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Objective: The incidence of childhood-onset type 1 diabetes (T1D) has been reported to be rising but there is also evidence that it has been attenuated in recent years. We described the time trends and the incidence of T1D in children in Hong Kong from 2008 to 2017 and compared with the previous local registry in 1997 to 2007., Methods: Data were extracted from the Hong Kong Childhood Diabetes Registry, which was established in 2016. It consists of a retrospective registry (including all childhood diabetes diagnosed in 2008 to 2015) and a prospective registry (including all T1D children diagnosed from 2016 onwards). All T1D children diagnosed at the age of less than 18 years from 1 January 2008 to 31 December 2017 and managed in the public system were included in this study., Results: For the incident years in the 2008 to 2017 period, a total of 498 children with T1D was identified. The crude incidence rate was 4.3 per 100 000 person/year (95% confidence interval 3.96-4.72), which was much higher than the last registry of 2.2 per 100 000 persons/year. Using general linear model, the increment is statistically significant (P = .02). When compared to the last registry, the rate of increment had attenuated, with annual increment in crude incidence in the two periods for T1D <15 years changing from 4.3% to 3.5% (P = .02)., Conclusions: The incidence of T1D children increased significantly in the past two decades in Hong Kong, but the rate of increase had attenuated in recent years., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The contribution of hyoid and laryngeal movement deficits to penetration or aspiration in dysphagia is unclear, partly due to large variations in normal hyolaryngeal kinematics for swallowing. In healthy volunteers, laryngeal and hyoid kinematics relate to the requirements for laryngeal vestibule closure suggesting a central schematic control of movement magnitude and patterning for airway protection. Our first aim was to determine if patients with severe dysphagia showed evidence of an impaired swallowing schema, by examining if their kinematic measures were related to their hyolaryngeal space before swallow onset, and if hyolaryngeal movement synchrony for vestibule closure was disrupted. Our second aim was to determine the kinematic measures that predicted bolus penetration and aspiration in dysphagia. The methods included two-dimensional measures of the hyoid and laryngeal anterior and superior displacement and velocity, and the change in laryngeal vestibule area made from videofluoroscopic swallow recordings of 21 healthy volunteers and 21 patients with dysphagia on tube feeding secondary to the stroke or head and neck cancer. The results demonstrated that the patients did not adapt their hyolaryngeal movements during swallowing to their initial hyolaryngeal space. Further, none of the patients' measures of hyoid or laryngeal peak velocity timing were synchronized with vestibule closure, demonstrating a disorganized movement patterning. Laryngeal elevation peak velocity independently predicted penetration and aspiration. In conclusion, the central schema for swallowing patterning was disturbed, impairing the integration of kinematic actions for airway protection in severe dysphagia, while laryngeal peak elevation velocity predicted penetration and aspiration on patient swallows.

Working memory impairment is associated with symptom severity and poor functional outcome in autistic individuals, and yet the neurobiology underlying such deficits is poorly understood. Neural oscillations are an area of investigation that can shed light on this issue. Theta and alpha oscillations have been found consistently to support working memory in typically developing individuals and have also been shown to be functionally altered in people with autism. While there is evidence, largely from functional magnetic resonance imaging studies, that neural processing underlying working memory is altered in autism, there remains a dearth of information concerning how sub-processes supporting working memory (namely encoding, maintenance and recognition) are impacted. In this study, we used magnetoencephalography to investigate inter-regional theta and alpha brain synchronization elicited during the widely used one-back task across encoding, maintenance and recognition in 24 adults with autism and 30 controls. While both groups performed comparably on the working-memory task, we found process- and frequency-specific differences in networks recruited between groups. In the theta frequency band, both groups used similar networks during encoding and recognition, but different networks specifically during maintenance. In comparison, the two groups recruited distinct networks across encoding, maintenance and recognition in alpha that showed little overlap. These differences may reflect a breakdown of coherent theta and alpha synchronization that supports mnemonic functioning, or in the case of alpha, impaired inhibition of task-irrelevant neural processing. Thus, these data provide evidence for specific theta and widespread alpha synchrony alterations in autism, and underscore that a detailed examination of the sub-processes that comprise working memory is warranted for a complete understanding of cognitive impairment in this population., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background: We previously reported that breast conservation was feasible for women with large or irregularly shaped breast cancers when tumor resection was guided by multiple localizing wires. We now report long-term outcomes of multiple-wire versus single-wire localized lumpectomies for breast cancer., Patients and Methods: We retrospectively reviewed wire-localized lumpectomies at our institution from May 2000 to November 2006. Rates of ipsilateral in-breast tumor recurrence, metastasis, and subsequent unplanned diagnostic imaging and biopsy were compared between multiple-wire and single-wire cohorts., Results: We identified 112 multiple-wire and 160 single-wire breast cancer lumpectomies that achieved clear margins. Median age was 64 years in the multiple-wire cohort and 57 years in the single-wire cohort. Mean lumpectomy volume was 75 mL in multiple-wire patients and 49 mL in single-wire patients (P = .003). Invasive tumor size, axillary node status, and use of radiation and systemic therapy were similar, but the multiple-wire group had more patients with ductal carcinoma-in-situ only (38% vs. 28%). At 108 months' median follow-up, there was no significant difference in local or distant recurrence rates between multiple-wire and single-wire cohorts. Six (5%) multiple-wire patients and 6 (4%) single-wire patients had local recurrences and 3 (3%) multiple-wire and 5 (3%) single-wire patients developed metastatic disease. Unplanned diagnostic imaging was required for 53 (47%) multiple-wire and 65 (41%) single-wire patients. Subsequent ipsilateral biopsy occurred in 15 (13%) multiple-wire and 19 (12%) single-wire patients., Conclusion: Breast-conserving surgery with multiple localizing wires is a safe alternative to mastectomy for breast cancer patients with large mammographic lesions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Cerebral intravoxel incoherent motion (IVIM) imaging assumes two components. However, more compartments are likely present in pathologic tissue. We hypothesized that spectral analysis using a nonnegative least-squares (NNLS) approach can detect an additional, intermediate diffusion component, distinct from the parenchymal and microvascular components, in lesion-prone regions., Purpose: To investigate the presence of this intermediate diffusion component and its relation with cerebral small vessel disease (cSVD)-related lesions., Study Type: Prospective cross-sectional study., Population: Patients with cSVD (n = 69, median age 69.8) and controls (n = 39, median age 68.9)., Field Strength/sequence: Whole-brain inversion recovery IVIM acquisition at 3.0T., Assessment: Enlarged perivascular spaces (PVS) were rated by three raters. White matter hyperintensities (WMH) were identified on a fluid attenuated inversion recovery (FLAIR) image using a semiautomated algorithm., Statistical Tests: Relations between IVIM measures and cSVD-related lesions were studied using the Spearman's rank order correlation., Results: NNLS yielded diffusion spectra from which the intermediate volume fraction f int was apparent between parenchymal diffusion and microvasular pseudodiffusion. WMH volume and the extent of MRI-visible enlarged PVS in the basal ganglia (BG) and centrum semiovale (CSO) were correlated with f int in the WMHs, BG, and CSO, respectively. f int was 4.2 ± 1.7%, 7.0 ± 4.1% and 13.6 ± 7.7% in BG and 3.9 ± 1.3%, 4.4 ± 1.4% and 4.5 ± 1.2% in CSO for the groups with low, moderate, and high number of enlarged PVS, respectively, and increased with the extent of enlarged PVS (BG: r = 0.49, P < 0.01; CSO: r = 0.23, P = 0.02). f int in the WMHs was 27.1 ± 13.1%, and increased with the WMH volume (r = 0.57, P < 0.01)., Data Conclusion: We revealed the presence of an intermediate diffusion component in lesion-prone regions of cSVD and demonstrated its relation with enlarged PVS and WMHs. In tissue with these lesions, tissue degeneration or perivascular edema can lead to more freely diffusing interstitial fluid contributing to f int ., Level of Evidence: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:1170-1180., (© 2019 The Authors. Journal of Magnetic Resonance Imaging published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)

Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 10 7 /g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium . In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops., Competing Interests: The authors declare no conflict of interest.

