bluuebunny/crossref_metadata_2025 huggingface dataset:
CLINICOPATHOLOGICAL ANALYSIS OF SECONDARY RETINAL VASOPROLIFERATIVE TUMOR/REACTIVE RETINAL ASTROCYTIC TUMOR SUCCESSFULLY TREATED BY ENDORESECTION
Authors: Hashimoto Izumi , Takase Hiroshi , Kase Satoru , Iwasaki Yuko , Kobayashi Daisuke , Ohno-Matsui Kyoko
Published: September 2022

Purpose:

To report the clinicopathological findings of retinal vasoproliferative tumor/reactive retinal astrocytic tumor (VPT/RRAT) with retinal vasculitis, treated by tumor resection.

Methods:

A retrospective single case report.

Patient:

A 29-year-old Japanese woman was referred with cystoid macular edema and retinal vasculitis in the right eye. Best-corrected visual acuity was 0.9. Results of fundus examination, optical coherence tomography, and fluorescein angiography demonstrated VPT/RRATs in the temporal retina surrounded by a subretinal exudate, serous retinal detachment and macular edema, and retinal vasculitis. Despite 3 months of oral prednisolone treatment, a full-thickness macular hole developed. Pars plana vitrectomy and endoresection of the VPT/RRATs were performed. Pathologic and immunohistochemical analyses with anti-CD34 antibody, antiglial fibrillary acidic protein antibody, anti-Ki67 antibody, and anti–vascular endothelial growth factor antibody were performed on the excised tissue. Inflammation was evaluated by immunohistological staining with leukocyte common antigen (LCA), anti-CD3 antibody, and anti-CD20 antibody.

Results:

After surgery, the macular hole closed, best-corrected visual acuity improved to 1.2, retinal vasculitis was ameliorated, and retinal exudate disappeared. There was no recurrence of VPT/RRAT or retinal vasculitis. Pathologic examination showed that antiglial fibrillary acidic protein and anti–vascular endothelial growth factor were widely expressed, irrespective of the distribution of blood vessels. Ki67-positive proliferating cells were detected in the perivascular area. Leukocyte common antigen–positive leukocytes and CD3-positive T cells were detected throughout the samples, whereas CD20-positive B cells were rarely detected.

Conclusion:

Endoresection of VPT/RRAT could be a good treatment option for secondary VPT/RRAT accompanied by retinal vasculitis. Pathologic findings revealed for the first time that inflammatory cells infiltrate the tissue in secondary VPT/RRAT.
Rhino-orbital mucormycosis
Authors: Ryu Byoung U. , Laylani Noor Adnan Rashid , Davila-Siliezar Pamela , Lee Andrew G.
Published: September 2022

Purpose of review

The current article will update and review the clinical and radiological manifestations and management of rhino-orbital mucormycosis (ROM).

Recent findings

There has been an increase in cases of ROM worldwide, especially in India. Immunosuppression (especially diabetes mellitus) is a known predisposing risk factor for ROM. Delayed diagnosis and treatment of ROM can be vision or life-threatening. This article reviews the clinical and radiologic features, treatment, and prognosis of ROM with special emphasis on new and emerging therapies.

ROM is an angioinvasive fungal infection that affects the sinuses and orbits and may present to ophthalmologists. Clinicians should have a high index of suspicion for ROM, especially in patients with poorly controlled diabetes mellitus or other immunosuppression. Corticosteroid treatment (including the recent COVID-19 pandemic) may be a predisposing risk factor for ROM.
Second-Generation Antipsychotic Long-Acting Injection Reduced the Time of Restrictive Interventions in Patients With Schizophrenia
Authors: Horikoshi Sho , Miura Itaru , Mui Akihiro , Hikichi Takeshi , Aono Tetsuro , Hoshino Ken-yo , Terayama Kenji , Yabe Hirooki
Published: September 2022

Background

Whether second-generation antipsychotic long-acting injection (SGA-LAI) reduces psychotic symptoms at relapse compared with oral antipsychotics remains unclear. The present study investigated the effects of SGA-LAI on the time (in hours) of restrictive interventions in hospitalization by conducting a retrospective observational 4-year mirror-image study at a single medical center in Japan.

