Proteomic analysis of brain metastatic lung adenocarcinoma reveals intertumoral heterogeneity and specific alterations associated with the timing of brain metastases.

BACKGROUND: Brain metastases are associated with considerable negative effects on patients' outcome in lung adenocarcinoma (LADC). Here, we investigated the proteomic landscape of primary LADCs and their corresponding brain metastases. MATERIALS AND METHODS: Proteomic profiling was conducted on 20 surgically resected primary and brain metastatic LADC samples via label-free shotgun proteomics. After sample processing, peptides were analyzed using an Ultimate 3000 pump coupled to a QExactive HF-X mass spectrometer. Raw data were searched using PD 2.4. Further data analyses were carried out using Perseus, RStudio and GraphPad Prism. Proteomic data were correlated with clinical and histopathological parameters and the timing of brain metastases. Mass spectrometry-based proteomic data are available via ProteomeXchange with identifier PXD027259. RESULTS: Out of the 6821 proteins identified and quantified, 1496 proteins were differentially expressed between primary LADCs and corresponding brain metastases. Pathways associated with the immune system, cell-cell/matrix interactions and migration were predominantly activated in the primary tumors, whereas pathways related to metabolism, translation or vesicle formation were overrepresented in the metastatic tumors. When comparing fast- versus slow-progressing patients, we found 454 and 298 differentially expressed proteins in the primary tumors and brain metastases, respectively. Metabolic reprogramming and ribosomal activity were prominently up-regulated in the fast-progressing patients (versus slow-progressing individuals), whereas expression of cell-cell interaction- and immune system-related pathways was reduced in these patients and in those with multiple brain metastases. CONCLUSIONS: This is the first comprehensive proteomic analysis of paired primary tumors and brain metastases of LADC patients. Our data suggest a malfunction of cellular attachment and an increase in ribosomal activity in LADC tissue, promoting brain metastasis. The current study provides insights into the biology of LADC brain metastases and, moreover, might contribute to the development of personalized follow-up strategies in LADC.

Alteración de los parámetros seminales y su asociación con la fragmentación del ADN espermático / Modification of seminal parameters and their association with sperm DNA fragmentation

Resumen ANTECEDENTES: el patrón de referencia para evaluar la infertilidad masculina es el análisis seminal, aunque es incapaz de detectar la fragmentación del ADN espermático relacionado con resultados reproductivos adversos. OBJETIVO: analizar la relación entre los parámetros seminales y la fragmentación del ADN espermático, y valorar la utilidad del seminograma como predictor de fragmentación. MATERIALES Y MÉTODOS: estudio retrospectivo y observacional efectuado en pacientes del Instituto de Ciencias en Reproducción Humana Vida con problemas de fertilidad. Análisis seminal e índice de fragmentación del ADN (DFI) mediante el test de dispersión de la cromatina espermática. RESULTADOS: se estudiaron 206 pacientes: 43 (20.9%) con espermas normales y 163 (79.1%) con algún parámetro seminal alterado. En los individuos con espermas normales, 8 (18.6%) tuvieron fragmentación moderada y quienes resultaron con al menos un parámetro seminal alterado 49 (30.1%) reportaron fragmentación moderada y 22 (13.5%) fragmentación crítica. Se encontró una correlación negativa entre el índice de fragmentación del ADN y la movilidad, vitalidad y concentración (p<0.0001). No se encontró correlación alguna con el resto de los parámetros seminales. Las medias de fragmentación aumentaron conforme se incrementó el número de parámetros alterados. La razón de momios de fragmentación moderada y crítica en pacientes con oligozoospermia fue de 2.2 y 1.9; con astenozoospermia 8.0 y 26.2; con necrozoospermia 10.5 y 18.8; y con ≥ 3 alteraciones seminales 4.6 y 14.2, respectivamente. CONCLUSIONES: en pacientes con astenozoospermia, necrozoospermia u oligozoospermia o con ≥ 3 parámetros seminales alterados se recomienda realizar el estudio de fragmentación del ADN.

