HCMV Infection Reduces Nidogen-1 Expression, Contributing to Impaired Neural Rosette Development in Brain Organoids.

Human cytomegalovirus (HCMV) is a leading cause of birth defects in humans. These birth defects include microcephaly, sensorineural hearing loss, vision loss, and cognitive impairment. The process by which the developing fetus incurs these neurological defects is poorly understood. To elucidate some of these mechanisms, we have utilized HCMV-infected induced pluripotent stem cells (iPSCs) to generate in vitro brain organoids, modeling the first trimester of fetal brain development. Early during culturing, brain organoids generate neural rosettes. These structures are believed to model neural tube formation. Rosette formation was analyzed in HCMV-infected and mock-infected brain organoids at 17, 24, and 31 days postinfection. Histological analysis revealed fewer neural rosettes in HCMV-infected compared to mock-infected organoids. HCMV-infected organoid rosettes incurred multiple structural deficits, including increased lumen area, decreased ventricular zone depth, and decreased cell count. Immunofluorescent (IF) analysis found that nidogen-1 (NID1) protein expression in the basement membrane surrounding neural rosettes was greatly reduced by virus infection. IF analysis also identified a similar downregulation of laminin in basement membranes of HCMV-infected organoid rosettes. Knockdown of NID1 alone in brain organoids impaired their development, leading to the production of rosettes with increased lumen area, decreased structural integrity, and reduced laminin localization in the basement membrane, paralleling observations in HCMV-infected organoids. Our data strongly suggest that HCMV-induced downregulation of NID1 impairs neural rosette formation and integrity, likely contributing to many of HCMV's most severe birth defects. IMPORTANCE HCMV infection in pregnant women continues to be the leading cause of virus-induced neurologic birth defects. The mechanism through which congenital HCMV (cCMV) infection induces pathological changes to the developing fetal central nervous system (CNS) remains unclear. Our lab previously reproduced identified clinical defects in HCMV-infected infants using a three dimensional (3D) brain organoid model. In this new study, we have striven to discover very early HCMV-induced changes in developing brain organoids. We investigated the development of neural tube-like structures, neural rosettes. HCMV-infected rosettes displayed multiple structural abnormalities and cell loss. HCMV-infected rosettes displayed reduced expression of the key basement membrane protein, NID1. We previously found NID1 to be specifically targeted in HCMV-infected fibroblasts and endothelial cells. Brain organoids generated from NID1 knockdown iPSCs recapitulated the structural defects observed in HCMV-infected rosettes. Findings in this study revealed HCMV infection induced early and dramatic structural changes in 3D brain organoids. We believe our results suggest a major role for infection-induced NID1 downregulation in HCMV-induced CNS birth defects.

Human Cytomegalovirus Utilizes Multiple Viral Proteins to Regulate the Basement Membrane Protein Nidogen 1.

Nidogen 1 (NID1) is an important basement membrane protein secreted by many cell types. We previously found that human cytomegalovirus (HCMV) infection rapidly induced chromosome 1 breaks and that the basement membrane protein NID1, encoded near the 1q42 break site, was downregulated. We have now determined that the specific breaks in and of themselves did not regulate NID1, rather interactions between several viral proteins and the cellular machinery and DNA regulated NID1. We screened a battery of viral proteins present by 24 hours postinfection (hpi) when regulation was induced, including components of the incoming virion and immediate early (IE) proteins. Adenovirus (Ad) delivery of the tegument proteins pp71 and UL35 and the IE protein IE1 influenced steady-state (ss) NID1 levels. IE1's mechanism of regulation was unclear, while UL35 influenced proteasomal regulation of ss NID1. Real-time quantitative PCR (RT-qPCR) experiments determined that pp71 downregulated NID1 transcription. Surprisingly, WF28-71, a fibroblast clone that expresses minute quantities of pp71, suppressed NID1 transcription as efficiently as HCMV infection, resulting in the near absence of ss NID1. Sequence analysis of the region surrounding the 1q42 break sites and NID1 promoter revealed CCCTC-binding factor (CTCF) binding sites. Chromatin immunoprecipitation experiments determined that pp71 and CTCF were both bound at these two sites during HCMV infection. Expression of pp71 alone replicated this binding. Binding was observed as early as 1 hpi, and colocalization of pp71 and CTCF occurred as quickly as 15 min postinfection (pi) in infected cell nuclei. In fibroblasts where CTCF was knocked down, Adpp71 infection did not decrease NID1 transcription nor ss NID1 protein levels. Our results emphasize another aspect of pp71 activity during infection and identify this viral protein as a key contributor to HCMV's efforts to eliminate NID1. Further, we show, for the first time, direct interaction between pp71 and the cellular genome. IMPORTANCE We have found that human cytomegalovirus (HCMV) utilizes multiple viral proteins in multiple pathways to regulate a ubiquitous cellular basement membrane protein, nidogen-1 (NID1). The extent of the resources and the redundant methods that the virus has evolved to affect this control strongly suggest that its removal provides a life cycle advantage to HCMV. Our discoveries that one of the proteins that HCMV uses to control NID1, pp71, binds directly to the cellular DNA and can exert control when present in vanishingly small quantities may have broad implications in a wide range of infection scenarios. Dysregulation of NID1 in an immunocompetent host is not known to manifest complications during infection; however, in the naive immune system of a developing fetus, disruption of this developmentally critical protein could initiate catastrophic HCMV-induced birth defects.