Hypoxia is one of the major challenges in aquaculture industry. Breeding of fish tolerant to hypoxia is an important task in genetic improvement of aquaculture species. The Asian seabass, Lates calcarifer, is an important foodfish species. We identified and characterized the hypoxia-inducible factor inhibitor (HIF1αn) gene in the Asian seabass. The full-length cDNA sequence of the HIF1αn was 3425 bp, with an ORF of 1065 bp, encoding 354 amino acids. The genomic sequence of the gene was 8667 bp in length, and contained eight exons and seven introns. Phylogenetic analysis of the gene in fish and tetrapods revealed that the HIF1αn in the Asian seabass was closely related to that of tilapia (Oreochromis niloticus). The HIF1αn was highly up-regulated in the gill, spleen and heart after 3.5-hours hypoxia treatment. We identified three SNPs in the third and fourth introns of the HIF1αn gene. The SNP (i.e. SNP 9332241 (C/T)) in the fourth intron was significantly (P < 0.01) associated with hypoxia tolerance. This SNP might be useful in selecting Asian seabass for improved tolerance to hypoxia. Our data also provide useful information for further detailed analysis of the function of the HIF1αn gene in hypoxia tolerance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Importance: Neoadjuvant systemic therapy (NST) is often administered to enable breast-conserving therapy (BCT) in stages II to III breast cancer., Objectives: To prospectively evaluate the role of NST in conversion from BCT ineligibility to BCT eligibility and to assess the association of response to NST, germline BRCA (gBRCA) status, and region of treatment with surgical choice in women with triple-negative breast cancer (TNBC)., Design, Setting, and Participants: This prespecified secondary analysis of a multicentered, phase 3, double-blind, randomized clinical trial (BrighTNess) enrolled 634 eligible women across 145 centers in 15 countries in North America, Europe, and Asia. Women with operable, clinical stages II to III TNBC who underwent gBRCA mutation testing before initiating NST were eligible to participate. Data were collected from April 1, 2014, to December 8, 2016. This preplanned analysis was performed from January 5, 2018, to October 28, 2019., Interventions: Study participants were randomized to receive 12 weeks of weekly paclitaxel alone or with the addition of carboplatin and/or veliparib, followed by 4 cycles of doxorubicin hydrochloride and cyclophosphamide., Main Outcomes and Measures: Surgeons assessed BCT candidacy by clinical and radiographic criteria before and after NST. Surgical choices and whether BCT eligibility was associated with the likelihood of pathologic complete response were then analyzed., Results: Among the 634 randomized patients (median age, 51 [range, 22-78] years), pre- and post-NST assessments were available for 604 patients. Of 141 patients deemed BCT ineligible at baseline, 75 (53.2%) converted to BCT eligible. Overall, 342 (68.1%) of 502 patients deemed BCT eligible after NST underwent BCT, including 42 (56.0%) of the 75 who converted to BCT eligible. Patients treated in Europe and Asia were more likely to undergo BCT (odds ratio, 2.66; 95% CI, 1.84-3.84) compared with those treated in North America. Among patients without gBRCA mutation undergoing mastectomy, those treated in North America were more likely to undergo contralateral prophylactic mastectomy (57 of 81 [70.4%] vs 6 of 30 [20.0%]; P < .001). Rates of pathologic complete response were similar between patients deemed BCT eligible at baseline and those who were BCT ineligible but converted to BCT eligibility after NST (55.3 [235 of 425] vs 49.3% [37 of 75]; P = .38)., Conclusions and Relevance: This prospective analysis of NST and BCT eligibility in TNBC demonstrates a conversion from BCT ineligibility to BCT eligibility of 53.2%. Lower BCT rates among eligible patients and higher bilateral mastectomy rates among patients without gBRCA mutation in North America merit investigation., Trial Registration: ClinicalTrials.gov identifier: NCT02032277.

A carboxylesterase (CXE) or carboxylic-ester hydrolase is an enzyme that catalyzes carboxylic ester and water into alcohol and carboxylate. In plants, CXEs have been implicated in defense, development, and secondary metabolism. We discovered a new CXE gene in Nicotiana benthamiana that is related to virus resistance. The transcriptional level of NbCXE expression was significantly increased after Tobacco mosaic virus (TMV) infection. Transient over-expression of NbCXE inhibited TMV accumulation in N. benthamiana plants. Conversely, when the NbCXE gene was silenced with a Tobacco rattle virus (TRV)-based gene silencing system, TMV RNA accumulation was increased in NbCXE-silenced plants after infection. NbCXE protein was shown to interact with TMV coat protein (CP ) in vitro. Additionally, the expressions of host defense-related genes were increased in transient NbCXE-overexpressed plants but decreased in NbCXE silenced N. benthamiana plants. In summary, our study showed that NbCXE is a novel resistance-related gene involved in host defense responses against TMV infection., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Introduction: The ability of breast magnetic resonance imaging (MRI) to predict pathologic complete response (pCR) to neoadjuvant systemic therapy (NST) varies across biological subtypes. We sought to determine how well breast MRI findings following initial treatment on the phase III BrighTNess trial correlated with pCR in patients with triple negative breast cancer (TNBC)., Methods: Baseline and mid-treatment imaging and pathologic response data were available in 519 patients with stage II-III TNBC who underwent NST as per protocol. MRI complete response (mCR) was defined as disappearance of all target lesion(s) and MRI partial response (mPR) as a ≥50% reduction in the largest tumor diameter., Results: Overall, mCR was demonstrated in 116 patients (22%), whereas 166 (32%) had mPR and 237 (46%) had stable/progressive disease (SD/PD). The positive predictive value (PPV), negative predictive value, and overall accuracy of the mid-treatment MRI for pCR were 78%, 56%, and 61%, respectively; accuracy did not differ significantly between gBRCA mutation carriers and non-carriers (52% vs. 63%, p = 0.10). When compared to patients with SD/PD, those with mPR or mCR were 3.35-fold (95% CI 2.07-5.41) more likely to have pCR at surgery. MRI response during NST was significantly associated with eligibility for breast-conserving surgery following completion of treatment (93.1% for mCR vs. 81.6% for SD/PD, p < 0.001)., Conclusions: Complete response on mid-treatment MRI in the BrighTNess trial had a PPV of 78% for demonstration of pCR after completion of NST in TNBC. However, a substantial proportion of patients with mPR or SD/PD also achieved a pCR., Clinical Trial Registration: NCT02032277., Competing Interests: Declaration of competing interest The following co-authors received funding from AbbVie: Mehra Golshan, Sibylle Loibl, Jens Bodo Huober, Joyce O'Shaughnessy, Hope S. Rugo, Norman Wolmark (institution only), Peter Ansell, David Maag, Danielle M. Sullivan, Otto Metzger-Filho, Gunter Von Minckwitz, Charles E. Geyer, Jr., William M. Sikov, Michael Untch. Jens Bodo Huober reports honoraria and is on advisory boards for Celgene, Roche, Novartis, Hexal, Pfizer, AstraZeneca, Lilly, Amgen, Eisai, MSD, and Abbvie; research grants from Celgene and Novartis; and travel grants from Roche, Novartis, Celgene, Pfizer and Daiichi-Sankyo. William Sikov reports honoraria for promotional talks from Lilly Oncology and Eisai in the last 12 months (all not related to this submission)., (Copyright © 2019 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Impairments in social functioning are characteristic of autism spectrum disorder (ASD). Differences in functional networks during face processing in ASD compared to controls have been reported; however, the spatial-temporal dynamics of networks underlying affective processing are still not well understood. The current magnetoencephalography study examined whole-brain functional connectivity to implicit happy and angry faces in 104 adults with and without ASD. A network of reduced gamma band (30-55 Hz) phase synchrony occurring 80-308 ms following angry face presentation was found in adults with ASD compared to controls. The network involved widespread connections primarily anchored in frontal regions, including bilateral orbitofrontal areas, bilateral inferior frontal gyri, and left middle frontal gyrus extending to occipital, temporal, parietal, and subcortical regions. This finding suggests disrupted long-range neuronal communication to angry faces. Additionally, reduced gamma band-specific connectivity may reflect altered E/I balance in brain regions critical for emotional face processing in ASD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

