Search

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

NPT520-34 is a clinical stage, small molecule being developed for the treatment of Parkinson's disease and other neurodegenerative disorders. The therapeutic potential of NPT520-34 was first suggested by findings from cell-based assays of alpha-synuclein clearance. As reported here, NPT520-34 was subsequently evaluated for therapeutically relevant actions in a transgenic animal model of Parkinson's disease that overexpresses human alpha-synuclein and in an acute lipopolysaccharide-challenge model using wild-type mice. Daily administration of NPT520-34 to mThy1-alpha-synuclein (Line 61) transgenic mice for 1 or 3 months resulted in reduced alpha-synuclein pathology, reduced expression of markers of neuroinflammation, and improvements in multiple indices of motor function. In a lipopolysaccharide-challenge model using wild-type mice, a single dose of NPT520-34 reduced lipopolysaccharide-evoked increases in the expression of several pro-inflammatory cytokines in plasma. These findings demonstrate the beneficial effects of NPT520-34 on both inflammation and protein-pathology end points, with consequent improvements in motor function in an animal model of Parkinson's disease. These findings further indicate that NPT520-34 may have two complementary actions: (i) to increase the clearance of neurotoxic protein aggregates; and (ii) to directly attenuate inflammation. NPT520-34 treatment may thereby address two of the predominate underlying pathophysiological aspects of neurodegenerative disorders such as Parkinson's disease., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The perception of robots as mindful enriches how humans relate to them. Given that congruence in perceived representations of the world enable humans to experience commonality in mental states (a shared reality), we propose that congruence between humans, and robots will encourage humans to attribute humanlike mental capacities to robots. To investigate this, we assessed the mental perceptions of a robot in a visual imagination task using Gray et al. mind perception scale, which evaluates experience (capacity to feel), and agency (capacity to plan and do). For each ambiguous picture in the designed task, humans, and a robot imagined an animal. The task was performed under six conditions (2 × 3: Lead/Follow for Low/Medium/High). In the Lead condition, the robot records its perceived animal first; in the Follow condition, the robot records after the human participant. The experiment had three different degrees of congruence: Low (0%), Medium (60%), and High (100%). The results showed that perceived experiences were higher in the Lead condition, suggesting that the robot is perceived to be empathetic. It is probable that the Follow condition was perceived as mimicry rather than shared reality. Therefore, the order of response may have played an important role in commonality in mental states. No differences were observed in the perceived agency across all conditions. These results suggest that the order of response affects how humans perceive the minds of robots. Additionally, we assessed a post-task questionnaire to evaluate the interpersonal closeness that the humans felt toward the android. The main effect was observed in the degrees of congruence. This result is in line with those of previous studies that report relationships across sharing of similarities and friendliness., (Copyright © 2019 Tatsukawa, Takahashi, Yoshikawa and Ishiguro.)

As the result of recent progress in technology of communication robot, robots are becoming an important social partner for humans. Behavioral synchrony is understood as an important factor in establishing good human-robot relationships. In this study, we hypothesized that biasing a human's attitude toward a robot changes the degree of synchrony between human and robot. We first examined whether eyeblinks were synchronized between a human and an android in face-to-face interaction and found that human listeners' eyeblinks were entrained to android speakers' eyeblinks. This eyeblink synchrony disappeared when the android speaker spoke while looking away from the human listeners but was enhanced when the human participants listened to the speaking android while touching the android's hand. These results suggest that eyeblink synchrony reflects a qualitative state in human-robot interactions.

Current flow-based blood counting devices require significant medical infrastructure and are not appropriate for field use. In this article we report on the development of a sample preparation, measurement, and analysis method that permits automated and accurate counting of red blood cells (RBCs), white blood cells (WBCs), and platelets, as well as allowing a 3-part differential of the WBCs to be performed on extremely small volumes of whole blood. This method is compatible with portable instrumentation that can be deployed in the field. The method consists of serially diluting blood samples first with sodium dodecyl sulfate dissolved in phosphate buffered saline, then in acridine orange dissolved in phosphate buffered saline, followed by fluorescence and dark field imaging with low magnification objectives. Image analysis is performed to extract cell counts and differentials. We performed a paired analysis of 20 volunteers with complete blood count values both within and beyond the normal reference range using a commercial automated hematology analyzer and the image-based method, with the new method achieving accuracies comparable to that of the commercial system. Because the sample preparation and imaging are simple and inexpensive to implement, this method has applications for pediatrics, clinician offices, and global health in regions that do not have access to central hematology laboratories.