Users Maintain Task Accuracy and Gait Characteristics During Missed Exoskeleton Actuations Through Adaptations In Joint Kinematics.

In operational settings, lower-limb active exoskeletons may experience errors, where an actuation that should be present is missed. These missed actuations may impact users' trust in the system and the adapted human-exoskeleton coordination strategies. In this study, we introduced pseudorandom catch trials, in which an assistive exoskeleton torque was not applied, to understand the immediate responses to missed actuations and how users' internal models to an exoskeleton adapt upon repeated exposure to missed actuations. Participants (N = 15) were instructed to complete a stepping task while wearing a bilateral powered ankle exoskeleton. Human-exoskeleton coordination and trust were inferred from task performance (step accuracy), step characteristics (step length and width), and joint kinematics at selected peak locations of the lower limb. Step characteristics and task accuracy were not impacted by the loss of exoskeleton torque as hip flexion was modulated to support completing the stepping task during catch trials, which supports an impacted human-exoskeleton coordination. Reductions in ankle plantarflexion during catch trials suggest user adaptation to the exoskeleton. Trust was not impacted by catch trials, as there were no significant differences in task performance or gait characteristics between earlier and later strides. Understanding the interactions between human-exoskeleton coordination, task accuracy, and step characteristics will support development of exoskeleton controllers for non-ideal operational settings.

Impact of Haptic Cues and an Active Ankle Exoskeleton on Gait Characteristics.

OBJECTIVE: This study examined the interaction of gait-synchronized vibrotactile cues with an active ankle exoskeleton that provides plantarflexion assistance. BACKGROUND: An exoskeleton that augments gait may support collaboration through feedback to the user about the state of the exoskeleton or characteristics of the task. METHODS: Participants (N = 16) were provided combinations of torque assistance and vibrotactile cues at pre-specified time points in late swing and early stance while walking on a self-paced treadmill. Participants were either given explicit instructions (N = 8) or were allowed to freely interpret (N=8) how to coordinate with cues. RESULTS: For the free interpretation group, the data support an 8% increase in stride length and 14% increase in speed with exoskeleton torque across cue timing, as well as a 5% increase in stride length and 7% increase in speed with only vibrotactile cues. When given explicit instructions, participants modulated speed according to cue timing-increasing speed by 17% at cues in late swing and decreasing speed 11% at cues in early stance compared to no cue when exoskeleton torque was off. When torque was on, participants with explicit instructions had reduced changes in speed. CONCLUSION: These findings support that the presence of torque mitigates how cues were used and highlights the importance of explicit instructions for haptic cuing. Interpreting cues while walking with an exoskeleton may increase cognitive load, influencing overall human-exoskeleton performance for novice users. APPLICATION: Interactions between haptic feedback and exoskeleton use during gait can inform future feedback designs to support coordination between users and exoskeletons.

Dosimetric advantages of volumetric modulated arc therapy (VMAT) with deep inspiration breath hold (DIBH) technique in Halcyon Linac for left breast cancer treatment.