There is an unmet need to develop robust predictive algorithms to preoperatively identify pediatric epilepsy patients who will respond to vagus nerve stimulation (VNS). Given the similarity in the neural circuitry between vagus and median nerve afferent projections to the primary somatosensory cortex, the current study hypothesized that median nerve somatosensory evoked field(s) (SEFs) could be used to predict seizure response to VNS. Retrospective data from forty-eight pediatric patients who underwent VNS at two different institutions were used in this study. Thirty-six patients ("Discovery Cohort") underwent preoperative electrical median nerve stimulation during magnetoencephalography (MEG) recordings and 12 patients ("Validation Cohort") underwent preoperative pneumatic stimulation during MEG. SEFs and their spatial deviation, waveform amplitude and latency, and event-related connectivity were calculated for all patients. A support vector machine (SVM) classifier was trained on the Discovery Cohort to differentiate responders from non-responders based on these input features and tested on the Validation Cohort by comparing the model-predicted response to VNS to the known response. We found that responders to VNS had significantly more widespread SEF localization and greater functional connectivity within limbic and sensorimotor networks in response to median nerve stimulation. No difference in SEF amplitude or latencies was observed between the two cohorts. The SVM classifier demonstrated 88.9% accuracy (0.93 area under the receiver operator characteristics curve) on cross-validation, which decreased to 67% in the Validation cohort. By leveraging overlapping neural circuitry, we found that median nerve SEF characteristics and functional connectivity could identify responders to VNS., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The need for cultural understanding is particularly important in end-of-life (EOL) care planning as the use of EOL care in minority populations is disproportionately lower than those who identify as Caucasian. Data regarding the use of EOL care services by Native Hawaiians in Hawai'i and the United States is limited but expected to be similarly disproportionate as other minorities. In a population with a lower life expectancy and higher prevalence of deaths related to chronic diseases such as cardiovascular disease, diabetes, and obesity, as compared to the state of Hawai'i as a whole, our objective was to review the current literature to understand the usage and perceptions of EOL care planning in the Native Hawaiian population. We searched ten electronic databases and after additional screening, seven articles were relevant to our research purpose. We concluded that limited data exists regarding EOL care use specifically in Native Hawaiians. The available literature highlighted the importance of understanding family and religion influences, educating staff on culturally appropriate EOL care communication, and the need for more research on the topic. The paucity of data in EOL care and decision-making in Native Hawaiians is concerning and it is evident this topic needs more study. From national statistics it looks as though this is another health disparate area that needs to be addressed and is especially relevant when considering the rapid increase in seniors in our population., Competing Interests: None of the authors identify a conflict of interest., (©Copyright 2019 by University Health Partners of Hawai‘i (UHP Hawai‘i).)

Widespread antisense long noncoding RNA (lncRNA) overlap with many protein-coding genes in mammals and emanate from gene promoter, enhancer, and termination regions. However, their origin and biological purpose remain unclear. We show that these antisense lncRNA can be generated by R-loops that form when nascent transcript invades the DNA duplex behind elongating RNA polymerase II (Pol II). Biochemically, R-loops act as intrinsic Pol II promoters to induce de novo RNA synthesis. Furthermore, their removal across the human genome by RNase H1 overexpression causes the selective reduction of antisense transcription. Consequently, we predict that R-loops act to facilitate the synthesis of many gene proximal antisense lncRNA. Not only are R-loops widely associated with DNA damage and repair, but we now show that they have the capacity to promote de novo transcript synthesis that may have aided the evolution of gene regulation., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been widely used to determine gene functions in Laodelphax striatellus (Fallén) (small brown planthopper). Selection of suitable reference gene(s) for normalizations of RT-qPCR data is critical for reliable results. To date, reports on identification of suitable L. striatellus reference genes are still very limited. L. striatellus is a destructive rice pest and it can transmit multiple viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV), to many important cereal crops worldwide. In this study, we examined the stablity of seven selected candidate reference genes in L. striatellus at different developmental stages, in different tissues, in RBSDV- or RSV-infected L. striatellus or in RBSDV-infected and Lssynaptojanin 1 ( LsSYNJ1 )-silenced L. striatellus . The RT-qPCR data representing individual candidate genes were analyzed using five different methods: the delta Ct method, geNorm, NormFinder, BestKeeper, and the RefFinder algorithm, respectively. The most stable reference gene for the specific condition was selected according to a comprehensive analysis using the RefFinder method. Ribosomal protein L5 ( LsRPL5 ) and LsRPL8 are the most stably expressed genes in L. striatellus at different developmental stages. Alpha-1-tubulin ( Lsα-TUB ) is the most stably expressed reference gene in different tissues of RBSDV viruliferous (RBSDV-V) or non-viruliferous (RBSDV-NV) L. striatellus . LsRPL8 is the most stably expressed reference gene in RBSDV-V or RSV viruliferous (RSV-V) L. striatellus , while beta-tubulin ( Lsβ-TUB ) is the most stably expressed reference gene in RBSDV-V and LsSYNJ1 -silenced L. striatellus . The selected reference genes were further investigated during analyses of RBSDV P5-1 and P10 gene expression in different tissues from RBSDV-V or RBSDV-NV L. striatellus . The stably expressed reference genes identified in this study will benefit future gene function studies using L. striatellus .

Purpose: Increase in breast cancer survivorship, advancements in diagnostic imaging and standardization of contralateral breast screening before breast cancer surgery have resulted in increased detection of contralateral breast cancer (CBC). The aim of this study was to assess national trends of synchronous bilateral breast cancer (sBBC) and metachronous bilateral breast cancer (mBBC) incidence in newly diagnosed breast cancer patients., Methods: The Surveillance, Epidemiology, and End Results (SEER) database (1973-2014) was used to identify 11,177 women diagnosed with CBC. CBC was classified as sBBC when primary breast cancer in both breasts is diagnosed in the same year, or as mBBC, when diagnosed more than one year from primary breast cancer. Temporal trends in sBBC incidence were then evaluated using the Cochran-Armitage test for trend., Results: Of the 11,177 women diagnosed with CBC, 4228 (38%) had sBBC and 6949 (62%) had mBBC. The incidence of sBBC increased significantly from 1.4% in 1975 to 2.9% in 2014 (p < 0.001). sBBC was more likely to be diagnosed as early stage in recent years (78% in 1975 vs. 90% in 2014 [p < 0.001]), and 69% of patients were treated with mastectomy in 2014., Conclusion: The number of sBBC has increased, and contralateral tumors are more likely to be detected at an early stage with the first primary breast cancer. Despite the early stage findings, most were treated with mastectomy. Further studies are needed to define the best therapy for patients with contralateral disease and optimal surveillance and detection methods.

Children born very preterm (VPT) often demonstrate selective difficulties in working memory (WM), which may underlie academic difficulties observed in this population. Despite this, few studies have investigated the functional networks underlying WM in young children born VPT, a period when cognitive deficits become apparent. Using magnetoencephalography, we examined the networks underlying the maintenance of visual information in 6-year-old VPT (n = 15) and full-term (FT; n = 20) children. Although task performance was similar, VPT children engaged different oscillatory mechanisms during WM maintenance. Within the FT group, we observed higher mean whole-brain connectivity in the alpha-band during the retention (i.e. maintenance) interval associated with correct compared to incorrect responses. VPT children showed reduced whole-brain alpha synchrony, and a different network organization with fewer connections. In the theta-band, VPT children demonstrated a slight increase in whole-brain connectivity during WM maintenance, and engaged similar network hubs as FT children in the alpha-band, including the left dorsolateral prefrontal cortex and superior temporal gyrus. These findings suggest that VPT children rely on the theta-band to support similar task performance. Altered oscillatory mechanisms may reflect a less mature pattern of functional recruitment underlying WM in VPT children, which may affect the processing in complex ecological situations.