A 58-year-old woman presented to her physician with rectal bleeding and intermittent diarrhea. Optical colonoscopy revealed a bulky tumor which was diagnosed as rectal cancer. She was referred to our institution for further evaluation and treatment. Slim optical colonoscopy showed an obstructive cancer in the rectosigmoid junction and no information of the proximal side of the obstruction. The patient then underwent computed tomographic (CT) colonography for further evaluation of the proximal side. Three-dimensional endoluminal 'fly-through' images revealed another protruded lesion in the proximal side of the obstruction. Diagnosis of synchronous double cancer was made by CT colonography. This CT data was not only used to create three-dimensional images but also to decide on a preoperative clinical staging. Laparoscopy-assisted high anterior resection was performed and T3 rectal cancer and T1 sigmoid colon cancer were confirmed in the resected specimen. Follow-up optical colonoscopy revealed no other tumors. CT colonography has recently become a popular preoperative examination tool with significant improvement in quality of image due to a rapid progress in computer technology. CT colonography correctly showed synchronous double cancer in our case and provided crucial information for planning surgery. We recommend that CT colonography should be used for evaluating the proximal side of obstructive colorectal cancer.

Background: In colorectal cancer (CRC) surgery, precise tumor localization is important for oncologically correct surgery and adequate tumor and lymph node resection margins. During laparoscopic surgery it is difficult to localize early CRC. The aim of this study was to compare the usefulness of two tumor localization techniques; intraoperative fluoroscopy and intraoperative laparoscopic ultrasonography., Methods: Seventeen patients with CRC necessitating preoperative marking were alternately allocated to either the fluoroscopy (F) group (n = 8) or the laparoscopic ultrasonography (LU) group (n = 9). A three-step technique was used. At first lesions were localized preoperatively by metallic clips that were colonoscopically applied proximally and distally to the tumor site. Second, computed tomography (CT) colonography was taken to obtain preoperative staging. The location of the metallic clips was confirmed by CT colonography, preoperatively. Third, in the F group, intraoperative fluoroscopy was performed to localize the applied clips. In the LU group, the applied clips were detected from the serosal aspect of the colon using intraoperative laparoscopic ultrasonography., Results: In all patients, colonoscopic metallic clips were successfully applied and preoperative CT colonography correctly detected the location of the tumor. Marking sites were detected precisely using intraoperative fluoroscopy or intraoperative laparoscopic ultrasonography in all cases, without complications. The mean detection time was 15.8 minutes in the F group and 7.0 minutes in the LU group (p = 0.005). In the LU group, two cases were technically difficult because of interruption of the ultrasound by intestinal air., Conclusions: Both intraoperative fluoroscopy and intraoperative laparoscopic ultrasonography are safe and accurate techniques for intraoperative localization of early CRC. With regard to detection time, intraoperative laparoscopic ultrasonography is superior to intraoperative fluoroscopy. However, when there is a massive amount of intestinal air, intraoperative laparoscopic ultrasonography is cumbersome in localizing the lesion. Computed tomography colonography is useful for preoperative tumor localization and might be effective for shortening detection time during surgery.

Background and Purpose: Stroke produces diaschisis in adjacent and connected regions. The sequential changes in diaschisis over time and the relationship of regions of diaschisis to functional cortical areas and regions of poststroke neuroplasticity have not been determined., Methods: Small cortical strokes were produced in the barrel cortex of rats. Relative glucose metabolism was determined in vivo over time with [18F]fluorodeoxyglucose small-animal positron emission tomography. Cerebral blood flow was measured with [14C]iodoantipyrine. Regions of hypometabolism and hypoperfusion were compared with histological damage in the same animals., Results: Small cortical strokes produce an initial network of hypometabolism in a broad region of cortex adjacent to the stroke and in the striatum and thalamus on day 1. Cerebral blood flow is diminished only immediately around the cortical infarct on day 1. A substantial area of cortex adjacent to the stroke remains hypometabolic on day 8. This persistent cortical hypometabolism occupies the somatosensory cortex, forelimb motor cortex, and second somatosensory area., Conclusions: Focal stroke produces ipsilateral diaschisis in connected cortical regions that is clearly distant from subtotal damage and may play a role in poststroke neuroplasticity.

