Endogenous galectin-3 is required for skeletal muscle repair.

Skeletal muscle has the intrinsic ability to self-repair through a multifactorial process, but many aspects of its cellular and molecular mechanisms are not fully understood. There is increasing evidence that some members of the mammalian ß-galactoside-binding protein family (galectins) are involved in the muscular repair process (MRP), including galectin-3 (Gal-3). However, there are many questions about the role of this protein on muscle self-repair. Here, we demonstrate that endogenous Gal-3 is required for: (i) muscle repair in vivo by using a chloride-barium myolesion mouse model and (ii) mouse primary myoblasts myogenic programming. Injured muscle from Gal-3 knockout mice (GAL3KO) showed persistent inflammation associated with compromised muscle repair and the formation of fibrotic tissue on the lesion site. In GAL3KO mice, osteopontin expression remained high even after 7 and 14 d of the myolesion, while Myoblast differentiation transcription factor (MyoD) and myogenin had decreased their expression. In GAL3KO mouse primary myoblast cell culture, Paired Box 7 (Pax7) detection seems to sustain even when cells are stimulated to differentiation and MyoD expression is drastically reduced. The detection and temporal expression levels of these transcriptional factors appear to be altered in Gal-3-deficient myoblast. Gal-3 expression in wild-type mice for GAL3KO states, both in vivo and in vitro, in sarcoplasm/cytoplasm and myonuclei; as differentiation proceeds, Gal-3 expression is drastically reduced, and its location is confined to the sarcolemma/plasma cell membrane. We also observed a change in the temporal-spatial profile of Gal-3 expression and muscle transcription factors levels during the myolesion. Overall, these results demonstrate that endogenous Gal-3 is required for the skeletal muscle repair process.

Nuclear PKR in retinal neurons in the early stage of diabetic retinopathy in streptozotocin­induced diabetic rats.

Retinal neuron apoptosis is a key component of diabetic retinopathy (DR), one of the most common complications of diabetes. Stress due to persistent hyperglycaemia and corresponding glucotoxicity represents one of the primary pathogenic mechanisms of diabetes and its complications. Apoptosis of retinal neurons serves a critical role in the pathogenesis of DR observed in patients with diabetes and streptozotocin (STZ)­induced diabetic rats. Retinal neuron apoptosis occurs one month after STZ injection, which is considered the early stage of DR. The molecular mechanism involved in the suppression of retinal neuron apoptosis during the early stage of DR remains unclear. RNA­dependent protein kinase (PKR) is a stress­sensitive pro­apoptotic kinase. Our previous study indicated that PKR­associated protein X, a stress­sensitive activator of PKR, is upregulated in the early stage of STZ­induced diabetes. In order to assess the role of PKR in DR prior to apoptosis of retinal neurons, immunofluorescence and western blotting were performed to investigate the cellular localization and expression of PKR in the retina in the early stage of STZ­induced diabetes in rats. PKR activity was indirectly assessed by expression levels of phosphorylated eukaryotic translation initiation factor 2α (p­eIF2­α) and the presence of apoptotic cells in the retina was investigated by TUNEL assay. The findings revealed that PKR was localized in the nucleus of retinal ganglion and inner nuclear layer cells from normal and diabetic rats. To the best of our knowledge, the present study is the first to demonstrate nuclear localization of PKR in retinal neurons. Immunofluorescence analysis demonstrated that PKR was expressed in the nuclei of retinal neurons at 3 and 6 days and its expression was decreased at 15 days after STZ treatment. In addition, p­eIF2­α expression and cellular localization followed the trend of PKR, suggesting that this pro­apoptotic kinase was active in the nuclei of retinal neurons. These findings are consistent with the hypothesis that nuclear translocation of PKR may be a mechanism to sequester active PKR, thus preventing upregulation of cytosolic signalling pathways that induce apoptosis in retinal neurons. Apoptotic cells were not detected in the retina in the early stage of DR. A model was proposed to explain the mechanism by which apoptosis of retinal neurons by PKR is suppressed in the early stage of DR. The possible role of mitochondrial RNA (mtRNA) and Alu RNA in this phenomenon is also discussed since it was demonstrated that the cellular stress due to prolonged hyperglycaemia induces the release of mtRNA and transcription of Alu RNA. Moreover, it mtRNA activates PKR, whereas Alu RNA inhibits the activation of this protein kinase.

Crosstalk between hnRNP K and SET in ATRA-induced differentiation in acute promyelocytic leukemia.