Abstract BACKGROUND: the reference pattern to evaluate male infertility is a semen analysis: although it is unable to detect sperm DNA fragmentation related to adverse reproductive outcomes. OBJECTIVE: to analyze the relationship between seminal parameters and sperm DNA fragmentation as well as assess the usefulness of semen analysis as predictor of fragmentation. MATERIALS AND METHODS: retrospective, observational study performed in patients of the Instituto de Ciencias en Reproducción Humana Vida with infertility problems. Semen analysis and sperm DNA fragmentation index (DFI) using the sperm chromatin dispersion test. RESULTS: 206 patients were studied: 43 (20.9%) with normal sperm and 163 (79.1%) with an impaired seminal parameter. Of the individuals with normal sperm, 8 (18.6%) had moderate fragmentation and those who had at least one seminal parameter impaired 49 (30.1%) reported moderate fragmentation and 22 (13.5%) critical fragmentation. We found a negative correlation between the DNA fragmentation index and the motility, vitality and concentration (p<0.0001). We found no correlation whatsoever with the remaining seminal parameters. The fragmentation measurements increased as the number of impaired parameters increased. The odds ratio of moderate and critical fragmentation in patients with oligozoospermia was 2.2 and 1.9; with asthenozoospermia 8.0 and 26.2; con necrozoospermia 10.5 and 18.8; and with ≥ 3 seminal impairments 4.6 and 14.2, respectively. CONCLUSIONS: in patients with asthenozoospermia, necrozoospermia or oligozoospermia or with ≥ 3 impaired seminal parameters, we recommend performing a DNA fragmentation test.

Repercusiones del índice de masa corporal masculina en los resultados de ICSI / Impact of male body mass index on ICSI outcome

Resumen OBJETIVO: primario, determinar la influencia de la obesidad paterna en la tasa de embarazo, implantación, aborto y de recién nacido en casa en pacientes que reciben tratamientos de fertilización in vitro con ICSI. El objetivo secundario: determinar el efecto del índice de masa corporal del padre en la calidad seminal y embrionaria y en la tasa de fertilización. MATERIALES Y MÉTODOS: estudio retrospectivo, comparativo y observacional al que se incluyeron ciclos ICSI con transferencia embrionaria. Los ciclos se dividieron en 3 grupos según el IMC paterno: peso normal (18.5-24.99 kg/m2), sobrepeso (25-29.99 kg/m2) y obesidad (≥ 30 kg/m2). RESULTADOS: se estudiaron 365 ciclos consecutivos en fresco en los que se encontró un incremento significativo en la tasa de aborto en pacientes con obesidad comparados con el grupo sano (39.0 vs 12.9, p = 0.02), y una significativa disminución en la tasa de recién nacido en casa (38.6 vs 23.6; p = 0.04), además de menor tasa de embriones de excelente calidad, sin afectar la tasa de fertilización con ICSI. CONCLUSIONES: los resultados demuestran que aun cuando las tasas de embarazo e implantación son elevadas en pacientes con sobrepeso y obesidad con técnicas de alta complejidad o ICSI, el desarrollo embrionario, la tasa de aborto y tasa de recién nacido en casa se ven afectados por el incremento del IMC paterno.

Abstract OBJECTIVE: The aim of the present study was primarily to determine the influence of paternal obesity on pregnancy, implantation, abortion and take home baby rates in patients undergoing in vitro fertilization with ICSI. Secondly, to determine the impact of paternal body mass index (BMI) on semen parameters, fertilization rate and embryo quality. MATERIALS AND METHODS: Retrospective, comparative and observational study. 352 cycles of ICSI with embryo transfer were included. They were divided in three groups based on male BMI: Normal (18.5-24.99 kg/m2), overweight (25-29.99 kg/m2), and obesity (≥30 kg/m2). RESULTS: Male obesity was associated with a significant increased miscarriage rate when compared with the normal group (39.0 vs 12.9, p = 0.02) and a significantly lower take home baby rate (23.6 vs 38.6, p = 0.04). We also found a lower rate of top quality embryos, without affecting fertilization with ICSI. CONCLUSION: Our results demonstrate that even though pregnancy and implantation rates are elevated when ICSI is performed in overweight and obese patients, embryo development, miscarriage and take home baby rates are affected with increased paternal BMI.