To evaluate the dosimetric advantages of incorporating the deep inspiration breath hold (DIBH) technique into left breast cancer volumetric modulated arc therapy (VMAT) treatment under Halcyon Linac and to investigate the correlation between mean heart dose (MHD) and distance from the heart to target volumes in left breast cancer VMAT treatment. Fifteen Post-lumpectomy, left-sided breast patients treated between January 2017 and October 2020 were selected. Two plans were generated for each patient using Eclipse treatment planning system (TPS) with the prescription of 50.4 Gy to planning target volume (PTV) breast and 58.8 Gy to PTV boost in 28 fractions. For each patient, DIBH and free breathing (FB) VMAT treatment plans under Halcyon Linac were generated. Dosimetric parameters, monitor unit and beam-on time of both DIBH and FB groups were compared. Three-dimensional distances from heart surface to each target volume were measured on computed tomography images using the TPS contouring tool and their correlation with MHD was evaluated by Pearson's correlation coefficient (r). Comparable target coverage was shown in both groups. Mean dose to heart, left anterior descending artery, and left ventricle in Halcyon-DIBH-VMAT group were significantly reduced by 0.49 Gy, 1.19 Gy, and 0.57 Gy, respectively, compared to Halcyon-FB-VMAT (p < 0.001). A significant lung dose reduction was also achieved in Halcyon-DIBH-VMAT group. There was also a strong negative correlation between MHD and distance from heart surface to PTV boost in both FB and DIBH group (r = -0.741, p < 0.001), but not observed for distance from heart surface to PTV breast. Incorporating DIBH into left breast cancer VMAT treatment under Halcyon Linac demonstrated significant cardiac and lung dose reduction. It was also demonstrated that MHD had a strong negative correlation with distance from heart surface to PTV boost but relatively independent of distance from heart surface to PTV breast. Recognizing the distance from the heart surface to PTV boost as the main factor in affecting MHD could potentially facilitate clinical treatment planning workflow and decision.

Actuation Timing Perception of a Powered Ankle Exoskeleton and Its Associated Ankle Angle Changes During Walking.

Robotic ankle exoskeletons have the potential to extend human ability, and actuation timing serves as one of the critical parameters in its controller design. While many experiments have investigated the optimal actuation timing values to achieve different objective functions (e.g. minimizing metabolic cost), studies on users' perception of control parameters are gaining interest as it gives information on people's comfort, coordination, and trust in using devices, as well as providing foundations on how the sensorimotor system detects the exoskeleton behavior changes. The purpose of this study was to evaluate people's sensitivity to changes in exoskeleton actuation timing and its associated exoskeleton ankle angle changes during walking. Participants (n =15) with little or no prior experience with ankle exoskeletons were recruited and performed a psychophysical experiment to characterize their just-noticeable difference (JND) thresholds for actuation timing. Participants wore a bilateral active ankle exoskeleton and compared pairs of torque profiles with different actuation timings and low peak torque (0.225 Nm/kg) while walking on the treadmill. The mean timing JND across participants was 2.8±0.6% stride period. Individuals exhibited different sensitivity towards actuation timing, and their associated exoskeleton ankle angle changes also varied. The variance in ankle angle changes might be explained by their differences in ankle stiffness and different ankle torques provided during walking. The results provide insights into how people perceive the changes in exoskeleton control parameters and show individual differences in exoskeleton usage. The actuation timing JND found in this study can also help determine the necessary controller precision.

Raman needle arthroscopy for in vivo molecular assessment of cartilage.

The development of treatments for osteoarthritis (OA) is burdened by the lack of standardized biomarkers of cartilage health that can be applied in clinical trials. We present a novel arthroscopic Raman probe that can "optically biopsy" cartilage and quantify key extracellular matrix (ECM) biomarkers for determining cartilage composition, structure, and material properties in health and disease. Technological and analytical innovations to optimize Raman analysis include (1) multivariate decomposition of cartilage Raman spectra into ECM-constituent-specific biomarkers (glycosaminoglycan [GAG], collagen [COL], water [H2 O] scores), and (2) multiplexed polarized Raman spectroscopy to quantify superficial zone (SZ) COL anisotropy via a partial least squares-discriminant analysis-derived Raman collagen alignment factor (RCAF). Raman measurements were performed on a series of ex vivo cartilage models: (1) chemically GAG-depleted bovine cartilage explants (n = 40), (2) mechanically abraded bovine cartilage explants (n = 30), (3) aging human cartilage explants (n = 14), and (4) anatomical-site-varied ovine osteochondral explants (n = 6). Derived Raman GAG score biomarkers predicted 95%, 66%, and 96% of the variation in GAG content of GAG-depleted bovine explants, human explants, and ovine explants, respectively (p < 0.001). RCAF values were significantly different for explants with abrasion-induced SZ COL loss (p < 0.001). The multivariate linear regression of Raman-derived ECM biomarkers (GAG and H2 O scores) predicted 94% of the variation in elastic modulus of ovine explants (p < 0.001). Finally, we demonstrated the first in vivo Raman arthroscopy assessment of an ovine femoral condyle through intraarticular entry into the synovial capsule. This study advances Raman arthroscopy toward a transformative low-cost, minimally invasive diagnostic platform for objective monitoring of treatment outcomes from emerging OA therapies.