HnRNP K protein is a heterogeneous nuclear ribonucleoprotein which has been proposed to be involved in the leukemogenesis of acute promyelocytic leukemia (APL), as well as in differentiation induced by all-trans retinoic acid (ATRA). We previously demonstrated a connection between SET and hnRNP K function in head and neck squamous cell carcinoma (HNSCC) cells related to splicing processing. The objective of this study was to characterize the participation of hnRNP K and SET proteins in ATRA-induced differentiation in APL. We observed higher (5- to 40-fold) levels of hnRNP K and SET mRNA in APL patients at the diagnosis phase compared with induction and maintenance phases. hnRNP K knockdown using short-hairpin RNA led to cell death in ATRA-sensitive NB4 and resistant NB4-R2 cells by apoptosis with SET cleavage. In addition, hnRNP K knockdown increased granulocytic differentiation in APL cells, mainly in NB4-R2 with ATRA. hnRNP K knockdown had an effect similar to that of treatment with U0126 (an meiosis-specific serine/threonine protein kinase/ERK inhibitor), mainly in NB4-R2 cells. SET knockdown in APL cells revealed that apoptosis induction in cells with hnRNP K knockdown occurred by SET cleavage rather than by reduction in SET protein. Transplantation of NB4-R2 cells into nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has higher potential against tumor progression when compared to ATO. Therefore, hnRNP K/SET and ERK are potential therapeutic targets for both antineoplastic leukemia therapy and relapsed APL patients with ATRA resistance.

Morpho-physiological performance of Mikania glomerata Spreng. and Mikania laevigata Sch. Bip ex Baker plants under different light conditions / Desempenho morfo-fisiológico de Mikania glomerata Spreng. e Mikania laevigata Sch. Bip ex Baker sob diferentes condições de luminosidade

In tropical and subtropical zones, lianas play important roles in the process of ecological succession. This study aims to evaluate the photosynthetic and morpho-physiological performance between two lianas species from Mikania genus in response to different levels of radiation: full sun (I0), 25% (I25), 50% (I50), and 75% (I75) retention of solar radiation flux. Plants grown under I75 showed a reduced net photosynthetic rate (A). We observed dynamic photoinhibition at I0 during hours of high irradiation and temperature. The highest and lowest leaf chlorophyll content occurred at I75 and I0, respectively, while carotenoids/total chlorophyll and leaf thickness increased under I0. Total dry mass was higher in plants grown at I0 and I25. However, A values and biomass production of Mikania laevigata were higher at I25, while for Mikania glomerata greater biomass accumulation was observed between I0-I50. Therefore, we concluded that M. laevigata and M. glomerata have different morpho-physiological performances under same the radiation conditions.

Em regiões tropical e subtropical, lianas desempenham um papel importante no processo de sucessão ecológica. O objetivo deste estudo foi avaliar as respostas fotossintéticas e morfo-fisiológicas entre duas espécies de Mikania em diferentes níveis de radiação: sol pleno (I0), 25% (I25), 50% (I50) e 75% (I75) de retenção do fluxo da radiação solar. Plantas crescidas sob I75 mostraram reduzida taxa fotossintética (A). Nós observamos fotoinibição dinâmica em plantas crescidas sob I0 durante as horas de alta irradiação e temperatura. O maior e menor conteúdo de clorofila ocorreu em plantas sob I75 e I0, respectivamente; enquanto carotenoides/clorofila total e espessuras da epiderme e mesofilo aumentaram sob I0. A massa seca total foi maior em plantas crescidas sob I0 e I25. No entanto, os valores de A e a produção de biomassa de M. laevigata foram maiores sob I25; enquanto para M. glomerata, maior acúmulo de biomassa foi observado entre I0-I50. Portanto, nós concluímos que M. laevigata e M. glomerata apresentaram diferentes respostas morfo-fisiológicas sob mesma condição de radiação.

Relative expression of KLK5 to LEKTI is associated with aggressiveness of oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) remains a challenging cancer to treat despite all the advances of the last 50 years. Kallikrein 5 (KLK5) is among the serine proteases implicated in OSCC development. However, whether the activity of KLK5 promotes carcinogenesis is still controversial. Moreover, knowledge regarding the role of the KLK5 cognate inhibitor, Lympho-Epithelial Kazal-Type related Inhibitor (LEKTI), in OSCC is scarce. We have, thus, sought to investigate the importance of KLK5 and LEKTI expression in premalignant and malignant lesions of the oral cavity. METHODS: KLK5 and LEKTI protein expression was evaluated in 301 human samples, which were comprised of non-malignant and malignant lesions of the oral cavity. Moreover, a bioinformatic analysis of the overall survival rate from 517 head and neck squamous cell carcinoma (HNSCC) samples was performed. Additionally, to mimic the uncovered KLK5 to serine peptidase inhibitor (SPINK5) imbalance, the KLK5 gene was abrogated in an OSCC cell line using CRISPR-Cas9 technology. The generated cell line was then used for in vivo and in vitro carcinogenesis related experiments. RESULTS: LEKTI was found to be statistically downregulated in OSCCs, with increased KLK5/SPINK5 mRNA ratio being associated with a shorter overall survival (p = 0.091). Indeed, disruption of KLK5 to SPINK5 balance through the generation of KLK5 null OSCC cells led to smaller xenografted tumors and statistically decreased proliferation rates following multiple time points of BrdU treatment in vitro. CONCLUSION: The association of increased enzyme/inhibitor ratio with poor prognosis indicates KLK5 to SPINK5 relative expression as an important prognostic marker in OSCC.