Nontypeable Haemophilus influenzae (NTHi) efficiently colonizes the human nasopharynx asymptomatically but also causes respiratory mucosal infections, including otitis media, sinusitis, and bronchitis. The lipooligosaccharide (LOS) on the cell surface of NTHi displays complex glycans that mimic host structures, allowing it to evade immune recognition. However, LOS glycans are also targets of host adaptive and innate responses. To aid in evasion of these responses, LOS structures exhibit interstrain heterogeneity and are also subject to phase variation, the random on/off switching of gene expression, generating intrastrain population diversity. Specific LOS modifications, including terminal sialylation of the LOS, which exploits host-derived sialic acid (Neu5Ac), can also block recognition of NTHi by bactericidal IgM and complement by mechanisms that are not fully understood. We investigated the LOS sialic acid-mediated resistance of NTHi to antibody-directed killing by serum complement. We identified specific LOS structures extending from heptose III that are targets for binding by naturally occurring bactericidal IgM in serum and are protected by sialylation of the LOS. Phase-variable galactosyltransferases encoded by lic2A and lgtC each add a galactose epitope bound by IgM that results in antibody-dependent killing via the classical pathway of complement. NTHi's survival can be influenced by the expression of phase-variable structures on the LOS that may also depend on environmental conditions, such as the availability of free sialic acid. Identification of surface structures on NTHi representing potential targets for antibody-based therapies as alternatives to antibiotic treatment would thus be valuable for this medically important pathogen., (Copyright © 2019 American Society for Microbiology.)

Background: The purpose of this study was to explore national patterns in the uptake of breast reconstruction and nipple-sparing mastectomy (NSM)., Methods: We used the National Cancer Database to identify all women who underwent mastectomy for stage 0-III breast cancer between 2005-2015. Multivariable logistic regression was used to determine factors associated with receipt of reconstruction, with subset analyses performed to determine trends and predictors of NSM in those who underwent mastectomy with reconstruction., Results: Our cohort consisted of 395,815 women, 238,568 (60.3%) who underwent mastectomy alone and 157,247 (39.7%) who underwent mastectomy followed by reconstruction. The use of breast reconstruction increased from 22.3% of mastectomy cases in 2005 to 49.7% of mastectomy cases in 2015 (odds ratio [OR] 9.7, 95% confidence interval [CI] 7.3-12.8). Among those receiving reconstruction, the use of NSM increased from 1.7% in 2005 to 14.3% in 2015 (OR 9.4, 95% CI 7.1-12.5), with increased utilization among those with early-stage and locally advanced disease, such that by 2015, NSM was performed in 15.3% of mastectomies with reconstruction for DCIS, 14.3% of mastectomies with reconstruction for stage I-II breast cancer, and 10.7% of mastectomies with reconstruction for stage III breast cancer. Factors strongly predicting receipt of NSM included age < 45 years, smaller clinical tumor size, clinically node negative disease, use of neoadjuvant therapy, and facility type., Conclusions: There has been a dramatic increase in the use of breast reconstruction and NSM between 2005-2015. Further prospective studies evaluating oncologic outcomes of NSM in locally advanced breast cancer are warranted.

Background: Surgery after neoadjuvant chemotherapy (NCT) is an accepted treatment approach for locally advanced and some early-stage breast cancers, even for patients with a clinical complete response (cCR) after NCT. This study sought to evaluate the survival outcomes for patients with cCR to NCT who did not undergo surgery., Methods: The National Cancer Data Base (NCDB) was used to identify 93,417 women age 18 years or older with a diagnosis of invasive breast cancer who received NCT between 2010 and 2015. The study identified 350 women with cT1-4, N0-3, and M0 tumors who underwent NCT and did not have surgery. A matched surgical cohort was extracted from the NCDB, and overall survival (OS) was compared between the surgical and nonsurgical patients after NCT., Results: Of the 350 NCT patients who did not undergo surgery, 45 (12.9%) had cCR, 51 (14.6%) had a partial response, 241 (68.9%) had a response but whether complete or partial was not recorded, and 13 (3.7%) had no response/progression. The 5-year OS was better in the cCR group than in the no-cCR group (96.8% vs 69.8%; p = 0.004). A 5-year OS analysis of the cCR patients without surgery (n = 45; median follow-up period, 37 months) compared with the patients with a pathologic complete response who underwent surgery (n = 3938; median follow-up period, 43 months) showed no statistically significant difference (96.8% vs 92.5%, respectively; p = 0.15)., Conclusion: This retrospective cohort study demonstrated that active surveillance or de-escalation therapy may be an option for patients who achieve cCR. Prospective studies are underway to determine whether a subgroup of patients may forgo surgery in the setting of cCR after NCT.

Background: The feasibility of sentinel lymph node biopsy (SLNB) in patients with clinically node-positive (cN+) disease who convert to clinically node-negative (cN0) disease following neoadjuvant chemotherapy (NAC) has been evaluated in several large clinical trials, but it remains unclear whether the approach has been broadly adopted in the United States., Methods: The National Cancer Database was used to identify women diagnosed with cN+ breast cancer who received NAC followed by surgery between 2012 and 2015. Trends in axillary surgery were evaluated and multivariable logistic regression analyses performed to determine factors associated with receipt of SLNB., Results: Of 12,965 women cN+ at baseline, the use of SLNB increased from 31.8% in 2012 to 49% in 2015 (p < 0.001). Using axillary pCR as a surrogate for patients who convert to cN0 following NAC, among 5127 (39.5%) ypN0 patients, SLNB increased from 38.2 to 58.4% over the study period (p < 0.001), resulting in avoidance of axillary dissection in 42.2% of ypN0 patients by 2015. In adjusted analyses, factors significantly associated with SLNB attempt included cN1 disease, age < 45 years, treatment facility type, triple-negative and HER2-positive subtypes, and year of diagnosis. In women with residual isolated tumor cells (ITCs), micrometastases, and ypN1 disease, SLNB was the only axillary procedure performed in 36.9%, 23.6%, and 13.0% of cases., Conclusions: The use of SLNB in cN+ patients receiving NAC increased significantly between 2012 and 2015. SLNB alone was performed in more than 10% of patients with ypN1 disease, 20% with micrometastases, and 35% with ITCs; the oncologic safety of omitting axillary dissection in these patients requires further evaluation.

Background: The prognostic significance of low-volume residual nodal disease following neoadjuvant chemotherapy (NAC) is unknown., Methods: Women with cT1-4N0-1 breast cancer treated with NAC were identified from Dana-Farber/Brigham and Women's Cancer Center (DFBWCC) and the National Cancer Database (NCDB). Disease-free survival (DFS) and overall survival (OS) estimates according to pathologic nodal status were calculated using the Kaplan-Meier method, with Cox proportional hazards regression used to assess the effect of clinical variables on survival outcomes., Results: Among 967 DFBWCC patients, 27 (2.8%) had residual isolated tumor cells (ITCs) and 61 (6.3%) had micrometastases. Five-year DFS was significantly worse in those with residual ITCs (73.5%) and micrometastases (74.7%) relative to those who were ypN0 following NAC (88.4%, p < 0.001). On adjusted analysis, those with residual ITCs (hazard ratio [HR] 2.4, 95% confidence interval [CI] 1.20-3.81) and micrometastases (HR 2.14, 95% CI 1.20-3.81) had increased risk of recurrence relative to ypN0 patients. Among 35,536 NCDB patients, 543 (1.5%) had ITCs and 1132 (3.2%) had micrometastases. Five-year OS estimates were significantly worse with increasing residual nodal burden: ypN0, 88.9%; ypN0[i+], 82.8%; ypN1mi, 79.5%; ypN1, 77.6% (p < 0.001). Compared with patients with ypN0 disease, NCDB patients with ITCs and micrometastases had 1.9- and 2.2-fold risk of death (p < 0.001). On subgroup analysis, the effect of low-volume residual disease on mortality was most pronounced in patients with triple-negative and human epidermal growth factor receptor 2 (HER2)-positive disease., Conclusions: Low-volume residual nodal disease following NAC is associated with poorer DFS and OS relative to those who are node negative.