Parathyroid hormone 2 receptor and its endogenous ligand tuberoinfundibular peptide of 39 residues are concentrated in endocrine, viscerosensory and auditory brain regions in macaque and human.

Parathyroid hormone receptor 2 (PTH2R) and its ligand, tuberoinfundibular peptide of 39 residues (TIP39) constitute a neuromodulator system implicated in endocrine and nociceptive regulation. We now describe the presence and distribution of the PTH2R and TIP39 in the brain of primates using a range of tissues and ages from macaque and human brain. In situ hybridization histochemistry of TIP39 mRNA, studied in young macaque brain, due to its possible decline beyond late postnatal ages, was present only in the thalamic subparafascicular area and the pontine medial paralemniscal nucleus. In contrast, in situ hybridization histochemistry in macaque identified high levels of PTH2R expression in the central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular and periventricular nuclei, medial geniculate, and the pontine tegmentum. PTH2R mRNA was also detected in several human brain areas by RT-PCR. The distribution of PTH2R-immunoreactive fibers in human, determined by immunocytochemistry, was similar to that in rodents, including dense fiber networks in the medial preoptic area, hypothalamic paraventricular, periventricular and infundibular (arcuate) nuclei, lateral hypothalamic area, median eminence, thalamic paraventricular nucleus, periaqueductal gray, lateral parabrachial nucleus, nucleus of the solitary tract, sensory trigeminal nuclei, medullary dorsal reticular nucleus, and dorsal horn of the spinal cord. Co-localization suggested that PTH2R fibers are glutamatergic, and that TIP39 may directly influence hypophysiotropic somatostatin containing and indirectly influence corticotropin releasing-hormone containing neurons. The results demonstrate that TIP39 and the PTH2R are expressed in the brain of primates in locations that suggest involvement in regulation of fear, anxiety, reproductive behaviors, release of pituitary hormones, and nociception.

Regional distribution and effects of postmortal delay on endocannabinoid content of the human brain.

Tissue levels of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have been determined in 16 regions and nuclei from human brains, using liquid chromatography/in-line mass spectrometry. Measurements in brain samples stored at -80 degrees C for 2 months to 13 years indicated that endocannabinoids were stable under such conditions. In contrast, the postmortal delay had a strong effect on brain endocannabinoid levels, as documented in brain samples microdissected and frozen 1-6 h postmortem, and in neurosurgical samples 0, 5, 30, 60, 180 and 360 min after their removal from the brain. The tissue levels of AEA increased continuously and in a region-dependent manner from 1 h after death, increasing about sevenfold by 6 h postmortem. In contrast, concentrations of 2-AG, which were 10-100 times higher in human brain regions than those of AEA, rapidly declined: within the first hour, 2-AG levels dropped to 25-35% of the initial ('0 min') value, thereafter they remained relatively stable. As analyzed in samples removed 1-1.5 h postmortem, AEA levels ranged from a high of 96.3 fmol/mg tissue in the nucleus accumbens to a low of 25.0 fmol/mg in the cerebellum. 2-AG levels varied eightfold, from 8.6 pmol/mg in the lateral hypothalamus to 1.1 pmol/mg in the nucleus accumbens. Relative levels of AEA and 2-AG varied from region to region, with the 2-AG:AEA ratio being high in the sensory spinal trigeminal nucleus (140:1), the spinal dorsal horn (136:1) and the lateral hypothalamus (98:1) and low in the nucleus accumbens (16:1) and the striatum (31:1). The results highlight the pitfall of analyzing endocannabinoid content in brain samples of variable postmortal delay, and document differential distribution of the two main endocannabinoids in the human brain.

Imaging of iron oxide nanoparticles by MR and light microscopy in patients with malignant brain tumours.