Activation of adipose tissue glycerokinase contributes to increased white adipose tissue mass in mice fed a high-fat diet.

PURPOSE: Investigate the pathways of glycerol-3-P (G3P) generation for triacylglycerol (TAG) synthesis in retroperitoneal (RWAT) and epididymal (EWAT) white adipose tissues from high-fat diet (HFD)-fed mice. METHODS: Mice were fed for 8 weeks a HFD and glycolysis, glyceroneogenesis and direct phosphorylation of glycerol were evaluated, respectively, by 2-deoxyglucose uptake, phosphoenolpyruvate carboxykinase (PEPCK-C) activity and pyruvate incorporation into TAG-glycerol, and glycerokinase activity and glycerol incorporation into TAG-glycerol in both tissues. RESULTS: HFD increased body and adipose tissue mass and serum levels of glucose and insulin, which were accompanied by glucose intolerance. RWAT and EWAT from HFD-fed mice had increased rates of de novo fatty acid (FA) synthesis (52% and 255%, respectively). HFD increased lipoprotein lipase (LPL) activity and content in EWAT (107%), but decreased in RWAT (79%). HFD decreased the lipolytic response to norepinephrine (57%, RWAT and 25%, EWAT), ß3-adrenoceptor content (50%), which was accompanied by a decrease in phosphorylated-hormone-sensitive lipase (~80%) and phosphorylated-adipocyte triacylglycerol lipase (~60%) in both tissues. HFD decreased the in vitro rates of glucose uptake (3.5- and 6-fold), as well as in glyceride-glycerol synthesis from pyruvate (~3.5-fold) without changes in PEPCK-C activity and content in RWAT and EWAT, but increased glycerokinase activity(~3-fold) and content (90 and 40%) in both tissues. CONCLUSION: The data suggest that direct phosphorylation of glycerol by glycerokinase may be responsible for maintaining the supply of G3P for the existing rates of FA esterification and TAG synthesis in RWAT and EWAT from HFD-fed mice, contributing, along with a lower lipolytic response to norepinephrine, to higher adiposity.

The topical use of insulin accelerates the healing of traumatic tympanic membrane perforations.

OBJECTIVE: In recent years, there has been a tendency to search for regulatory substances that can optimize the healing process of perforated tympanic membranes (TMs). The purpose of this study was to determine the effects of using topical insulin on the healing process of traumatic TMs perforations. STUDY DESIGN: Experimental. METHODS: Tympanic membranes of 20 Wistar rats were perforated in two sections, anterior and posterior to the malleus. The rats were divided into two groups: control and insulin. The insulin group was treated with topical regular insulin. The TMs were histologically examined 3, 5, and 7 days after the perforation through a morphometric analysis of the epithelial layer thickness, perforation size, TM cross-sectional area, semiquantitative and qualitative evaluation of the collagen production by polarization microscopy, and immunohistochemical evaluation of epithelial cells and myofibroblasts markers. RESULTS: Insulin accelerated the healing process of the perforated TMs (P < 0.01); stimulated early thickening of the outer epithelial layer (P < 0.01); contributed to a larger identification of the antipankeratin antibody as epithelial marker (P < 0.05); and increased labeling of smooth muscle anti-alpha-actin antibody (P < 0.05), indicating greater proliferation of myofibroblasts. When the topical insulin was used, it resulted in a greater formation of collagen tissue (P < 0.05), with thicker and better-organized collagen fibers (P < 0.05). CONCLUSION: Insulin accelerated the healing process of TMs traumatic perforations.

Spontaneous healing of the tympanic membrane after traumatic perforation in rats.