Previously, vibratory stimulation increased spontaneous swallowing rates in healthy volunteers indicating that sensory stimulation excited the neural control of swallowing. Here, we studied patients with severe chronic dysphagia following brain injury or radiation for head and neck cancer to determine if sensory stimulation could excite an impaired swallowing system. We examined (1) if laryngeal vibratory stimulation increased spontaneous swallowing rates over sham (no stimulation); (2) the optimal rate of vibration, device contact pressure, and vibratory mode for increasing swallowing rates; and (3) if vibration altered participants' urge to swallow, neck comfort, and swallow initiation latency. Vibration was applied to the skin overlying the thyroid lamina bilaterally in thirteen participants to compare vibratory rates 30, 70, 110, 150, or 70 + 110 Hz, different devices to neck pressures (2, 4, or 6 kilopascals), and pulsed versus continuous vibration. Swallows were confirmed from recordings of laryngeal accelerometry and respiratory apneas and viewing neck movement. Participants' swallowing rates, urge to swallow, discomfort levels, and swallow initiation latencies were measured. Vibration at 70 Hz and at 110 Hz significantly increased swallowing rates over sham. All vibratory frequencies except 70 + 100 Hz increased participants' urge to swallow, while no pressures or modes were optimal for increasing urge to swallow. No conditions increased discomfort. Vibration did not reduce measures of swallow initiation latency using accelerometry. In conclusion, as non-invasive neck vibration overlying the larynx increased swallowing rates and the urge to swallow without discomfort in patients with chronic dysphagia, the potential for vibratory stimulation facilitating swallowing during dysphagia rehabilitation should be investigated.

Aims: Overexpression of the mechanistic target of rapamycin (mTOR), a member of the PIKK (phosphoinositide kinase-related kinase) family, protects cardiomyocytes from cell death induced by pathological stimuli such as ischemia. We previously reported that posttranslational modification of mTOR plays an important role in regulating cardiac mTOR expression. The aim of this study was to see if Tel2 (telomere maintenance 2), a protein that regulates the abundance of PIKKs, confers similar cardioprotective effects as mTOR. Tel2 is not well-characterized in cardiomyocytes, therefore we examined the effects of Tel2 on cardiomyocyte viability under ischemic stress conditions., Materials and Methods: We overexpressed Tel2 or silenced Tel2 with siRNA in the HL-1 cardiomyocyte cell line to survey the effects of Tel2 overexpression and downregulation on cell survival during hypoxia. Adult mouse cardiomyocytes transfected with Tel2 adenoviruses were used to test whether Tel2 sufficiently prevented cardiomyocyte cell death against hydrogen peroxide (H 2 O 2 )., Key Findings: Overexpressing Tel2 increased mTOR expression with a concomitant increase in mTOR Complex 1 (mTORC1) and mTORC2 activity in HL-1 cells. Tel2 deletion decreased mTOR expression, and mTORC1 and mTORC2 activity accordingly. In both HL-1 cells and adult mouse cardiomyocytes, Tel2 overexpression protected cardiomyocytes under ischemic stress. These effects were mTOR-dependent, as mTOR inhibitors blunted the effects of Tel2. While gene silencing of Tel2 did not affect cell survival under normoxia, Tel2 silencing made cardiomyocytes more vulnerable to cell death under hypoxia., Significance: Upregulating Tel2 expression increases mTOR-mediated cardiomyocyte survival and targeting Tel2 could be another therapeutic strategy against ischemic heart disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Streptococcus pneumoniae is among the top causes of bacterial endophthalmitis, an infectious disease of the intraocular fluids. The mechanisms by which S. pneumoniae grows and thrives in the intraocular cavity are not well understood. We used a bacterial genome-wide assessment tool (transposon insertion site sequencing) to determine genes essential for S. pneumoniae growth in vitreous humor. The results indicated that an ascorbic acid (AA) transport system subunit was important for growth. We created an isogenic gene deletion mutant of the AA transcriptional activator, ulaR2 , in 2 strains of S. pneumoniae . Growth curve analysis indicated that ulaR2 deletion caused attenuated growth in vitro for both strains. However, in vivo vitreous humor infection in rabbits with either strain determined that ulaR2 was necessary for growth in one strain but not the other. These results demonstrate that ulaR2 may be important for fitness during S. pneumoniae endophthalmitis depending on the background of the strain.

Hand, foot, and mouth disease (HFMD), a highly contagious disease in children, is caused by human enteroviruses, including enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus A6 (CVA6). Although HFMD is usually mild and self-limiting, EV71 infection occasionally leads to fatal neurological disorders. Currently, no commercial antiviral drugs for HFMD treatment are available. Here, numerous sulfonated azo dyes, widely used as food additives, were identified as having potent antiviral activities against human enteroviruses. Among them, brilliant black BN (E151) was able to inhibit all EV71, CVA16, and CVA6 strains tested. In rhabdomyosarcoma cells, the 50% inhibitory concentrations of the dye E151 for various strains of EV71 ranged from 2.39 μM to 28.12 μM, whereas its 50% cytotoxic concentration was 1,870 μM. Food azo dyes, including E151, interacted with the vertex of the 5-fold axis of EV71 and prevented viral entry. Their efficacy in viral inhibition was regulated by amino acids at VP1-98, VP1-145, and/or VP1-246. Dye E151 not only prevented EV71 attachment but also eluted attached viruses in a concentration-dependent manner. Moreover, E151 inhibited the interaction between EV71 and its cellular uncoating factor cyclophilin A. In vivo studies demonstrated that E151 at a dose of 200 mg/kg of body weight/day given on the initial 4 days of challenge protected AG129 mice challenged with 10× the 50% lethal dose of wild-type EV71 isolates. Taken together, these data highlight E151 as a promising antiviral agent against EV71 infection. IMPORTANCE Human enterovirus 71 (EV71) is one of the causative agents of hand, foot, and mouth disease in children and is responsible for thousands of deaths in the past 20 years. Food azo dyes have been widely used since the nineteenth century; however, their biological effects on humans and microbes residing in humans are poorly understood. Here, we discovered that one of these dyes, brilliant black BN (E151), was particularly effective in inhibiting the infectivity of EV71 in both cell culture and mouse model studies. Mechanistic studies demonstrated that these sulfonated dyes mainly competed with EV71 attachment factors for viral binding to block viral attachment/entry to host cells. As no commercial antiviral drugs against EV71 are currently available, our findings open an avenue to exploit the development of permitted food dye E151 as a potential anti-EV71 agent., (Copyright © 2019 American Society for Microbiology.)