Perception of Powered Ankle Exoskeleton Actuation Timing During Walking: A Pilot Study.

Actuation timing is an important parameter in powered ankle exoskeleton control that can significantly influence user experience and human-system performance. Previous studies have investigated the actuation timing through optimization under different objective functions, such as minimizing metabolic cost. However, little is known about people's psychological sense of actuation timing. This pilot study measured two subjects' sensitivity to small changes in actuation timing during walking. The just-noticeable difference (JND) threshold was determined via a fitted psychometric function, which quantified subjects' performance in discriminating between a pair of actuation timings. Subjects could detect changes of 3.6% and 6.8% stride period in actuation timing respectively, showing the difference in perception between individuals. The results from this pilot study provide a preliminary understanding of human perception towards exoskeleton control parameters, which offers insight on individual differences in exoskeleton usage and informs exoskeleton precision requirements to minimize undesired human-system interaction.

Indoor nocturnal noise is associated with body mass index and blood pressure: a cross-sectional study.

BACKGROUND: Studies have demonstrated that noise is associated with various health problems, such as obesity and hypertension. Although the evidence of the associations of noise with obesity and hypertension is inconsistent, there seems to be a stronger association of the latter. This study aimed to investigate the associations of noise with body mass index (BMI) and blood pressure in adults living in multi-story residential buildings. METHODS: A cross-sectional study was conducted in Hong Kong from February 2018 to September 2019. The Weinstein Noise Sensitivity Scale, Pittsburgh Sleep Quality Index, ENRICHD Social Support Instrument, Patient Health Questionnaire, Perceived Stress Scale, and Hospital Anxiety and Depression Scale were administered to the participants. BMI and blood pressure were assessed. Nocturnal noise exposure and total sleep duration were measured for a week. RESULTS: Five hundred adults (66.4% female), with an average age of 39 years (range: 18-80), completed the study. The average levels of nocturnal noise, BMI, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were 51.3 dBA, 22.2 kg/m2, 116.0 mmHg, and 75.4 mmHg, respectively. After adjusting for sociodemographic characteristics, nocturnal noise was associated with BMI (b = 0.54, 95% CI: 0.01 to 1.06, p = 0.045) and SBP (b = 2.90, 95% CI: 1.12 to 4.68, p = 0.001). No association was detected between nocturnal noise and DBP (b = 0.79, 95% CI: - 0.56 to 2.13, p = 0.253). Specifically, higher nocturnal noise was associated with higher BMI (b = 0.72, 95% CI: 0.07 to 1.38, p = 0.031) and SBP (b = 3.91, 95% CI: 2.51 to 5.31, p < 0.001) in females but only higher SBP (b = 3.13, 95% CI: 1.35 to 4.92, p < 0.001) in males. The association between noise and SBP remained significant (b = 2.41, 95% CI: 0.62 to 4.20, p = 0.008) after additionally adjusting for lifestyle, diagnosis of hypertension, psychometric constructs, and sleep. CONCLUSIONS: Indoor nocturnal noise was associated with BMI and blood pressure in females but only blood pressure in males. It is important to control nocturnal noise or use soundproofing materials in buildings to reduce noise exposure.

Human Cytomegalovirus Interactions with the Basement Membrane Protein Nidogen 1.