Method

We performed a retrospective observational mirror-image study conducted between November 2013 and January 2018. Data were initially retrieved from 101 patients. The 38 patients with schizophrenia who met the inclusion criteria were enrolled in the analysis. The primary outcome was the time of restrictive interventions and the secondary outcomes included the number of hospitalizations (total, voluntary, and involuntary) and bed days compared 2 years before and after initiating SGA-LAI. The restrictive interventions were defined as seclusion and physical restraints.

Results

The mean time of restrictive interventions significantly decreased from 43.7 to 3.03 (P = 0.021). The number of admissions and the total number of bed days in post–SGA-LAI fell from 1.03 to 0.61 (P = 0.011) and 130 to 39.3 (P = 0.003), respectively, compared with pre–SGA-LAI. In particular, the number of involuntary admissions was significantly reduced (0.50–0.26, P = 0.039).

Conclusions

The findings indicate that SGA-LAI reduced the time of restrictive interventions and the number of involuntary admissions. Moreover, SGA-LAI may contribute to mild psychiatric symptoms during relapse.
Cognitive Function in Adults With Acute Myeloid Leukemia Treated With Chemotherapy
Authors: Chan Ya-Ning , Betancur Stephanie , Conklin Jamie L. , Hirschey Rachel , Piepmeier Aaron , Foster Matthew , Anderson Ruth A. , Bender Catherine M. , Bryant Ashley Leak
Published: September 2022

Background

Chemotherapy is essential for treating acute myeloid leukemia (AML). Previous studies concluded that survivors of cancer who were treated with chemotherapy experience cognitive impairment. Therefore, it is important to understand cognitive function in survivors of AML.

Objective

The aim of this study was to explore distributions and correlates of cognitive function, and prediction of cognitive function on other outcomes in adults with AML who were treated with chemotherapy.

Methods

A health science librarian systematically searched PubMed, CINAHL, PsycINFO, and EMBASE databases. Two reviewers independently conducted the title, abstract, and full-text screening. Data were extracted and synthesized based on the aims of the review.

Results

A total of 10 articles were included. Findings indicate that up to 62.2% of adults with AML experienced impaired cognitive function after starting chemotherapy. Three studies found cognitive function remained stable over time. Education and cytokines were potential correlates of cognitive function. Worse cognitive function may predict lower physical performance and higher mortality, although the results were inconsistent across studies.

Conclusion

Impaired cognitive function was observed in adults with AML who were treated with chemotherapy. However, no study used a validated subjective cognitive-function–specific patient-reported questionnaire, and previous studies focusing on cognitive function included relatively young samples. Hence, further research on cognitive function in older adults with AML is needed.

Implications for Practice

Because of the high prevalence of cognitive impairment identified, it is important to screen cognitive function in adults with AML who are planning to receive chemotherapy to intervene and provide support earlier.
Maintenance of Functional Gains Following a Goal-Directed and FES-Assisted Cycling Program for Children With Cerebral Palsy
Authors: Armstrong Ellen L. , Boyd Roslyn N. , Horan Sean A. , Kentish Megan J. , Ware Robert S. , Carty Christopher P.
Published: September 2022

Purpose:

This study investigated whether the functional improvements associated with functional electrical stimulation-assisted cycling, goal-directed training, and adapted cycling in children with cerebral palsy were maintained 8 weeks after the intervention ceased.

Methods:

The intervention (2 × 1-hour supervised sessions and 1-hour home program/week) ran for 8 weeks. Primary outcomes were the Gross Motor Function Measure (GMFM-88) and the Canadian Occupational Performance Measure (COPM). Secondary outcomes included the GMFM-66 and goal scores, 5 times sit-to-stand test (FTSTS), Participation and Environment Measure-Children and Youth (PEM-CY), Pediatric Evaluation of Disability Inventory Computer Adaptive Test (PEDI-CAT), and cycling power output (PO). Outcomes were assessed at baseline, 8 and 16 weeks.

Results:

Twenty children participated (mean age = 10 years 3 months; SD = 2 years 11 months; Gross Motor Function Classification System II = 5, III = 6, and IV = 9). Improvements were retained above baseline at 16 weeks on the GMFM and COPM. Improvements in cycling PO, PEDI-CAT scores, PEM-CY environmental barriers and FTSTS were also retained.