OBJECTIVE: Ferumoxtran-10 (Combidex), a dextran-coated iron oxide nanoparticle, provides enhancement of intracranial tumours by magnetic resonance (MR) for more than 24 h and can be imaged histologically by iron staining. Our goal was to compare ferumoxtran imaging and histochemistry vs. gadolinium enhancement in malignant brain tumours on preoperative and postoperative MR. METHODS: Seven patients with primary and metastatic malignant tumours underwent MR imaging with gadolinium and ferumoxtran both pre- and postoperatively. Normalized signal intensities on the ferumoxtran-enhanced scans were determined in representative regions of interest. Resected tissue from six ferumoxtran patients and from three patients who did not receive ferumoxtran was assessed for localization of iron in tumour and reactive brain. RESULTS: All malignant tumours (all of which enhanced by gadolinium MR) showed ferumoxtran accumulation with T1 and T2 signal changes, even using a 0.15 T intraoperative MR unit in one patient. Iron staining was predominantly in reactive cells (reactive astrocytes and macrophages) and not tumour cells. In five of the seven patients, including two patients who showed additional lesions, areas enhancing with ferumoxtran but not with gadolinium were observed. Comparison of the pre- and postoperative MR revealed residual ferumoxtran-enhancing areas in four of seven cases. CONCLUSION: In malignant tumours, ferumoxtran may show areas of enhancement, even with a 0.15 T intraoperative MR, that do not enhance with gadolinium. Ferumoxtran-enhancing lesions have persistent increased T1 signal intensity for 2-5 days, which may provide advantages over gadolinium for postoperative imaging. Histochemistry for iron shows uptake of ferumoxtran in reactive cells (astrocytes and macrophages) rather than tumour cells.

Early experiences with image-guided transoral surgery for the pathologies of the upper cervical spine.

STUDY DESIGN: Technical note. OBJECTIVES: Three years of convincing experience with cranial neuronavigation suggested the application of the cranial software and registration method for the transoral access to the C1-C2 vertebrae. BACKGROUND DATA: The C1-C2 vertebrae are located in close vicinity to the cranial base. If the intersegmental movements of the C0-C1/C1-C2 segments are prevented with HALO fixation, the upper cervical spine can be considered as a caudal part of the skull base and included in the extended navigation space of the skull. METHODS: Three patients were selected for navigation-assisted transoral odontoidectomy. Before surgery the patients were fixed and scanned in a HALO device. The fiducials were attached supraorbitally and to both mastoids, determining a wide registration area and allowing the caudal extension of the navigation space. The BrainLAB VectorVision navigation system was used in cranial mode during the operations. RESULTS: Neuronavigation and fluoroscopy-controlled transoral surgery were performed with success in all three cases. The registration accuracy was 1.5, 2.7, and 3.1 mm. CONCLUSION: Image guidance during transoral exposure of the upper cervical spine offered excellent three-dimensional guidance on the ventral surface of the craniocervical junction, allowing a safer, more controlled surgery. As the targets of the transoral spinal surgery are fixed bony and ligamentous structures, no shifting occurs and continuous high navigation accuracy can be achieved. The use of the navigation can reduce the significance of the intraoperative fluoroscopy, diminishing the radiograph load of the patient and the operating room team.

[Neuronavigation in pediatric neurosurgery]. / Neuronavigáció a gyermek-idegsebészetben.

The precise orientation in the intracranial space is essential for the minimal invasive neurosurgical interventions. The CT and MR based neuro-navigation permits small, targeted exposures on the skull, and intraoperatively gives exact graphic-interactive guidance to the targeted intracranial lesions. The use of neuro-navigation can shorten the time of surgery and diminish the surgical mortality and morbidity. The favourable experiences of the first 21 neuro-navigation aided operations in pediatric patients performed in the National Institute of Neurosurgery (Budapest, Hungary) with the Vector Vision Neuro-navigation System (BrainLAB Gmbh, Germany) are discussed.

[Chiari I. malformation, a new childhood disease?]. / Chiari I. malformáció, egy "új" gyermekbetegség?