In 2000, we reported that human cytomegalovirus (HCMV) induced specific damage on chromosome 1. The capacity of the virus to induce DNA breaks indicated potent interaction between viral proteins and these loci. We have fine mapped the 1q42 breaksite. Transcriptional analysis of genes encoded in close proximity revealed virus-induced downregulation of a single gene, nidogen 1 (NID1). Beginning between 12 and 24 hours postinfection (hpi) and continuing throughout infection, steady-state (ss) NID1 protein levels were decreased in whole-cell lysates and secreted supernatants of human foreskin fibroblasts. Addition of the proteasomal inhibitor MG132 to culture medium stabilized NID1 in virus-infected cells, implicating infection-activated proteasomal degradation of NID1. Targeting of NID1 via two separate pathways highlighted the virus' emphasis on NID1 elimination. NID1 is an important basement membrane protein secreted by many cell types, including the endothelial cells (ECs) lining the vasculature. We found that ss NID1 was also reduced in infected ECs and hypothesized that virus-induced removal of NID1 might offer HCMV a means of increased distribution throughout the host. Supporting this idea, transmigration assays of THP-1 cells seeded onto NID1-knockout (KO) EC monolayers demonstrated increased transmigration. NID1 is expressed widely in the developing fetal central and peripheral nervous systems (CNS and PNS) and is important for neuronal migration and neural network excitability and plasticity and regulates Schwann cell proliferation, migration, and myelin production. We found that NID1 expression was dramatically decreased in clinical samples of infected temporal bones. While potentially beneficial for virus dissemination, HCMV-induced elimination of NID1 may underlie negative ramifications to the infected fetus.IMPORTANCE We have found that HCMV infection promotes the elimination of the developmentally important basement membrane protein nidogen 1 (NID1) from its host. The virus both decreased transcription and induced degradation of expressed protein. Endothelial cell (EC) secretion of basement membrane proteins is critical for vascular wall integrity, and infection equivalently affected NID1 protein levels in these cells. We found that the absence of NID1 in an EC monolayer allowed increased transmigration of monocytes equivalent to that observed after infection of ECs. The importance of NID1 in development has been well documented. We found that NID1 protein was dramatically reduced in infected inner ear clinical samples. We believe that HCMV's attack on host NID1 favors viral dissemination at the cost of negative developmental ramifications in the infected fetus.

Laparoscopic anatomical liver resection using indocyanine green fluorescence imaging.

BACKGROUND: Anatomical liver resections guided by a demarcation line after portal staining or inflow clamping of the target area have been established as essential methods for curative treatment of hepatocellular carcinoma (HCC) and have subsequently been applied to other malignancies. However, laparoscopic anatomical liver resection (LALR) procedures are very difficult to reproduce, and the confirmation of demarcation of the hepatic segment on a monitor is also challenging. Recently, indocyanine green (ICG) fluorescence imaging has been used to identify hepatic tumors and segmental boundaries during hepatectomy. Herein, we describe LALR using ICG fluorescence imaging. METHODS: Three patients underwent pure LALR using ICG fluorescence imaging at our institute. One patient underwent anatomical partial liver resection for HCC, another underwent segmentectomy 3 for metastatic liver cancer, and the third underwent right anterior sectionectomy for HCC. To visualize hepatic perfusion and the demarcation line by negative staining using an optical imaging system, 2.5 mg ICG was injected intravenously during surgery following clamping or closure of the proximal Glissonean pedicles. RESULTS: For all three cases, ICG fluorescent imaging clearly delineated the demarcation lines and allowed identification of intersegmental planes to some extent because the tumor-bearing hepatic region became non-fluorescing parenchyma during parenchymal transection. This allowed surgeons to recognize the direction and guide the transection of the liver parenchyma when performing LALR. CONCLUSION: LALR using ICG fluorescence imaging is a feasible procedure for resection of the tumor-bearing hepatic region and facilitates visualization of the demarcation line and identification of the boundaries of the hepatic sections.

Laparoscopic transabdominal preperitoneal approach for giant inguinal hernias.