Conclusion:

Functional improvements in children with cerebral palsy were retained 8 weeks post-intervention.
Application of the E-Value to Assess Bias in Observational Research in Plastic Surgery
Authors: Baxter Natalie B. , Kocheril Alex , Chung Kevin C.
Published: September 2022

Background:

The E-value is a statistical measure that is used to quantify the degree of unmeasured confounding that is necessary to undermine the treatment-outcome associations established in observational studies. Despite the substantial amount of observational research that informs evidence-based practice in plastic surgery, sensitivity analyses based on the E-value have not been conducted in the field.

Methods:

We performed a systematic search of the literature to identify meta-analyses of observational studies in plastic surgery. We calculated E-values for various treatment-outcome associations based on the risk, odds, or hazard ratios in each study, to assess unmeasured confounding effects that may influence the validity of the conclusions. We then analyzed the distribution of E-values from pooled versus individual studies.

Results:

We identified 45 meta-analyses that met the inclusion criteria, with each containing an average of 3 pooled assessments of observational data. The E-value of the pooled effect estimates ranged from 1.11 to 19.49, with an average value of 3.82. As for the individual effect estimates from each primary study within the meta-analyses, the E-values ranged from 1.00 to 321.50, with an average value of 8.74.

Conclusions:

We determined that E-values vary substantially across the literature and that unmeasured confounding may be present in a substantial number of observational studies. Although extant statistical techniques will continue to be necessary to control for measured confounding, the E-value is a novel concept that can facilitate more robust sensitivity analyses in plastic surgery research.
A novel method of fibula flap in situ distraction osteogenesis prior to flap transfer
Authors: Purnell Chad A , Aasen Margaret , Alkureishi Lee WT , Dumanian Gregory A , Patel Pravin K
Published: September 2022

Purpose:

The fibula flap is the workhorse for mandibular reconstruction, but fibula bone width is not ideal to match mandibular height. In this study, in-situ widening of the fibula with distraction osteogenesis prior to transfer is evaluated as a solution. We present a proof of concept of this technique with a patient series, including one patient who has undergone subsequent orthognathic surgery of their reconstructed mandible.

Methods:

A retrospective review of patients undergoing our technique was performed. A longitudinal fibula osteotomy is made in situ, and distraction is performed in the leg to widen the fibula. After distraction and consolidation periods, flaps were osteotomized and transferred to the mandible.

Results:

This technique was applied to three patients (ages 9, 11, and 13) with Pruzansky III mandibular hypoplasia at our institution over 15 years. In all cases, bony union was achieved. Mean surgical followup was 5 years. No significant morbidity occurred at the donor sites. Partial flap resorption was observed a number of years postoperatively in one patient. One patient developed TMJ ankylosis after closed treatment of an unrelated mandible fracture. One patient developed a sinus tract requiring debridement of a partial flap necrosis. One patient has gone on to have orthognathic surgery, including osteotomy of the fibula.

Conclusions:

In-situ fibula distraction osteogenesis is a novel technique to “pre-laminate" a fibula flap prior to transfer to the mandible. This method allows for the reconstruction of challenging mandibular defects without compromising bone height, pedicle length, or the ability to perform orthognathic surgery.
“A Multicenter Randomized Controlled Clinical Trial Evaluating Two Application Regimens of Dehydrated Human Amniotic Membrane and Standard of Care vs Standard of Care Alone in the Treatment of Venous Leg Ulcers”
Authors: Serena Thomas E. , Orgill Dennis P. , Armstrong David G. , Galiano Robert D. , Glat Paul , Carter Marissa , Kaufman Jarrod , Li William , Zelen Charles M.
Published: September 2022

Background:

An aseptically processed dehydrated human amnion and chorion allograft(dHACA) without terminal sterilization has been shown to improve healing rates in chronic wounds. This randomized controlled trial(RCT) evaluated the safety and effectiveness of weekly and biweekly applications of dHACA plus Standard of Care (SOC) compared to SOC alone on chronic venous leg ulcers (VLUs).

Methods:

This open-label RCT included patients with chronic VLUs at 8 wound care centers across the United States. The primary endpoint was the proportion of healed ulcers at 12 weeks. Secondary endpoints included the proportion of ulcers achieving 40% closure at 4 weeks and the incidence of adverse events (AEs).