Tobacco mosaic virus (TMV) is a positive, single-stranded RNA virus. It encodes two replicases (126 kDa and 183 kDa), a movement protein and a coat protein. These proteins interact with host proteins for successful infection. Some host proteins such as eEF1α, Tm-1, TOM1, 14-3-3 proteins directly interact with Tobamovirus replication proteins. There are host proteins in the virus replication complex which do not interact with viral replicases directly, such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. We have used Proximity-dependent biotin identification (BioID) technique to screen for transient or weak protein interactions of host proteins and viral replicase in vivo. We transiently expressed BirA* tagged TMV 126 kDa replicase in TMV infected Nicotiana benthamiana plants. Among 18 host proteins, we identified NbSGT1 as a potential target for further characterization. Silencing of NbSGT1 in N. benthamiana plants increased its susceptibility to TMV infection, and overexpression of NbSGT1 increased resistance to TMV infection. There were weak interactions between NbSGT1 and TMV replicases but no interaction between them was found in Y2H assay. It suggests that the interaction might be transient or indirect. Therefore, the BioID technique is a valuable method to identify weak or transient/indirect interaction(s) between pathogen proteins and host proteins in plants. BIOLOGICAL SIGNIFICANCE: TMV is a well characterized positive-strand RNA virus model for study of virus-plant host interactions. It infects >350 plant species and is one of the significant pathogens of crop loss globally. Many host proteins are involved in TMV replication complex formation. To date there are few techniques available for identifying interacting host proteins to viral proteins. There is limited knowledge on transient or non-interacting host proteins during virus infection/replication. In this study, we used agroinfiltration-mediated in planta BioID technique to identify transiently or non-interacting host proteins to viral proteins in TMV-infected N. benthamiana plants. This technique allowed us to identify potential candidate proteins in the vicinity of TMV 126 kDa replicase. We have selected NbSGT1 and its overexpression suppresses TMV replication and increase plant resistance. NbSGT1 is believed to interact transiently or indirectly with TMV replicases in the presence of Hsp90/Hsp70. BioID is a novel and powerful technique to identify transiently or indirectly interacting proteins in the study of pathogen-host interactions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Autophagy is a conserved, highly-regulated catabolic process that plays important roles in growth, development and innate immunity in plants. In this study, we compared the rate of autophagy induction in Nicotiana benthamiana plants infected with Tobacco mosaic virus or the TMV 24A + UPD mutant variant, which replicates at a faster rate and induces more severe symptoms. Using a BirA* tag and proximity-dependent biotin identification (BioID) analysis, we identified host proteins that interact with the core autophagy protein, ATG8 in TMV 24A + UPD infected plants. By combining the use of a fast replicating TMV mutant and an in vivo protein-protein screening technique, we were able to gain functional insight into the role of autophagy in a compatible virus-host interaction., Results: Our study revealed an increased autophagic flux induced by TMV 24A + UPD, as compared to TMV in N. benthamiana. Analysis of the functional proteome associated with ATG8 revealed a total of 67 proteins, 16 of which are known to interact with ATG8 or its orthologs in mammalian and yeast systems. The interacting proteins were categorized into four functional groups: immune system process, response to ROS, sulphur amino acid metabolism and calcium signalling. Due to the presence of an ubiquitin-associated (UBA) domain, which is demonstrated to interact with ATG8, the Huntingtin-interacting protein K-like (HYPK) was selected for validation of the physical interaction and function. We used yeast two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and subcellular localization to validate the ATG8-HYPK interaction. Subsequent down-regulation of ATG8 by virus-induced gene silencing (VIGS) showed enhanced TMV symptoms, suggesting a protective role for autophagy during TMV 24A + UPD infection., Conclusion: This study presents the use of BioID as a suitable method for screening ATG8 interacting proteins in planta. We have identified many putative binding partners of ATG8 during TMV 24A + UPD infection in N. benthamiana plants. In addition, we have verified that NbHYPK is an interacting partner of ATG8. We infer that autophagy plays a protective role in TMV 24A + UPD infected plants.

In pathogens that produce lipooligosaccharide (LOS), sugar residues within the surface-exposed LOS outer core mediate interactions with components of the host immune system, promoting bacterial infection. Many LOS structures are controlled by phase variation mediated by random slipped-strand base mispairing, which can reversibly switch gene expression on or off. Phase variation diversifies the LOS, however its adaptive role is not well-understood. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen that causes a range of illnesses in the upper and lower respiratory tract. In NTHi a phase variable galactosyltransferase encoded by lic2A initiates galactose chain extension of the LOS outer core. The donor substrate for Lic2A, UDP-galactose, is generated from UDP-glucose by UDP-galactose epimerase encoded by galE . Our previous fitness profiling of H. influenzae mutants in a murine lung model showed that the galE mutant had a severe survival defect, while the lic2A mutant's defect was modest, leading us to postulate that unidentified factors act as suppressors of potential defects in a lic2A mutant. Herein we conducted a genome-wide genetic interaction screen to identify genes epistatic on lic2A for survival in the murine lung. An unexpected finding was that galE mutants exhibited restored virulence properties in a lic2A mutant background. We identified an alternative antibody epitope generated by Lic2A in the galE mutant that increased sensitivity to classical complement mediated killing in human serum. Deletion of lic2A or restoration of UDP-galactose synthesis alleviated the galE mutant's virulence defects. These studies indicate that when deprived of its galactosyl substrate, Lic2A acquires an alternative activity leading to increased recognition of NTHi by IgM and decreased survival in the lung model. Biofilm formation was increased by deletion of galE and by increased availability of UDP-GlcNAc precursors that can compete with UDP-galactose production. NTHi's ability to reversibly inactivate lic2A by phase-variation may influence survival in niches of infection in which UDP-Galactose levels are limiting.

Viruses are metastable macromolecular assemblies that toggle between multiple conformational states through molecular rearrangements that are critical for mediating viral host entry. Viruses respond to different host specific environmental cues to form disassembly intermediates for the eventual release of genomic material required for replication. Although static snapshots of these intermediates have been captured through structural techniques such as X-ray crystallography and cryo-EM, the mechanistic details of these conformational rearrangements underpinning viral metastability have been poorly understood. Amide hydrogen deuterium exchange mass spectrometry (HDXMS) is a powerful tool that measures hydrogen bonding propensities to probe changes in the dynamics of different macromolecular interactions. Chaotropic agents such as urea can be used to disrupt hydrogen bonds between different subunits, thereby ranking regions of the virus that are critical in maintaining viral stability. By controlled urea denaturation with HDXMS, we have identified specific loci in a Turnip Crinkle Virus (TCV) model showing increased deuterium exchange with even minimally disruptive concentrations of urea. These loci represent dynamic disassembly hotspots. These hotspots are predominantly present at the quaternary contacts at the 3-fold and 5-fold axes. This approach can be applied to detect vulnerabilities in virus icosahedral structures to uncover the molecular mechanism of viral disassembly., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To investigate the link between blood-brain-barrier (BBB) permeability and cerebral blood flow (CBF) and the relation with white matter hyperintensities (WMH) in cerebral small vessel disease (cSVD)., Methods: Twenty-seven patients with cSVD received dynamic susceptibility contrast and dynamic contrast-enhanced MRI to determine CBF and BBB permeability (expressed as leakage rate and volume), respectively. Structural MRI were segmented into normal-appearing white matter (NAWM) and WMH, for which a perilesional zone was defined. In these regions, we investigated the BBB permeability, CBF, and their relation using Pearson correlation r ., Results: We found a decrease in CBF of 2.2 mL/min/100 g ( p < 0.01) and an increase in leakage volume of 0.7% ( p < 0.01) per mm closer to the WMH in the perilesional zones. Lower CBF values correlated with higher leakage measures in the NAWM and WMH (-0.53 < r < -0.40, p < 0.05). This relation was also observed in the perilesional zones, which became stronger in the proximity of WMH ( p = 0.03)., Conclusion: BBB impairment and hypoperfusion appear in the WMH and NAWM, which increase in the proximity of the WMH, and are linked. Both BBB and CBF are regulated in the neurovascular unit (NVU) and the observed link might be due to the physiologic regulation mechanism of the NVU. This link may suggest an early overall deterioration of this unit., (© 2019 American Academy of Neurology.)

Blood-brain barrier (BBB) leakage increases with age and is involved in the pathophysiology of cerebral small vessel disease (cSVD). We examined the relationship between BBB leakage and white matter hyperintensity (WMH) volume and cognition, in cSVD patients and healthy controls. Seventy-seven patients with clinically overt cSVD and thirty-nine age matched healthy controls underwent dynamic contract-enhanced and structural brain MRI and neuropsychological assessment. We quantified BBB leakage volume and rate in normal appearing white matter (NAWM), WMH and cortical grey matter (CGM). Larger leakage volume and lower leakage rate in WMH were associated with larger WMH volume in cSVD but not in controls. Higher leakage rate in NAWM was associated with lower scores on executive function and information processing speed in healthy controls, whereas no relation with cognition was found in cSVD patients. Our findings support the involvement of BBB leakage in cSVD and aging. They also suggest that the mechanism of cognitive dysfunction in cSVD is more complex and multifactorial in cSVD compared with normal aging.