BACKGROUND: Many surgical techniques have been developed to treat inguinal hernia. In recent years, the laparoscopic transabdominal preperitoneal (TAPP) approach has been widely performed to repair inguinal hernia. Giant inguinal hernia (GIH) is an extremely rare disease that is a challenge for general surgeons. GIH appears when patients neglect the treatment for many years and it is defined as an inguinal hernia that extends below the midpoint of inner thigh in standing position. According to previous publications, the Lichtenstein tension-free hernioplasty is recommended to repair GIH. In this article, we describe consecutive four cases of GIH repaired via the TAPP approach. METHODS: From April 2015 to March 2017, 200 patients underwent hernioplasty against inguinal hernia at our hospital. Inguinal hernias were treated via the TAPP approach in principle. We performed hernioplasty via the TAPP approach in all 4 patients (2%) who met the definition of Type 1 GIH. Demographic information, maximum diameter of hernia sac, hernia orifice size, and surgical data were obtained. RESULTS: The mean operative time was 135 min. No intraoperative complications were encountered. All patients could walk from postoperative day 1 and were discharged home early, but they all had scrotal seromas. Three patients did not need puncture or drainage, but one of them required puncture. All seromas disappeared within 6 months. There was no recurrence in the 8- to 24-month follow-up. CONCLUSION: The TAPP approach is a feasible, safe therapeutic option that may reduce wound size and pain following surgical treatment of Type 1 GIH.

A resected case of gallbladder metastasis with symptoms of acute cholecystitis in multiple metastatic ductal carcinoma of the breast.

Gallbladder metastasis from breast cancer, especially from ductal carcinoma, is rare. Herein, we report a rare case of gallbladder metastasis from ductal carcinoma of the breast that was diagnosed after laparoscopic cholecystectomy (LC) for acute cholecystitis. A 78-year-old woman presented with right upper abdominal tenderness and positive Murphy's sign during chemotherapy for advanced multiple metastases of the breast cancer. Abdominal ultrasonography and computed tomography showed a slightly thickened gallbladder wall and two calculi. After a diagnosis of acute calculous cholecystitis was established, LC was performed. Pathological examination revealed poorly differentiated adenocarcinoma infiltrating the submucosal and subserosal layer over the entire gallbladder, and a lymph node metastasis in the gallbladder neck. Immunohistochemical examination revealed that the tumor cells tested positive for estrogen receptor and negative for progesterone receptor, which was consistent with primary breast cancer. The patient was uneventfully discharged without abdominal pain 7 days later. Although she subsequently underwent several chemotherapies, she died 16 months later. In conclusion, gallbladder metastasis should be considered in patients with multiple metastatic breast cancer who present with signs or symptoms of cholecystitis. Moreover, LC should be considered to relieve the symptoms of cholecystitis for improved prognosis, even in a patient with multiple metastases.

Optimization of steroid injection intervals for prevention of stricture after esophageal endoscopic submucosal dissection: A randomized controlled trial.

BACKGROUND: Esophageal endoscopic submucosal dissection enables en bloc resection of large superficial esophageal cancer; however, this procedure may induce severe stricture. Intralesional steroid injection is an effective treatment for prevention of stricture after endoscopic resection; however, there have been no studies assessing the duration of such treatment. The aim of this study was to reduce treatment duration and to evaluate the effectiveness of weekly and biweekly steroid injections in preventing esophageal stricture after endoscopic resection. PATIENTS METHOD: We performed a randomized controlled trial comparing patients receiving weekly or biweekly intralesional triamcinolone injections. Patients with a mucosal defect greater than 75% (3/4) of the luminal circumference after esophageal endoscopic submucosal dissection for superficial esophageal cancers were enrolled. The primary endpoint was the duration of steroid injection treatment. RESULTS: The median duration of treatment was 37.0 days in the weekly group and 34.2 days in the biweekly group (P = 0.059). Among patients with a mucosal defect larger than 50 mm, there was a significant difference in the median duration of treatment between the weekly and biweekly groups (42.5 days vs 29.0 days, P = 0.013). CONCLUSION: Biweekly steroid injection of triamcinolone reduces treatment duration, particularly in those with mucosal defects larger than 50 mm. (Acta gastro-enterol. belg., 2016, 79, 315-320).

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress.

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introduction of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion.

Human Cytomegalovirus nuclear egress and secondary envelopment are negatively affected in the absence of cellular p53.