From July 1, 1994 to July 1, 1997 a total of 17 children with Chiari I malformation diagnosed by MRI was seen in our hospital. Six of them underwent craniocervical decompression and tonsilar resection. The major complaints of all these children were gait ataxia and persisting (longer than 6 month) headache, and one child had acutely presenting vertigo and problem of swallowing. The MRI showed that in all cases the tonsils were displaced below the level of CI. Hydrocephalus or syrinx was not seen. At surgery the tonsils were resected. All the patients can be considered cured and symptom free at an average follow up of 13 month.

Capacitative Ca2+ influx in adrenal glomerulosa cells: possible role in angiotensin II response.

We examined the effect of the depletion of intracellular Ca2+ stores on Ca2+ influx in rat glomerulosa cells. Depletion of intracellular Ca2+ stores was achieved by inhibiting sarco/endoplasmic reticulumtype Ca(2+)-ATPase with thapsigargin or 2,5,di-(t-butyl)-1,4-benzohydroquinone (t-BHQ). Both inhibitors induced a sustained rise in cytoplasmic Ca2+ concentration. The initial rise was observed also in Ca(2+)-free medium, while the sustained phase disappeared, indicating that the latter requires Ca2+ influx. In Ca(2+)-free medium, the readdition of Ca2+ induced a steeper and higher rise in intracellular Ca2+ concentration in thapsigargin-treated cells than in controls, supporting the role of Ca2+ influx. In normal medium, the addition of Cd2+ (80 microM) evoked an immediate inhibition of the sustained phase of thapsigargin response. The response to thapsigargin was insensitive to nifedipine. Thapsigargin failed to enhance Mn2+ quenching of fura 2. Our results provide evidence for the existence of capacitative Ca2+ influx in rat glomerulosa cells and indicate that dihydropyridine-sensitive Ca2+ channels do not participate in capacitative Ca2+ entry. High concentrations of thapsigargin and t-BHQ, similar to the reported effects of angiotensin II and vasopressin, inhibited K(+)-induced Ca2+ signals. These effects appear, however, to be independent of the depletion of internal Ca2+ stores.

Dihydropyridine-sensitive initial component of the ANG II-induced Ca2+ response in rat adrenal glomerulosa cells.

The Ca2+ signal induced by an increase in extracellular K+ concentration from 3.6 to 5.6 mM or angiotensin II (ANG II) was inhibited by the dihydropyridine (DHP) Ca2+ channel blocker, nifedipine, and enhanced by the DHP Ca2+ channel agonist, BAY K 8644. The DHP sensitivity of the ANG II-induced Ca2+ response was already detectable during the peak phase, suggesting that the DHP receptor plays an important role during the initial phase of ANG II stimulation. K+ and ANG II stimulated a nifedipine-sensitive Mn2+ influx pathway, further promoting the role of a DHP receptor in their mechanism of action. Fluorescent membrane potential measurements showed that, in contrast to the rapid depolarization induced by K+, the ANG II-induced depolarization had a lag time of > 30 s. The slow kinetics of depolarization compared with the immediate effect of ANG II on Mn2+ influx and the DHP sensitivity of the initial Ca2+ peak indicates that ANG II initiates the activation of the DHP-sensitive Ca2+ channel by a mechanism other than depolarization.

Angiotensin II exerts its effect on aldosterone production and potassium permeability through receptor subtype AT1 in rat adrenal glomerulosa cells.

The stimulatory effect of angiotensin II (AT) on the accumulation of inositol phosphates and on aldosterone production is abolished by the AT1 selective receptor antagonist DuP753 while PD123177, an AT2 antagonist, is ineffective. Similarly, a depolarizing effect of AT (inhibition of K+/86Rb efflux) is prevented by DuP753. While mediators derived from phospholipase C activation have a central role in the stimulation of aldosterone production by AT, the effect of AT on K+ permeability is mimicked neither by elevation of cytoplasmic [Ca2+] by ionomycin nor by kinase C activation with phorbol ester. Our results suggest that AT stimulates phospholipase C and the subsequent steroid production by glomerulosa cells through AT1 receptors. In addition some events induced by the activation of AT1 receptors may not be mediated by the activation of phospholipase C.