Results:

Among 101 patients screened for eligibility, 60 were eligible and enrolled. At 12 weeks, significantly more VLUs healed in the two dHACA-treated groups (30/40, 75%) than in the SOC group (6/20, 30%), p = 0.001. Treatment with dHACA remained significant after adjustment for wound area (p = 0.002), with an odds ratio of 8.7 (95% confidence interval: 2.2-33.6). Only six VLUs (30%) healed in the SOC group compared to 15 (75%) in the weekly dHACA group (p = 0.02) and 15 (75%) in the biweekly dHACA group (p =0.02). There were no significant differences in the proportion of wounds with percent area reduction (PAR ) ≥40% at 4 weeks among all groups. The AE rate was 63.5%. Among the 38 AEs, none were graft- or procedure-related, and all were resolved with appropriate treatment.

Conclusions:

The adjunctive treatment of chronic VLUs with dHACA and SOC, regardless of weekly or biweekly application, significantly healed more VLUs than SOC alone, suggesting that the use of aseptically processed dHACA is advantageous as well as a safe and effective treatment option in the healing of chronic VLUs.
CRF neurons establish resilience via stress-history dependent BNST modulation
Authors: Haynes Sherod E , Lacagnina Anthony , Seong Hyun Seo , Afzal Muhammad , Morel Carole , Menigoz Aurelie , Rajan Kanaka , Clem Roger L , Mayberg Helen S , Rannie Donald G. , Young Larry J. , Han Ming-Hu
Published: September 2022

Cumulative stress is a major risk factor for developing major depressive disorder (MDD), yet not everyone experiencing chronic stress develops MDD. In those who do not, it is unclear at what point, or by what mechanism, a trajectory of stable resiliency emerges. Utilizing a 10-day repeated social defeat stress model (RSDS) for MDD, we observed that a critical period between 7 and 10 daily defeats marks the phenotypical divergence of resilient from susceptible mice. In response to ongoing stress, corticotropin-releasing factor (CRF) neurons of the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) display a sustained increased firing rate in resilient, but not susceptible mice. This neurophysiological adaptation was self-sustaining, but only after 7 critical stress exposures, indicating that the process of developing resilience is dependent on stress history. Our study reveals a novel process by which individuals might persist in the face of adversity by way of stress-provoked activation, not inhibition of a key CRF limbic region that establishes a pathway to resilience.
Classification Of Mushrooms Using Artificial Neural Network
Authors: Gupta Aaditya Prasad
Published: September 2022

The artificial neural network (ANN) has had remarkable success in pattern recognition in recent years. It stands for a new learning paradigm in artificial intelligence (AI) and machine learning and has been applied to problems ranging from speech recognition to the prediction of protein secondary structure, cancers, and gene prediction. Recent breakthrough results in image analysis and speech recognition have generated massive interest in this field. However, the mathematical and computational methodology underlying deep learning models is very challenging, especially for interdisciplinary scientists. In this manuscript, a Neural Network model is used to classify whether a given mushroom is edible or poisonous using Tensorflow in Python based on the attributes present in the dataset. The dataset includes descriptions of hypothetical samples corresponding to 23 species of gilled mushrooms in the Agaricus and Lepiota Family Mushroom, drawn from The Audubon Society Field Guide to North American Mushrooms (1981).
Plasticity in Na+/K+-ATPase thermal kinetics drives variation in the critical thermal minimum of adult Drosophila melanogaster
Authors: Andersen Mads Kuhlmann , Robertson R. Meldrum , MacMillan Heath A.
Published: September 2022

The majority of insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. Here, we focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated D. melanogaster. After first confirming cold acclimation altered SD onset, we investigated the dependency of the SD event on Na+/K+-ATPase activity by injecting an inhibitor, ouabain, into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, we found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.
CIRKO: A chemical-induced reversible gene knockout system for studying gene function in situ
Authors: Shi Hui , Jin Qin , Chen Fangbing , Ouyang Zhen , Gou Shixue , Liu Xiaoyi , Li Lei , Mu Shuangshuang , Lai Chengdan , Zhang Quanjun , Ye Yinghua , Wang Kepin , Lai Liangxue
Published: September 2022

