Prepubertal exposure to low doses of sodium arsenite impairs spermatogenesis and epididymal histophysiology in rats

For the first time, juvenile toxicity of inorganic arsenic (As) was investigated in male rats, focusing on reproductive effects. As is a metalloid naturally occurring in the environment, being the inorganic forms the most toxics. Contaminated drinking water and agricultural products are the main prospectors of intoxication for general population. In the present study, Wistar male rats (21 days old) were distributed into three groups (n = 10/group): control (received vehicle‐filtered drinking water), As1 (received AsNaO₂ at 0.01 mg L^?1) and As2 (received AsNaO₂ at 10 mg L^?1). The animals were euthanized on PND 53. Testicular damages increased in As1 and As2 compared to control (ie, presence of vacuolization, acidophilic cells, and epithelium degeneration). Testicular interstitium of As1 and As2 presented fluid's increase and intense inflammatory infiltration. In the epididymis there was reduction of sperm amount in the lumen, besides epithelium areas presenting cribriform aspect in As1 and As2, exfoliation of cells in the light (in As1) and vacuoles (in As2). In epididymis interstitium, inflammatory infiltrates were observed in initial segment of As1 and As2. AsNaO₂ changed immunolabeling pattern for androgen receptor in epididymis of As2, although serum testosterone levels was statistically comparable to control. Mass spectrometry revealed higher As concentrations in testis and epididymis of As2 compared to As1 and Control. These results indicate compromise of spermatogenesis and epididymal histophysiology in AsNaO₂‐treated animals, possibly impairing sperm quality and fertility in long‐term, even at low levels of exposure. Investigations about the reversibility of reproductive damages are necessary to better understand the mechanisms of As reproductive toxicity.     

Pembrolizumab with R-CHOP in previously untreated diffuse large B-cell lymphoma: potential for biomarker driven therapy

Tumor programmed death-ligand 1 (PD-L1) expression in diffuse large B-cell lymphoma (DLBCL) is associated with inferior outcomes. The first-line immunologically-replete setting may be an opportune time for PD-1 inhibition. We evaluated pembrolizumab in combination with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) in untreated patients with DLBCL. Eligible patients were age 18 or older, had adequate organ function, and had DLBCL requiring full-course therapy. Patients received pembrolizumab 200 mg/cycle with R-CHOP, primarily to assess toxicity. Response assessment utilized standard criteria, and PD-L1 staining was performed at a validated central laboratory. Among 30 patients, toxicity was comparable to standard R-CHOP but with two grade ≥3 immune related adverse events (rash, pneumonitis). The overall and complete response rate was 90% and 77%. With 25·5 months of median follow-up, 2-year progression-free survival (PFS) is 83%. PD-L1 expression was associated with non-GCB subtype, and improved PFS and survival. Pembrolizumab can safely be added to R-CHOP, and is associated with a high CR rate and 2-year PFS. Improved PFS with PR-CHOP in PD-L1 expressing tumors contradicts historical data in R-CHOP treated patients, supporting evaluation of PD-L1 as a biomarker to identify DLBCL patients who may benefit from this first-line strategy.     

Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms

The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases’ homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.Since its discovery, c-Src (1) has been subject to intensive research into its cellular functions and regulation. Whereas c-Src is the best-studied protooncogene, less is known about the other, closely related Src family kinase (SFK) members. Their high degree of similarity in structure and regulation suggests that SFKs can partially compensate for each other in vivo. Indeed, knockout studies have shown that only mice deficient in all three genes (src, yes, and fyn) show embryonic lethality (2). Early studies demonstrated that disruption of Src or Fyn genes individually resulted only in subtle changes in function of a few cell types (e.g., osteoclasts for src^−/−, and T cells for fyn^−/−) (3, 4). Roche et al. provided strong evidence that Src, Yes, and Fyn substitute for each other during cell cycle progression (5). These studies suggested that there is a high degree of functional redundancy among Src family kinases.Nonetheless, emerging evidence indicates that Src and Fyn regulate distinct processes in the same cell. Down-regulation of Fyn expression enhances VEGF-stimulated migration of endothelial cells, whereas down-regulation of Src does not (6). Differences in the transforming capacity of SFKs are thought to depend on their affinity for cholesterol-enriched membrane microdomains, which is determined in part by their N-terminal lipid modifications (7, 8). Src has higher tumorigenic potential than Fyn in prostate epithelium, and this is differently affected by alterations in N-terminal palmitoylation (9). Previous studies have shown that Src localizes to perinuclear endosomal compartments and translocates to the plasma membrane upon activation (10–12), whereas Fyn localizes to the plasma membrane regardless of its activity (13, 14). Although these studies suggest that localization is important in differentiating the actions of the two kinases, they do not identify specific roles associated with particular subcellular locations.Various techniques have been applied to elucidate the differences in signaling specificity of SFKs. Kinase–substrate interactions have been examined using purified substrates (15). Mutated kinases with selectivity for radiolabeled ATP analogs have identified directly phosphorylated substrates of Src (16). These methods were restricted to cell lysates or purified proteins, and so were unable to address the role of cellular localization in substrate specificity.To dissect the unique role of different SFK isoforms (2–4, 17, 18) in living cells, we engineered regulatable analogs of Fyn, Yes, and LynA kinases using our rapamycin-regulated activation (RapR) strategy, which has been developed using Src as a prototype (19, 20). Insertable FKBP12 (iFKBP, a truncated form of FKBP) was inserted into the catalytic domain of each SFK, which abolished their kinase activity. Activity was rescued by treating cells with rapamycin in the presence of the FKBP12-rapamycin binding domain (FRB) (Fig. 1A). Molecular dynamics studies have indicated that heterodimerization of the inserted iFKBP with FRB likely reduces the conformational mobility of the kinase G loop, restoring ATP binding (3, 21).Open in a separate windowFig. 1.Design of RapR kinases. (A) Schematic representation of the approach used to regulate catalytic activity of SFKs. The insertion of iFKBP at a highly conserved site in the catalytic domain of each kinase resulted in loss of kinase activity. Catalytic activity was restored by rapamycin, which induced binding of iFKBP and coexpressed FRB. (B) Sequence alignment of SFKs shows that there is a well-defined loop where iFKBP is inserted (blue). It is linked to the G loop (red) through a β-sheet in each SFK.These analogs enabled activation of each isoform specifically, within minutes, resulting in clear phenotypic differences. Unlike genetic modifications of cell populations, there was little time for the cell to compensate for kinase activation before observation. The induced cell behaviors occurred in a succession of stages, associated with changes in the subcellular distribution of each kinase. We focused on Src and Fyn, developing quantitative tools to carefully characterize the kinetics of induced behaviors and associated localization dynamics. Our results indicated that Src’s unique ability to induce polarized movement shortly after kinase activation results from its localization in a perinuclear compartment, where it phosphorylates substrates that traffic on microtubules to the cell perimeter. Both the localization dynamics and phenotype differences between Src and Fyn were dependent on N-terminal lipid modifications, and not on SH2 and SH3 domain interactions.     

Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA–like structural element in the ribosomal decoding center

In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA–like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.Protein synthesis relies on precise placement of the ORF within the ribosome during translation initiation. Canonical initiation in eukaryotes depends on a 7-methylguanosine cap at the 5′ terminus of mRNA and on extraribosomal initiation factors (1). Following a stepwise assembly, the 80S initiation complex contains the initiator methionyl-tRNA^Met and the AUG start codon in the peptidyl (P) site. Some viral mRNAs use alternative cap-independent mechanisms that involve internal ribosome entry sites (IRESs) (2). IRESs are folded RNA structures in the 5′ UTR that promote formation of the 80S initiation complex in the presence of fewer initiation factors than required for cap-dependent initiation (3).The ribosomal P-site employment in initiation is thought to be ubiquitous for cap-dependent and IRES-dependent translation (4). Of the four groups of known IRESs, the most streamlined mechanism has been described for IRESs from the Dicistroviridae family of arthropod-infecting viruses. The Dicistroviridae genome has two ORFs separated by an intergenic region (IGR). The IGR contains an IRES that drives translation of the second ORF without the aid of initiation factors (4). Based on phylogenetic analyses of the structural polyprotein ORF2 and IGR IRES, the Dicistroviridae viruses are divided into the genus Cripavirus [including cricket paralysis virus (CrPV), Drosophila C virus, and Plautia stali intestine virus (PSIV)] and Aparavirus [including Taura syndrome virus (TSV), Kashmir bee virus, and acute bee paralysis virus] (4). Biochemical studies suggest that despite differences between some secondary structure elements of Cripavirus and Aparavirus IRESs, the molecular mechanisms of translation initiation are similar (5). IGR IRESs can initiate translation on ribosomes from yeast, wheat, human, and other eukaryotic organisms, indicating that the molecular mechanism of IGR IRES-driven initiation in eukaryotes is conserved and is not species-specific (6–10).In contrast to cap-dependent initiation and initiation from other groups of IRESs, translation from IGR IRESs starts from a non-AUG start codon and does not involve initiator methionyl-tRNA^Met. Translation from the majority of IGR IRESs, including the CrPV and TSV IRESs, initiates with alanyl-tRNA^Ala (7, 9, 10). IGR IRESs contain three pseudoknots. At the 5′ region, pseudoknot II (PKII) and PKIII, which are critical for formation of the 40S•IRES and 80S•IRES complexes (8, 9), form a double-nested pseudoknot (11, 12). PKI, located immediately upstream of the start codon, forms a separate domain at the 3′ region of the IRES. This domain is essential for the function of IGR IRESs (13). The crystal structure of an isolated PKI of the CrPV IGR IRES shows that the pseudoknot resembles the anticodon stem loop of tRNA bound to a cognate mRNA codon (14, 15). Isolated PKI of CrPV and PSIV IRESs binds to the P site of the bacterial 70S ribosome, demonstrating that PKI has an affinity to the highly conserved tRNA binding sites on the ribosome (16).The molecular mechanism of translation initiation by IGR IRESs is not fully understood. The current view is that upon formation of the 80S•IRES complex, the PKI is placed in the P site on the small subunit, in a manner mimicking the initiator methionyl-tRNA^Met and the AUG codon (6–8, 10, 17). In this mode, the IRES would position the ORF on the ribosome by presenting the initiating alanine codon in the A (aminoacyl) site. The structural studies of the mechanism, however, have been inconclusive. Previous electron cryomicroscopy (cryo-EM) reconstruction of the CrPV IRES bound to human ribosomal 40S subunit revealed the IRES density spanning from the A site to beyond the exit (E) site (18). The interpretation of the 40S•IRES map favored a model in which PKI interacts with the P-site region (18), although the 20-Å map lacked detailed features in this location. Cryo-EM studies of the 80S ribosome-bound CrPV IRES suggested that upon subunit joining and 80S•IRES complex formation, the IRES may rearrange relative to the 40S subunit (18), and/or reposition PKI in the vicinity of the A and P sites, yet potentially present the downstream alanine codon in the A site (19). The density for the PKI region in these 20-Å and 7.3-Å cryo-EM reconstructions was, however, significantly weaker than that for the rest of the CrPV IRES (18, 19), and it remained unclear how the IGR IRESs initiate translation by accurately positioning the ORF on the 80S ribosome. We report here ∼6-Å cryo-EM structures of the initiation 80S complex bound with an intergenic IRES, which provide structural insights into the mechanism of IGR IRES-driven initiation.     