Cross-protection is a phenomenon in which infection with a mild virus strain protects host plants against subsequent infection with a closely related severe virus strain. This study showed that a mild strain mutant virus, Tobacco mosaic virus (TMV)-43A could cross protect Nicotiana benthamiana plants against wild-type TMV. Furthermore, we investigated the host responses at the proteome level to identify important host proteins involved in cross-protection. We used the isobaric tags for relative and absolute quantification (iTRAQ) technique to analyze the proteome profiles of TMV, TMV-43A and cross-protected plants at different time-points. Our results showed that TMV-43A can cross-protect N. benthamiana plants from TMV. In cross-protected plants, photosynthetic activities were augmented, as supported by the increased accumulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) enzymes, which are crucial for chlorophyll biosynthesis. The increased abundance of ROS scavenging enzymes like thioredoxins and L-ascorbate peroxidase would prevent oxidative damage in cross-protected plants. Interestingly, the abundance of defence-related proteins (14-3-3 and NbSGT1) decreased, along with a reduction in virus accumulation during cross-protection. In conclusion, we have identified several important host proteins that are crucial in cross-protection to counter TMV infection in N. benthamiana plants. BIOLOGICAL SIGNIFICANCE: TMV is the most studied model for host-virus interaction in plants. It can infect wide varieties of plant species, causing significant economic losses. Cross protection is one of the methods to combat virus infection. A few cross-protection mechanisms have been proposed, including replicase/coat protein-mediated resistance, RNA silencing, and exclusion/spatial separation between virus strains. However, knowledge on host responses at the proteome level during cross protection is limited. To address this knowledge gap, we have leveraged on a global proteomics analysis approach to study cross protection. We discovered that TMV-43A (protector) protects N. benthamiana plants from TMV (challenger) infection through multiple host pathways: secondary metabolite biosynthesis, photosynthesis, defence, carbon metabolism, protein translation and processing and amino acid biosynthesis. In the secondary metabolite biosynthesis pathway, enzymes 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) play crucial roles in chlorophyll biosynthesis during cross protection. In addition, accumulation of ROS scavenging enzymes was also found in cross-protected plants, providing rescues from excessive oxidative damage. Reduced abundance of plant defence proteins is correlated to reduced virus accumulation in host plants. These findings have increased our knowledge in host responses during cross-protection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Detailed characterization of typical human neurodevelopment is key if we are to understand the nature of mental and neurological pathology. While research on the cellular processes of neurodevelopment has made great advances, in vivo human imaging is crucial to understand our uniquely human capabilities, as well as the pathologies that affect them. Using magnetoencephalography data in the largest normative sample currently available (324 participants aged 6-45 years), we assess the developmental trajectory of resting-state oscillatory power and functional connectivity from childhood to middle age. The maturational course of power, indicative of local processing, was found to both increase and decrease in a spectrally dependent fashion. Using the strength of phase-synchrony between parcellated regions, we found significant linear and nonlinear (quadratic and logarithmic) trajectories to be characterized in a spatially heterogeneous frequency-specific manner, such as a superior frontal region with linear and nonlinear trajectories in theta and gamma band respectively. Assessment of global efficiency revealed similar significant nonlinear trajectories across all frequency bands. Our results link with the development of human cognitive abilities; they also highlight the complexity of neurodevelopment and provide quantitative parameters for replication and a robust footing from which clinical research may map pathological deviations from these typical trajectories., Competing Interests: Competing Interests: The authors have declared that no competing interests exist.

Tobacco mosaic virus (TMV) is a positive single-stranded RNA virus. Its 5' end ORF codes for the replicase proteins, namely 126 kDa and 183 kDa, respectively. These proteins interact with many host proteins to form a virus replication complex (VRC). This study aims to dissect the proteome profile of TMV-infected Nicotiana tabacum in host cellular and molecular pathways. We used the isobaric tags for relative and absolute quantification (iTRAQ) technique to analyse the differential global proteomic profile of TMV infected and mock infected plants. Out of 1897 total proteins, we identified 407 differentially abundant proteins and grouped them into three functional categories, namely metabolism, cellular processes and signalling processing. Our results showed that photosynthesis, carbon metabolism, plant defence, protein synthesis, and protein processing in the endoplasmic reticulum were significantly altered. Carbon metabolism and photosynthesis were present in very low abundance, whereas accumulation of reactive oxygen species and misfolded proteins lead to the accumulation of thioredoxin H-type 1. In conclusion, we identified several key host proteins that are involved in TMV infection/replication in N. tabacum plants. SIGNIFICANCE OF THE STUDY: TMV is one of the most widely studied plant virus. It is used as a tool to study host-virus interaction. There are several host proteins reported that facilitate VRC formation and replication of TMV. However, there is limited knowledge in the expression regulation of these host proteins upon TMV infection. This study is the first report that investigates the response of host protein expression involved in TMV infection through a quantitative proteomics technique iTRAQ, combined with LC-MS/MS analysis. We used TMV-infected Nicotiana tabacum plants to investigate the effects of TMV infection on host proteins. Our results revealed differential abundance of proteins involving various pathways in protein translation, protein processing, photosynthesis and plant defence. There was a high abundance of thioredoxin H-type 1, a protein that counters oxidative stress and accelerated regulation of fatty acid synthesis to provide additional lipid molecules for VRC formation. There was a significant reduction in abundance of psaA and psbB proteins in the photosynthetic pathways. Our results identified key candidate host proteins involved in TMV-infected N. tabacum for functional studies in future., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Research on Autism Spectrum Disorder (ASD) has focused on processing of socially-relevant stimuli, such as faces. Nonetheless, before being 'social', faces are visual stimuli. The present magnetoencephalography study investigated the time course of brain activity during an implicit emotional task in visual emotion-related regions in 19 adults with ASD (mean age 26.3 ± 4.4) and 19 typically developed controls (26.4 ± 4). The results confirmed previously-reported differences between groups in brain responses to emotion and a hypo-activation in the ASD group in the right fusiform gyrus around 150 ms. However, the ASD group also presented early enhanced activity in the occipital region. These results support that impaired face processing in ASD might be sustained by atypical responses in primary visual areas.

During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three-stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single-stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here, we show in vivo how R-loop formation drives a short purine-rich repeat, (GAA) 10 , to become a replication impediment that engages the repriming activity of the primase-polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R-loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R-loops promote the creation of replication blocks at susceptible structure-forming sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication., (© 2018 MRC Laboratory of Molecular Biology Published under the terms of the CC BY 4.0 license.)

Purpose: The combined use of anatomical magnetic resonance imaging (MRI), cellular MRI, and bioluminescence imaging (BLI) allows for sensitive and improved monitoring of brain metastasis in preclinical cancer models. By using these complementary technologies, we can acquire measurements of viable single cell arrest in the brain after systemic administration, the clearance and/or retention of these cells thereafter, the growth into overt tumours, and quantification of tumour volume and relative cancer cell viability over time. While BLI is very useful in measuring cell viability, some considerations have been reported using cells engineered with luciferase such as increased tumour volume variation, changes in pattern of metastatic disease, and inhibition of in vivo tumour growth., Procedures: Here, we apply cellular and anatomical MRI to evaluate in vivo growth differences between iron oxide labeled naïve (4T1BR5) and luciferase-expressing (4T1BR5-FLuc-GFP) murine brain-seeking breast cancer cells. Balb/C mice received an intracardiac injection of 20,000 cells and were imaged with MRI on days 0 and 14. Mice that received 4T1BR5-FLuc-GFP cells were also imaged with BLI on days 0 and 14., Results: The number of signal voids in the brain (representing iron-labeled cancer cells) on day 0 was significantly higher in mice receiving 4T1BR5 cells compared to mice receiving 4T1BR5-FLuc-GFP cells ( p < 0.0001). Mice that received 4T1BR5 cells also had significantly higher total brain tumour burden and number of brain metastases than mice that received 4T1BR5-FLuc-GFP cells ( p < 0.0001)., Conclusions: By employing highly sensitive cellular MRI tools, we demonstrate that engineered cells did not form tumours as well as their naïve counterparts, which appear to primarily be due to a reduction in cell arrest. These results indicate that engineering cancer cells with reporter genes may alter their tropism towards particular organs and highlight another important consideration for research groups that use reporter gene imaging to track metastatic cancer cell fate in vivo .