Functional imaging by repeated noninvasive scans of specific (18)F tracer distribution using a high-resolution small-animal PET scanner, the microPET, assessed the time course of alterations in energy utilization and dopamine receptors in rats with unilateral striatal quinolinic acid lesions. Energy utilization ipsilateral to the lesion, determined using scans of 2-deoxy-2-[(18)F]fluoro-d-glucose uptake, was compromised severely 1 week after intrastriatal excitotoxin injections. When the same rats were imaged 5 and 7 weeks postlesion, decrements in energy metabolism were even more prominent. In contrast, lesion-induced effects on dopamine D(2) receptor binding were more progressive, with an initial upregulation of [3-(2'-(18)F]fluoroethyl)spiperone binding apparent 1 week postlesion followed by a decline 5 and 7 weeks thereafter. Additional experiments revealed that marked upregulation of dopamine D(2) receptors consequent to quinolinic acid injections could be detected as early as 3 days after the initial insult. Postmortem markers of striatal GABAergic neurons were assessed in the same rats 7 weeks after the lesion: expression of glutamic acid decarboxylase and dopamine D(1) receptor mRNA, as well as [(3)H]SCH-23,390 and [(3)H]spiperone binding to dopamine D(1) and D(2) receptors, respectively, detected prominent decrements consequent to the lesion. In contrast, by 7 weeks postlesion [(3)H]WIN-35,428 binding to dopamine transport sites within the striatum appeared to be enhanced proximal to the quinolinic acid injection sites. The results demonstrate that functional imaging using the microPET is a useful technique to explore not only the progressive neurodegeneration that occurs in response to excitotoxic insults, but also to examine more closely the intricacies of neurotransmitter activity in a small animal model of HD., (Copyright 2000 Academic Press.)

The study of neural repair and neuroplasticity in rodents would be enhanced by the ability to assess neuronal function in vivo. Positron emission tomography (PET) is used to study brain plasticity in humans, but the limited resolution and sensitivity of conventional scanners have generally precluded the use of PET to study neuroplasticity in rodents. We now demonstrate that microPET, a PET scanner developed for use with small animals, can be used to assess metabolic activity in different regions of the conscious rodent brain using [18F]fluorodeoxyglucose (FDG) as the tracer, and to monitor changes in neuronal activity. Limbic seizures result in dramatically elevated metabolic activity in the hippocampus, whereas vibrissal stimulation results in more modest increases in FDG uptake in the contralateral neocortex. We also show that microPET can be used to study lesion-induced plasticity of the brain. Cerebral hemidecortication resulted in diminished relative glucose metabolism in the neostriatum and thalamus ipsilateral to the lesion, with subsequent, significant recovery of metabolic function. These studies demonstrate that microPET can be used for serial assessment of metabolic function of individual, awake rats with a minimal degree of invasiveness, and therefore, has the potential for use in the study of brain disorders and repair.

A hypothetical mechanism for the partial sparing of visual function in the contralateral visual field following cerebral hemispherectomy early in life is the formation of a new corticotectal pathway arising from the remaining primary visual cortex (areas 17 and 18) that projects to the contralateral superior colliculus. To test this hypothesis, the left superior colliculus of intact adult and neonatal (5-15 days old) cats and of adult cats with a left cerebral hemispherectomy sustained neonatally (7-9 days old) or in adulthood, was injected with WGA-HRP and the brains were processed for combined TMB/DAB histochemistry. The primary visual cortex was examined, labelled neurons were counted and the cross sectional area of their somata was measured. The left primary visual cortex of intact adult animals exhibited a mean of 959.68 labelled cells +/- 406.5 (S.E.), with a mean soma size of 366.7 microns2 +/- 131.2. For the neonatal intact cats, there was a mean of 75.31 +/- 21.08 cells within the left primary visual cortex which exhibited a mean soma size of 249.56 microns2 +/- 68.18. The peak cell size distribution for both intact groups was similar at 300 microns2. Virtually no labelled neurons were detected in the right primary visual cortex of intact animals (neonatal or adult). For neonatal-hemispherectomized cats, the remaining right primary visual cortex exhibited a mean cell count of 351.09 +/- 126.3 cells, with a mean soma size of 436.1 microns2 +/- 131.5, and a peak cell size distribution of 400 microns2. Finally, for adult-hemispherectomized animals, the contralateral primary visual cortex exhibited 68.27 +/- 20.13 neurons having a mean soma size of 486.6 microns2 +/- 143.2 with a peak cell size distribution of 500 microns2. These results indicate that reorganization of the corticotectal pathway occurs in both adult- and neonatal-hemispherectomized cats but is more pronounced in neonatal-lesioned animals. In addition, the cells of origin of this reorganized pathway tended to be larger, perhaps in response to a greater axonal arborization.