Vanadium doped few-layer ultrathin MoS2 nanosheets on reduced graphene oxide for high-performance hydrogen evolution reaction

In this paper, we demonstrate a facile solvothermal synthesis of a vanadium(v) doped MoS₂-rGO nanocomposites for highly efficient electrochemical hydrogen evolution reaction (HER) at room temperature. The surface morphology, crystallinity and elemental composition of the as-synthesized material have been thoroughly analyzed. Its fascinating morphology propelled us to investigate the electrochemical performance towards the HER. The results show that it exhibits excellent catalytic activity with a low onset potential of 153 mV versus reversible hydrogen electrode (RHE), a small Tafel slope of 71 mV dec⁻¹, and good stability over 1000 cycles under acidic conditions. The polarization curve after the 1000^th cycle suggests there has been a decrement of less than 5% in current density with a minor change in onset potential. The synergistic effects of V-doping at S site in MoS₂ NSs leading to multiple active sites and effective electron transport route provided by the conductive rGO contribute to the high activity for the hydrogen evolution reaction. The development of a high-performance catalyst may encourage the effective application of the as-synthesized V-doped MoS₂-rGO as a promising electrocatalyst for hydrogen production.

In this paper, we demonstrate a facile solvothermal synthesis of a vanadium(v) doped MoS₂-rGO nanocomposites for highly efficient electrochemical hydrogen evolution reaction (HER) at room temperature.     

Deep neural network for automatic characterization of lesions on 68Ga-PSMA-11 PET/CT

European Journal of Nuclear Medicine and Molecular Imaging - This study proposes an automated prostate cancer (PC) lesion characterization method based on the deep neural network to determine tumor...     

Variability of magnetoencephalographic sensor sensitivity measures as a function of age,brain volume and cortical area

Objective

To assess the feasibility and appropriateness of magnetoencephalography (MEG) for both adult and pediatric studies, as well as for the developmental comparison of these factors across a wide range of ages.

Methods

For 45 subjects with ages from 1 to 24 years (infants, toddlers, school-age children and young adults), lead fields (LFs) of MEG sensors are computed using anatomically realistic boundary element models (BEMs) and individually-reconstructed cortical surfaces. Novel metrics are introduced to quantify MEG sensor focality.

Results

The variability of MEG focality is graphed as a function of brain volume and cortical area. Statistically significant differences in total cerebral volume, cortical area, MEG global sensitivity and LF focality are found between age groups.

Conclusions

Because MEG focality and sensitivity differ substantially across the age groups studied, the cortical LF maps explored here can provide important insights for the examination and interpretation of MEG signals from early childhood to young adulthood.

Significance

This is the first study to (1) investigate the relationship between MEG cortical LFs and brain volume as well as cortical area across development, and (2) compare LFs between subjects with different head sizes using detailed cortical reconstructions.     