UNLABELLED: The most common etiologies of tympanic membrane perforation are infections and trauma. OBJECTIVE: The objective of the present study was to assess the healing of traumatic tympanic membrane perforation in rats. METHODS: The tympanic membrane from male Wistar rats was perforated in the anterior and posterior portions to the handle of the malleus. Five tympanic membranes were evaluated 3 days after tympanic perforation; 5 after 5 days; 5 after 7 days; 3 after 10 days; and 4 after 14 days. The tympanic membranes were submitted to histopathological evaluation after hematoxylin-eosin staining. RESULTS: Tympanic membrane closure occurred at about 7-10 days after injury and the healing process was complete by day 14. The proliferative activity of the outer epithelial layer was present close to the handle of the malleus and to the tympanic annulus. CONCLUSION: The spontaneous healing process of the tympanic membrane starts from the outer epithelial layer, with later healing of the lamina propria and the mucosal layer.

Spontaneous healing of the tympanic membrane after traumatic perforation in rats / Cicatrização espontânea da membrana timpânica após sua perfuração traumática: estudo experimental em ratos,

OBJECTIVE: The objective of the present study was to assess the healing of traumatic tympanic membrane perforation in rats. METHODS: The tympanic membrane from male Wistar rats was perforated in the anterior and posterior portions to the handle of the malleus. Five tympanic membranes were evaluated 3 days after tympanic perforation; 5 after 5 days; 5 after 7 days; 3 after 10 days; and 4 after 14 days. The tympanic membranes were submitted to histopathological evaluation after hematoxylin-eosin staining. RESULTS: Tympanic membrane closure occurred at about 7-10 days after injury and the healing process was complete by day 14. The proliferative activity of the outer epithelial layer was present close to the handle of the malleus and to the tympanic annulus. CONCLUSION: The spontaneous healing process of the tympanic membrane starts from the outer epithelial layer, with later healing of the lamina propria and the mucosal layer. .

OBJETIVOS: Avaliar o reparo cicatricial de perfurações traumáticas da membrana timpânica em ratos. MÉTODO: A membrana timpânica de ratos Wistar machos foram perfuradas nas porções anterior e posterior ao cabo do martelo. Cinco membranas timpânicas foram avaliadas 3 dias após perfuração timpânica; 5 após 5 dias; 5 após 7 dias; 3 após 10 dias; e 4 após 14 dias. As membranas timpânicas foram submetidas à avaliação histopatológica após coloração com hematoxilina- eosina. RESULTADOS: O fechamento da membrana timpânica ocorreu em torno de 7 a 10 dias após perfuração traumática, e o processo de cicatrização estava completo no 14° dia. A atividade proliferativa da camada epitelial externa foi identificada próxima ao cabo do martelo e ao ânulus timpânico. CONCLUSÃO: O processo de cicatrização espontânea da membrana timpânica se inicia com a camada epitelial externa, com posterior cicatrização da lâmina própria e da camada mucosa. .

Serotonergic neural links from the dorsal raphe nucleus modulate defensive behaviours organised by the dorsomedial hypothalamus and the elaboration of fear-induced antinociception via locus coeruleus pathways.

Decrease of γ-aminobutyric acid (GABA)-mediated neurotransmission in the dorsomedial hypothalamus (DMH) evokes instinctive fear-like responses. The aim of the present study was to investigate the involvement of the serotonin (5-HT)- and norepinephrine-mediated pathways of the endogenous pain inhibitory system, including the dorsal raphe nucleus (DRN) and the locus coeruleus (LC), in the defensive responses and antinociceptive processes triggered by the blockade of GABAergic receptors in the DMH. The intra-hypothalamic microinjection of the GABA(A) receptor antagonist bicuculline (40 ng/200 nL) elicited elaborate defensive behaviours interspersed with exploratory responses. This escape behaviour was followed by significantly increased pain thresholds, a phenomenon known as fear-induced antinociception. Furthermore, at 5 and 14 days after DRN serotonin-containing neurons were damaged using the selective neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), the frequency and duration of alertness and escape behaviour evoked by the GABA(A) receptor blockade in the DMH decreased, as well as fear-induced antinociception. Pre-treatment with the non-selective 5-HT receptor antagonist methysergide, the 5-HT(2A/2C) receptor antagonist ketanserin and the 5-HT(2A) receptor selective antagonist R-96544 in the LC also decreased fear-induced antinociception, without significant changes in the expression of defensive behaviours. These data suggest that the serotonergic neurons of the DRN are directly involved in the organisation of defensive responses as well as in the elaboration of the innate fear-induced antinociception. However, serotonin-mediated inputs from the NDR to the LC modulate only fear-induced antinociception and not the defensive behaviours evoked by GABA(A) receptor blockade in the DMH.