‐photoproducts. [ABSTRACT FROM AUTHOR]