Association of sleep apnea with clinically silent microvascular brain tissue changes in acute cerebral ischemia

The aim of this study was to determine the importance of sleep apnea in relation to clinically silent microvascular brain tissue changes in patients with acute cerebral ischemia. Patients with acute cerebral ischemia prospectively underwent nocturnal respiratory polygraphy within 5 days from symptom-onset. Sleep apnea was defined as apnea–hypopnea-index (AHI) ≥5/h. Experienced readers blinded to clinical and sleep-related data reviewed brain computed tomography and magnetic resonance imaging scans for leukoaraiosis and chronic lacunar infarctions. Ischemic lesions were considered clinically silent when patients did not recall associated stroke-like symptoms. Functional outcome was assessed with modified Rankin Scale at discharge, 6 and 12 months. Fifty-one of 56 (91 %) patients had sleep apnea of any degree. Patients with moderate-to-severe leukoaraiosis (Wahlund score ≥5) were found to have higher mean AHI than those with none or mild leukoaraiosis (34.4 vs. 12.8/h, p < 0.001). Moderate-to-severe sleep apnea (AHI ≥15/h) was found to be an independent predictor of moderate-to-severe leukoaraiosis (adjusted OR 6.03, 95 % CI 1.76–20.6, p = 0.0042) and of moderate-to-severe leukoaraiosis associated with clinically silent chronic lacunar infarctions (adjusted OR 10.5, 95 % CI 2.19–50.6, p = 0.003). The higher the Wahlund score and the AHI, the more likely unfavorable functional outcome resulted over time (p = 0.0373). In acute cerebral ischemia, sleep apnea is associated with clinically silent microvascular brain tissue changes and may negatively influence functional outcome. Routine sleep apnea screening and further investigation of possible long-term effects of non-invasive ventilatory treatment of sleep apnea appear warranted in this at-risk population.     

Regional neurovascular coupling and cognitive performance in those with low blood pressure secondary to high-level spinal cord injury: improved by alpha-1 agonist midodrine hydrochloride

Individuals with high-level spinal cord injury (SCI) experience low blood pressure (BP) and cognitive impairments. Such dysfunction may be mediated in part by impaired neurovascular coupling (NVC) (i.e., cerebral blood flow responses to neurologic demand). Ten individuals with SCI >T6 spinal segment, and 10 age- and sex-matched controls were assessed for beat-by-beat BP, as well as middle and posterior cerebral artery blood flow velocity (MCAv, PCAv) in response to a NVC test. Tests were repeated in SCI after 10 mg midodrine (alpha₁-agonist). Verbal fluency was measured before and after midodrine in SCI, and in the control group as an index of cognitive function. At rest, mean BP was lower in SCI (70±10 versus 92±14 mm Hg; P<0.05); however, PCAv conductance was higher (0.56±0.13 versus 0.39±0.15 cm/second/mm Hg; P<0.05). Controls exhibited a 20% increase in PCAv during cognition; however, the response in SCI was completely absent (P<0.01). When BP was increased with midodrine, NVC was improved 70% in SCI, which was reflected by a 13% improved cognitive function (P<0.05). Improvements in BP were related to improved cognitive function in those with SCI (r²=0.52; P<0.05). Impaired NVC, secondary to low BP, may partially mediate reduced cognitive function in individuals with high-level SCI.     

The True Incidence of Gastric GIST—a Study Based on Morbidly Obese Patients Undergoing Sleeve Gastrectomy

Background

Gastrointestinal stromal tumor (GIST) is a rare neoplasm of the alimentary tract. Previous reports described an incidence of 1 per 100,000. Laparoscopic sleeve gastrectomy (LSG) provides pathological specimens of the majority of the stomach. We examined the pathology from LSG and the incidence and location of GIST. The aim of this study was to study the incidence of asymptomatic GISTs found during LSG at our institution.

Methods

A search was conducted in a prospectively maintained bariatric registry. Data collected included the following: gender, age, body mass index (BMI), and concomitant hypertension or diabetes mellitus. Histopathology reports were reviewed for incidental GIST. We compared the patients with incidental GIST to the rest of the cohort.

Results

Pathology reports of 827 patients that underwent LSG between 2007 and 2014 were reviewed. Five patients had GIST in the resected stomach, an incidence of 0.6 %. The group of patients with GIST had lower BMI and older age compared to the remaining 822 patients. All tumors were located close to the lesser curvature.

Conclusions

The incidence of GIST found in this cohort is significantly higher than previously reported. This may be due to an association between these tumors and obesity or because asymptomatic GISTs are underdiagnosed in the general population. These tumors are particularly common in older patients and special attention must be given when performing LSG on this subpopulation. The stomach should be inspected thoroughly before resection. A tumor on the lesser curvature may necessitate changing the surgical plan or aborting the procedure.