Ana P. Pinto

In vitro organogenesis of Passiflora alata

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO3 incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA3 and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.

Excessive eccentric exercise-induced overtraining model leads to endoplasmic reticulum stress in mice skeletal muscles

AIMS The present study verified the responses of selected endoplasmic reticulum (ER) stress proteins (i.e., BiP, ATF-6, pIRE1, pPERK, and peIF2alpha) in mice skeletal muscles after three different running overtraining (OT) protocols with same external load (i.e., intensity vs. volume), but performed in downhill, uphill and without inclination. MATERIALS AND METHODS The rodents were randomly divided into control (CT; sedentary mice), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without inclination (OTR) groups. The incremental load test and exhaustive test were used as performance parameters. Forty hours after the exhaustive test performed at the end of the OT protocols (i.e., at the end of week 8) and after a 2-week total recovery period (i.e., at the end of week 10), the extensor digitorum longus (EDL) and soleus muscles were removed and used for immunoblotting. KEY FINDINGS For both skeletal muscle types, the OTR/down protocol increased the pIRE-1, pPERK and peIF2alpha, which were not normalized after the total recovery period. At the end of week 8, the other two OT protocols up-regulated the BiP, pPERK and peIF2alpha levels only for the soleus muscle. These ER stress proteins were not normalized after the total recovery period for the OTR/up group. SIGNIFICANCE The above findings suggest that the OTR/down protocol-induced skeletal muscle ER stress may be linked to a pathological condition in EDL and soleus muscles.

Excessive training impairs the insulin signal transduction in mice skeletal muscles

The main aim of this investigation was to verify the effects of overtraining (OT) on the insulin and inflammatory signaling pathways in mice skeletal muscles. Rodents were divided into control (CT), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up), and overtrained by running without inclination (OTR) groups. Rotarod, incremental load, exhaustive, and grip force tests were used to evaluate performance. Thirty-six hours after the grip force test, the extensor digitorum longus (EDL) and soleus were extracted for subsequent protein analyses. The three OT protocols led to similar responses of all performance evaluation tests. The phosphorylation of insulin receptor beta (pIRβ; Tyr), protein kinase B (pAkt; Ser473), and the protein levels of plasma membrane glucose transporter-4 (GLUT4) were lower in the EDL and soleus after the OTR/down protocol and in the soleus after the OTR/up and OTR protocols. While the pIRβ was lower after the OTR/up and OTR protocols, the pAkt was higher after the OTR/up in the EDL. The phosphorylation of IκB kinase alpha and beta (pIKKα/β; Ser180/181), stress-activated protein kinases/Jun amino-terminal kinases (pSAPK-JNK; Thr183/Tyr185), factor nuclear kappa B (pNFκB p65; Ser536), and insulin receptor substrate 1 (pIRS1; Ser307) were higher after the OTR/down protocol, but were not altered after the two other OT protocols. In summary, these data suggest that OT may lead to skeletal muscle insulin signaling pathway impairment, regardless of the predominance of eccentric contractions, although the insulin signal pathway impairment induced in OTR/up and OTR appeared to be muscle fiber-type specific.

Genetic transformation of sweet passion fruit ( Passiflora alata ) and reactions of the transgenic plants to Cowpea aphid borne mosaic virus

Passion fruit woodiness disease, which is caused by the potyvirus Cowpea aphid borne mosaic virus (CABMV), is the primary constraint for passion fruit production in Brazil. Transgenic Passiflora alata lines that contain a CABMV-derived coat protein gene fragment in a hairpin configuration were obtained via Agrobacterium tumefaciens-mediated transformation. The plants were propagated and the reaction to CABMV infection was evaluated after three mechanical and one viruliferous vector inoculations. After three mechanical inoculations, two lines from a total of 21 transgenic lines tested maintained all four propagated clones symptomless. After the fourth inoculation, all transgenic lines presented at least one propagated clone infected with CABMV. However, 20 propagated clones from different transgenic lines remained asymptomatic. These asymptomatic plants were analyzed by RT-PCR and CABMV was detected in 17 plants. The estimated viral titers in these plants, which were determined by RT-qPCR, were consistently low compared with those of the positive control (non-transgenic inoculated plants). A biological virus recovery test was performed using leaf extracts from the three RT-PCR negative propagated clones and the absence of the CABMV was confirmed. The results of the present study indicate that the incorporation of CABMV-gene fragments into the Passiflora genome may influence the resistance of these plants to the pathogen.