Transforming growth factor alpha (TGF alpha) is a mitogenic polypeptide which acts at the epidermal growth factor receptor to produce its biologic effects. Recent studies have demonstrated that TGF alpha may act as a neurotrophic factor. Cerebral hemispherectomy (hemidecortication) is performed on some children with intractable epilepsy. Prior studies have demonstrated improved functional recovery in both children and animals when the surgery is performed at a very early age. In order to test whether TGF alpha may be involved in the functional recovery of the neostriatum following cerebral hemidecortication, we performed in situ hybridization for TGF alpha mRNA on brains of rats which underwent hemispherectomy at postnatal day (P) 6 or P12 or in adulthood, and sacrificed one, 7, or 30 days following surgery. Normal striatal expression in control animals was very high at P6 and then decreased throughout development. In animals undergoing lesion at earlier ages (P6 and P12), TGF alpha mRNA expression was first depressed in the ipsilateral neostriatum one day after surgery and then elevated to supranormal levels 7 and 30 days after surgery. Maximal decreases (40% below contralateral neostriatum) were seen in animals lesioned at P12 and sacrificed the next day. Maximal elevations (60% greater than opposite neostriatum) were seen in animals operated on at P6 and sacrificed 30 days post surgery. Expression in the adult animal was only mildly affected, with a 20% increase found in the ipsilateral caudate 7 days after the lesion, but no significant changes after one or 30 days survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Human pituitary adenoma tissues were not tumorigenic in the hormonally manipulated nude mouse. Mouse fibroblast cells (C3H 10T1/2) also did not form tumors when inoculated alone into nude mice. When these two tissues were cocultured and coinoculated into nude mice however, the majority of inocula developed progressively enlarging tumors which could be established in tissue culture and passaged through the nude mouse. These tumors were sarcomatous histologically and thus did not resemble any human pituitary adenoma tissue injected. In order to detect any human cells in these tumors, tumor genomic DNA was subjected to Southern analysis using human repetitive Alu and HGH DNA sequence probes. Southern blot analysis of the nude mouse derived tumor genomic DNA revealed no sizeable human DNA in the mouse tumor cell genome indicating the absence of significant numbers of human cells in the tumors or the transfer of human DNA to the mouse cells. The tumors therefore arose from transformed C3H 10T1/2 cells after coculture with the human pituitary adenoma cells. These results implied that the tumorigenic transformation of susceptible C3H 10T1/2 cells in the cocultures occurred as a result of the secretion by the adenoma cells of transforming substances in the culture media or the induction of tumorigenicity through direct cell-cell contact between the two cell types.

The contribution of phagocytes and antibody to protection against Salmonella typhimurium during the early phase of infection in mice was analysed. Following intravenous injection, most of the bacteria were trapped in the liver and spleen within 10 to 60 min and killed within 6 h; surviving organisms began to multiply in these tissues after 24 h and reached a maximum at 5 to 7 d. The transient killing phase was abrogated by treatment with carrageenan, a macrophage blocker, but not by whole-body X-irradiation. These observations suggest that carrageenan-sensitive, but radio-resistant macrophages play an important role in the early phase of the infection. Actively immunized mice showed accelerated trapping and killing; the protection observed at the early stage of infection in immunized mice could be passively transferred to normal mice, whereas carrageenan-treated mice did not kill the bacterial even after receiving immune serum. It seems that the synergistic action of macrophages and antibody provided the main initial primary defence in immune animals.

It has been suggested that human neutrophils exposed to performed immune complexes or activated complement fragments generate O2- anions in extracellular medium. In vivo studies have revealed that oxygen intermediates produced by immune complex-activated neutrophils play an important role in subsequent tissue damage. Since it is difficult to obtain direct evidence that O2- is released into plasma in patients with systemic lupus erythematosus (SLE), we studied the capacities of their sera to stimulate O2- release by human neutrophils in vitro. Sera from patients with SLE significantly enhanced O2- generation by neutrophils compared to normal sera. The enhancing activity of serum in the induction of increased O2- generation correlated positively with the presence of serum immune complexes and negatively with serum complement levels. The enhancing factors were analyzed by serum fractionation on Sephadex G-200 gel filtration, and were concluded to be immune complexes of intermediate size containing an activated complement fragment.