Human Cytomegalovirus (HCMV) infection is compromised in cells lacking p53, a transcription factor that mediates cellular stress responses. In this study we have investigated compromised functional virion production in cells with p53 knocked out (p53KOs). Infectious center assays found most p53KOs released functional virions. Analysis of electron micrographs revealed modestly decreased capsid production in infected p53KOs compared to wt. Substantially fewer p53KOs displayed HCMV-induced infoldings of the inner nuclear membrane (IINMs). In p53KOs, fewer capsids were found in IINMs and in the cytoplasm. The deficit in virus-induced membrane remodeling within the nucleus of p53KOs was mirrored in the cytoplasm, with a disproportionately smaller number of capsids re-enveloped. Reintroduction of p53 substantially recovered these deficits. Overall, the absence of p53 contributed to inhibition of the formation and function of IINMs and re-envelopment of the reduced number of capsids able to reach the cytoplasm.

Clinical significance of the muscle-retracting sign during colorectal endoscopic submucosal dissection.

BACKGROUND AND STUDY AIMS: During colorectal endoscopic submucosal dissection (ESD), the feature of a muscle layer being pulled toward a neoplastic tumor is sometimes detected. We call this feature the muscle-retracting sign (MR sign). The aim of this study was to evaluate whether the MR sign is associated with particular types of neoplastic lesions and whether it has any clinical significance for ESD sessions. PATIENTS AND METHODS: A total of 329 patients underwent ESD for 357 colorectal neoplasms. The frequency of positivity for the MR sign was evaluated in different morphologic and histopathologic types of neoplasm. The success rate of complete resection and the incidence of complications were also evaluated according to whether lesions were positive or negative for the MR sign. RESULTS: The rates of positivity for the MR sign in the various lesion types were as follows: laterally spreading tumor - granular nodular mixed type (LST-G-M), 9.6 %; laterally spreading tumor - granular homogeneous type (LST-G-H) and laterally spreading tumor - nongranular type (LST-NG), 0 %; sessile type, 41.2 %. The resection rate was 100 % (329 /329) in lesions negative for the MR sign; however, it was 64.3 % (18 /28) in lesions positive for the MR sign, which was significantly lower (P < 0.001). CONCLUSIONS: The MR sign was present only in some protruding lesions, and more importantly, it was associated with a high risk of incomplete tumor removal by ESD. Our data indicate that lesions positive for the MR sign lesions should be dissected with great caution; alternatively, based on the features of the individual case, a switch to surgery should be considered for the benefit of the patient.

Totally intracorporeal delta-shaped B-I anastomosis following laparoscopic distal gastrectomy using the Tri-Staple™ reloads on the manual Ultra handle: a prospective cohort study with historical controls.

BACKGROUND: A delta-shaped anastomosis in totally laparoscopic Billroth I gastrectomy could be performed easily and sufficiently using only laparoscopic linear staplers. However, the restricted maneuverability and severe blurring of these staplers along with their limited hemostability induced strain. In this study, we determined the feasibility and safety of performing delta-shaped anastomosis using the Endo GIA™ Reloads with Tri-Staple™ Technology combined with Endo GIA™ Ultra Universal stapler (Tri-Staple) with a particular focus on short-term surgical outcomes. METHODS: We performed a single-institutional prospective interventional study (UMIN 000008014). The Tri-Staple was prospectively used on 23 consecutive patients who underwent a curative totally laparoscopic Billroth I gastrectomy with delta-shaped anastomosis. These patients were matched with the 19 patients previously treated using the ENDOPATH(®) ETS Articulating Linear Cutters (ETS) on clinical and demographic characteristics. RESULTS: There were no differences between the groups in anastomosis-related local complications, morbidity, non-anastomosis-related local complications, total systemic complications, and short-term outcomes with the exception of significantly reduced blood loss in the Tri-Staple group (ETS vs. Tri-Staple: 37 [10-306] vs. 15 [5-210] mL, p = 0.02). Intraoperative bleeding from the staple line was significantly reduced in the Tri-Staple group. The postoperative drain indwelling period (ETS vs. Tri-Staple, 6 [4-10] vs. 4 [2-43] days, p = 0.032), fasting period (5 [3-7] vs. 3 [3-24] days, p = 0.022), and hospital stay (14 [10-47] vs. 11 [6-58] days, p = 0.025) were significantly shorter in the Tri-Staple group. There was no mortality in this series. Acceleration assessed as indices of blurring of stapler tip might have a significant adverse influence on staple-line bleeding at stapling sites. CONCLUSION: Totally laparoscopic Billroth I distal gastrectomy using Tri-Staple was feasible and safe with favorable short-term surgical outcomes. Reduced blurring while stapling may be a novel endpoint which newly developed stapling devices should target.