Exhaustive Training Leads to Hepatic Fat Accumulation

Recently, we demonstrated that an overtraining (OT) protocol for mice based on downhill running sessions increased the hepatic phosphorylation of 70‐kDa ribosomal protein S6 kinase 1 (S6K1; Thr389), a downstream target of the mammalian target of rapamycin complex 1 (mTORC1). In liver, the overactivation of the Akt/mTORC1 pathway induces lipogenesis via regulation of the action of sterol regulatory element binding protein‐1 (SREBP‐1) at multiple steps. Herein, we verified the effects of three running OT models with same external load (i.e., the product between intensity and volume of training), but performed in downhill, uphill and without inclination, on the proteins related to the mTORC1 signaling pathway, the protein content of the SREBP‐1, ACC, and FAS, and the morphological characteristics of C57BL/6 mouse livers. In summary, the downhill running‐induced OT model up‐regulated the levels of major proteins of the mTORC1 signaling pathway, the protein levels of SREBP‐1 (p125 precursor) and induced signs of cell swelling accompanied by acute inflammation. The other two OT protocols performed uphill and without inclination did not modulate the most analyzed molecular proteins, but induced hepatic morphological alterations, suggesting an acute pathological adaptation. The three OT models induced hepatic fat accumulation. J. Cell. Physiol. 232: 2094–2103, 2017.

Treadmill Slope Modulates Inflammation, Fiber Type Composition, Androgen, and Glucocorticoid Receptors in the Skeletal Muscle of Overtrained Mice

Overtraining (OT) may be defined as an imbalance between excessive training and adequate recovery period. Recently, a downhill running-based overtraining (OTR/down) protocol induced the nonfunctional overreaching state, which is defined as a performance decrement that may be associated with psychological and hormonal disruptions and promoted intramuscular and systemic inflammation. To discriminate the eccentric contraction effects on interleukin 1beta (IL-1β), IL-6, IL-10, IL-15, and SOCS-3, we compared the release of these cytokines in OTR/down with other two OT protocols with the same external load (i.e., the product between training intensity and volume), but performed in uphill (OTR/up) and without inclination (OTR). Also, we evaluated the effects of these OT models on the muscle morphology and fiber type composition, serum levels of fatigue markers and corticosterone, as well as androgen receptor (AR) and glucocorticoid receptor (GR) expressions. For extensor digitorum longus (EDL), OTR/down and OTR groups increased the cytokines and exhibited micro-injuries with polymorphonuclear infiltration. While OTR/down group increased the cytokines in soleus muscle, OTR/up group only increased IL-6. All OT groups presented micro-injuries with polymorphonuclear infiltration. In serum, while OTR/down and OTR/up protocols increased IL-1β, IL-6, and tumor necrosis factor alpha, OTR group increased IL-1β, IL-6, IL-15, and corticosterone. The type II fibers in EDL and soleus, total and phosphorylated AR levels in soleus, and total GR levels in EDL and soleus were differentially modulated by the OT protocols. In summary, the proinflammatory cytokines were more sensitive for OTR/down than for OTR/up and OTR. Also, the specific treadmill inclination of each OT model influenced most of the other evaluated parameters.

Excessive training induces molecular signs of pathologic cardiac hypertrophy: DA ROCHA et al.

Chronic exercise induces cardiac remodeling that promotes left ventricular hypertrophy and cardiac functional improvement, which are mediated by the mammalian or the mechanistic target of rapamycin (mTOR) as well as by the androgen and glucocorticoid receptors (GRs). However, pathological conditions (i.e., chronic heart failure, hypertension, and aortic stenosis, etc.) also induce cardiac hypertrophy, but with detrimental function, high levels of proinflammatory cytokines and myostatin, elevated fibrosis, reduced adenosine monophosphate‐activated protein kinase (AMPK) activation, and fetal gene reactivation. Furthermore, recent studies have evidenced that excessive training induced an inflammatory status in the serum, muscle, hypothalamus, and liver, suggesting a pathological condition that could also be detrimental to cardiac tissue. Here, we verified the effects of three running overtraining (OT) models on the molecular parameters related to physiological and pathological cardiac hypertrophy. C57BL/6 mice performed three different OT protocols and were evaluated for molecular parameters related to physiological and pathological cardiac hypertrophy, including immunoblotting, reverse transcription polymerase chain reaction, histology, and immunohistochemistry analyses. In summary, the three OT protocols induced left ventricle (LV) hypertrophy with signs of cardiac fibrosis and negative morphological adaptations. These maladaptations were accompanied by reductions in AMPKalpha (Thr172) phosphorylation, androgen receptor, and GR expressions, as well as by an increase in interleukin‐6 expression. Specifically, the downhill running–based OT model reduced the content of some proteins related to the mTOR signaling pathway and upregulated the β‐isoform of myosin heavy‐chain gene expression, presenting signs of LV pathological hypertrophy development.