Hibiscus latent Singapore virus (HLSV) mutant HLSV-22A could not express coat protein (CP) nor infect plants systemically (Niu et al., 2015). In this study, a serine- and threonine-rich motif TTTSTTT at the C-terminus of HLSV CP was found to be involved in virus replication and systemic movement. Deletion the last amino acid residue in HLSV-22A led to a more rapid virus replication, but with delayed systemic movement. When the RNA structure in TTTSTTT motif was altered, while keeping its amino acids unchanged, mutants HLSV-87A-mmSL and HLSV-22A-mmSL showed no change in viral replication. These results indicated that the unique TTTSTTT motif is associated with virus replication and systemic movement. Deletion but not substitution of amino acid(s) at the C-terminus of TTTSTTT motif of HLSV CP with short internal poly(A) track enhanced virus replication, whereas the virus with a longer internal poly(A) tract of 87 A showed delayed systemic movement (147 words)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Long intergenic noncoding RNAs (lincRNAs), which possess diverse features such as remodeling chromatin and genome architecture, RNA stabilization, and genome architecture, are important regulatory factors in plant genomes. They serve to fine-tune the expression of neighboring genes. Here, we describe a procedure of discovery, identification, and functional characterization of plant lincRNAs after virus infection. From high-throughput RNA-Seq transcriptome analysis, the noncoding RNA transcripts with significant fold changes (upregulation or downregulation) will be discovered and identified. The lincRNA of interest will be further confirmed and validated using rapid amplification of cDNA ends (RACE). In addition, functional characterization of the lincRNA will be followed up through overexpression and knockdown strategies.

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Working Memory (WM) supports a wide range of cognitive functions, and is positively associated with academic achievement. Although fMRI studies have revealed WM networks in adults, little is known about how these networks develop to support successful WM performance in children. Using magnetoencephalography, we examined the networks underlying the maintenance of visual information in 6-year-old children. We observed an increase in mean whole-brain connectivity that was specific to the alpha frequency band during the retention interval associated with correct compared to incorrect responses. Additionally, our network analysis revealed elevated alpha synchronization during WM maintenance in a distributed network of frontal, parietal and temporal regions. Central hubs in the network were lateralized to the left hemisphere with dominant fronto-temporal connections, including the dorsolateral prefrontal cortex, middle temporal and superior temporal gyri, as well as other canonical language areas. Local changes in power were also analysed for seeds of interest, including the left inferior parietal lobe, which revealed an increase in alpha power after stimulus onset that was sustained throughout the retention period of WM. Our results therefore implicate sustained fronto-temporal alpha synchrony during the retention interval with subsequent successful WM responses in children, which may be aided by subvocal rehearsal strategies., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Individuals with autism spectrum disorder (ASD) demonstrate poor social functioning, which may be related to atypical emotional face processing. Altered functional connectivity among brain regions, particularly involving limbic structures may be implicated. The current magnetoencephalography (MEG) study investigated whole-brain functional connectivity of eight a priori identified brain regions during the implicit presentation of happy and angry faces in 20 7 to 10-year-old children with ASD and 22 typically developing controls. Findings revealed a network of increased alpha-band phase synchronization during the first 400 ms of happy face processing in children with ASD compared to controls. This network of increased alpha-band phase synchronization involved the left fusiform gyrus, right insula, and frontal regions critical for emotional face processing. In addition, greater connectivity strength of the left fusiform gyrus (maximal 85 to 208 ms) and right insula (maximal 73 to 270 ms) following happy face presentation in children with ASD compared to typically developing controls was found. These findings reflect altered neuronal communication in children with ASD only to happy faces during implicit emotional face processing.

The management of isolated in-breast tumor recurrence is complex, requiring careful consideration of prior local therapies to plan future multimodality treatment. Options for surgical management have evolved from standard salvage mastectomy with axillary clearance and now include repeat breast conservation with axillary staging in select patients. Reattempting sentinel lymph node biopsy may avoid the morbidity of extensive axillary surgery and has been shown to be feasible in clinically node-negative patients with oncologically safe outcomes. In the adjuvant setting, partial breast irradiation has emerged as a valuable means to improve local control rates with limited associated toxicity and acceptable overall cosmesis. Furthermore, results from prospective trials are now available to support the use of chemotherapy in hormone-receptor negative subgroups, which is associated with improvements in long-term, disease-free, and overall survival.

Vagus nerve stimulation (VNS) is increasingly considered for the treatment of intractable epilepsy and holds potential for the management of a variety of neuropsychiatric conditions. The emergence of the field of connectomics and the introduction of large-scale modeling of neural networks has helped elucidate the underlying neurobiology of VNS, which may be variably expressed in patient populations and related to responsiveness to stimulation. In this report, the authors outline current data on the underlying neural circuitry believed to be implicated in VNS responsiveness in what the authors term the "vagus afferent network." The emerging role of biomarkers to predict treatment effect is further discussed and important avenues for future work are highlighted.

Nontypeable Haemophilus influenzae (NTHi) is a major cause of community acquired pneumonia and exacerbation of chronic obstructive pulmonary disease. A current effort in NTHi vaccine development has focused on generating humoral responses and has been greatly impeded by antigenic variation among the numerous circulating NTHi strains. In this study, we showed that pulmonary immunization of mice with killed NTHi generated broad protection against lung infection by different strains. While passive transfer of immune antibodies protected only against the homologous strain, transfer of immune T cells conferred protection against both homologous and heterologous strains. Further characterization revealed a strong Th17 response that was cross-reactive with different NTHi strains. Responding Th17 cells recognized both cytosolic and membrane-associated antigens, while immune antibodies preferentially responded to surface antigens and were highly strain specific. We further identified several conserved proteins recognized by lung Th17 cells during NTHi infection. Two proteins yielding the strongest responses were tested as vaccine candidates by immunization of mice with purified proteins plus an adjuvant. Immunization induced antigen-specific Th17 cells that recognized different strains and, upon adoptive transfer, conferred protection. Furthermore, immunized mice were protected against challenge with not only NTHi strains but also a fully virulent, encapsulated strain. Together, these results show that the immune mechanism of cross-protection against pneumonia involves Th17 cells, which respond to a broad spectrum of antigens, including those that are highly conserved among NTHi strains. These mechanistic insights suggest that inclusion of Th17 antigens in subunit vaccines offers the advantage of inducing broad protection and complements the current antibody-based approaches., Competing Interests: The authors declare no conflict of interest.

Post-traumatic stress disorder (PTSD) is associated with heightened responses to threatening stimuli, particularly aggression-related emotional facial expressions. The stability over time of this neurophysiological 'hyperactive' threat response has not been determined. We studied implicit emotional face processing in soldiers with and without PTSD at two time-points (roughly 2 years apart) using magnetoencephalography to determine the response of oscillations and synchrony to happy and angry faces, and the reliability of this marker for PTSD over time. At the initial time-point we had 20 soldiers with and 25 without PTSD; 35 returned for follow-up testing 2 years later, and included 13 with and 22 without PTSD. A mixed-effects analysis was used. There were no significant differences (albeit a slight reduction) in the severity of PTSD between the two time-points. MEG contrasts of the neurophysiological networks involved in the processing of angry vs. happy faces showed that the PTSD group had elevated oscillatory connectivity for angry faces. Maladaptive hypersynchrony in PTSD for threatening faces was seen in subcortical regions, including the thalamus, as well as the ventromedial prefrontal cortex, cingulum gyri, inferior temporal and parietal regions. These results are generally consistent with prior studies and our own, and we demonstrate that this hyperconnectivity was stable over a two year period, in line with essentially stable symptomatology. Together, these results are consistent with the theory that hypervigilance in PTSD is driven by bottom-up, rapid processing of threat-related stimuli that engage a widespread network working in synchrony.