Excessive training is associated with endoplasmic reticulum stress but not apoptosis in the hypothalamus of mice

Downhill running-based overtraining model increases the hypothalamic levels of IL-1β, TNF-α, SOCS3, and pSAPK-JNK. The aim of the present study was to verify the effects of 3 overtraining protocols on the levels of BiP, pIRE-1 (Ser724), pPERK (Thr981), pelF2α (Ser52), ATF-6, GRP-94, caspase 4, caspase 12, pAKT (Ser473), pmTOR (Ser2448), and pAMPK (Thr172) proteins in the mouse hypothalamus. The mice were randomized into the control, overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up), and overtrained by running without inclination (OTR) groups. After the overtraining protocols (i.e., at the end of week 8), hypothalamus was removed and used for immunoblotting. The OTR/down group exhibited increased levels of all of the analyzed endoplasmic reticulum stress markers in the hypothalamus at the end of week 8. The OTR/up and OTR groups exhibited increased levels of BiP, pIRE-1 (Ser724), and pPERK (Thr981) in the hypothalamus at the end of week 8. There were no significant differences in the levels of caspase 4, caspase 12, pAKT (Ser473), pmTOR (Ser2448), and pAMPK (Thr172) between the experimental groups at the end of week 8. In conclusion, the 3 overtraining protocols increased the endoplasmic reticulum stress at the end of week 8.

Excessive treadmill training enhances the insulin signaling pathway and glycogen deposition in mice hearts: OLIVEIRA et al.

Exhaustive and chronic physical exercise leads to peripheral inflammation, which is one of the molecular mechanisms responsible for the impairment of the insulin signaling pathway in the heart. Recently, 3 different running overtraining models performed downhill (OTR/down), uphill (OTR/up), and without inclination (OTR) increased the serum levels of proinflammatory cytokines. This proinflammatory status induced insulin signaling impairment in the skeletal muscle; however, the response of this signaling pathway in the cardiac muscle of overtrained mice was still unknown. Thus, we investigated the effects of OTR/down, OTR/up, and OTR protocols on the protein levels of phosphorylation of insulin receptor β (pIRβ) (Tyr), phosphorylation of protein kinase B (pAkt) (Ser473), plasma membrane glucose transporter‐1 (GLUT1) and GLUT4, phosphorylation of insulin receptor substrate‐1 (pIRS‐1) (Ser307), phosphorylation of IκB kinase α/β) (pIKKα/β (Ser180/181), phosphorylation of p38 mitogen‐activated protein kinase (p‐p38MAPK) (Thr180/Tyr182), phosphorylation of stress‐activated protein kinases‐Jun amino‐terminal kinases (pSAPK‐JNK) (Thr183/Tyr185), and glycogen content in mice hearts. The rodents were divided into naïve (N, sedentary mice), control (CT, sedentary mice submitted to performance evaluations), trained (TR, performed the training protocol), OTR/down, OTR/up, and OTR groups. After the grip force test, the cardiac muscles (ie, left ventricle) were removed and used for immunoblotting and histology. Although the OTR/up and OTR groups exhibited higher cardiac levels of pIRβ (Tyr), only the OTR group exhibited higher cardiac levels of pAkt (Ser473) and plasma membrane GLUT4. On the contrary, the OTR/down group exhibited higher cardiac levels of pIRS‐1 (Ser307). The OTR model enhanced the cardiac insulin signaling pathway. All overtraining models increased the left ventricle glycogen content, with this probably acting as a compensatory organ in response to skeletal muscle insulin signaling impairment.

Positive effects of total recovery period on anti- and pro-inflammatory cytokines are not linked to performance re-establishment in overtrained mice.

Abstract The association between excessive training sessions (i.e., overtraining/OT) and periods of inadequate recovery is linked to the nonfunctional overreaching (NFOR) state, which is defined as an unexplained decrement or stagnation of performance. The cytokine hypothesis of OT considers that pro‐inflammatory cytokines are responsible by the NFOR state‐induced performance decrement. Investigations using rodent models of OT verified increased levels of pro‐inflammatory cytokines in hypothalamus, liver, serum and skeletal muscle samples. Recently, our research group observed that a 2‐week total recovery period was not able to re‐establish the NFOR state‐induced performance decrement. As the responses of anti‐ and pro‐inflammatory cytokines were not measured, we aimed to investigate the effects of 2‐week total recovery period on the protein contents of IL‐1beta, IL‐6, IL‐10, IL‐15, TNF‐alpha and SOCS‐3 in serum and skeletal muscle samples of overtrained mice. Also, a bioinformatics analysis was performed to investigate the correlations of IL‐1beta, IL‐6, IL‐10, IL‐15, TNF‐alpha and SOCS‐3 in skeletal muscle with locomotor activity. In summary, the 2‐week total recovery period upregulated the anti‐inflammatory cytokines and normalized the pro‐inflammatory cytokines without a concomitant re‐establishment of performance.