Conditional loss and restoration of function are becoming important approaches for investigating gene function. Given that reversible conditional gene knockouts in cells required complicated manipulation, conditional inactivation and reactivation of a gene in primary somatic cells with limited proliferative capacity and in animal models remain difficult to achieve. Here, we first developed a reportable and reversible conditional intronic cassette (ReCOIN), wherein inactivation and reactivation of the gene are mediated via sequential expression of Cre and Flp recombinases, respectively. The expression pattern of the target gene can be monitored by direct visualization. To simply and tightly control temporal expression of the recombinases, on the basis of ReCOIN, we further presented a dual chemical-induced reversible gene knockout system (CIRKO) by insertion of reverse tetracycline transcriptional activator (rtTA) and tetracycline response element (TRE)-controlled Cre and FlpoERT2 recombinases cassettes into Rosa26 and Hipp11 loci of cells, respectively, in which transcription termination of the target gene can be induced at a specific stage in the presence of doxycycline, while gene restoration is achieved in the presence of doxycycline and tamoxifen simultaneously. This system provides a simple, rapid, and flexible gene switch for studying gene function in situ both in vitro and in vivo.

Impact statement

A novel chemical-induced reversible gene knockout system provides a simple, rapid, and flexible gene switch to facilitate the study of gene function in primary somatic cells in vitro, embryos in vitro or in vivo, and animals in vivo.
Magnetic Resonance Spectroscopy Spectral Registration Using Deep Learning
Authors: Ma David J. , Yang Yanting , Harguindeguy Natalia , Tian Ye , Guo Jia
Published: September 2022

A novel convolutional neural network based spectral registration (CNN-SR) approach is introduced to achieve efficient and accurate simultaneous frequency-and-phase correction (FPC) of single-voxel MEGA-PRESS magnetic resonance spectroscopy (MRS) data. For this approach, one neural network was trained and validated using a published simulated and in vivo MEGA-PRESS MRS dataset with a wide-range of artificial frequency (0-20 Hz) and phase (0-90°) offsets applied. The proposed CNN-SR approach was subsequently tested and compared to the sequential FPC deep learning approaches and demonstrated more effective and accurate performance. Furthermore, a large random Gaussian signal-to-noise ratio (SNR 20 and SNR 2.5) and line broadening (0-20 ms) was introduced to the original simulated dataset to investigate our model performance compared to the other deep learning models. The testing showed that CNN-SR was a more accurate quantification tool and resulted in a lower SNR when compared with the other deep learning methods, due to having smaller mean absolute errors in both frequency and phase offset predictions. For Off spectra, the CNN-SR model was capable of correcting frequency offsets with 0.014 ± 0.010 Hz and phase offsets with 0.104 ± 0.076° absolute errors on average for unseen simulated data with SNR 20 and correcting frequency offsets with 0.678 ± 0.883 Hz and phase offsets with 2.367 ± 2.616° absolute errors on average at very low SNR (2.5) and line broadening (0-20 ms) introduced. In addition, we tested the simulated dataset with additional SNR and line broadening on a more refined model (CNN-SR+) where the pre-trained CNN-SR was further optimized by minimizing the difference between individual spectra and a common template with unsupervised learning. The performance on Off spectra was improved to 0.058 ± 0.050 Hz for correcting frequency offsets and to 0.416 ± 0.317° for correcting phase offsets. We further evaluated the ability of our model to process the published Big GABA in vivo dataset and CNN-SR+ achieved the best performance. Moreover, additional frequency and phase offsets (i.e., small, moderate, large) were applied to the in vivo dataset, and CNN-SR+ also demonstrated better performance for FPC when compared to the other deep learning models. These results indicate the utility of using deep learning for spectral registration and demonstrate the application of unsupervised learning in further improving the model to achieve state-of-the-art performance.
Polygenic risk score-based phenome-wide association study identifies novel associations for Tourette syndrome
Authors: Jain Pritesh , Miller-Fleming Tyne , Topaloudi Apostolia , Yu Dongmei , Drineas Petros , Georgitsi Marianthi , Yang Zhiyu , Rizzo Renata , Müller-Vahl Kirsten R. , Tumer Zeynep , Mol Debes Nanette , Hartmann Andreas , Depienne Christel , Worbe Yulia , Mir Pablo , Cath Danielle C. , Boomsma Dorret I. , Roessner Veit , Wolanczyk Tomasz , Janik Piotr , Szejko Natalia , Zekanowski Cezary , Barta Csaba , Nemoda Zsofia , Tarnok Zsanett , Buxbaum Joseph D. , Grice Dorothy , Glennon Jeffrey , Stefansson Hreinn , Hengerer Bastian , Benaroya-Milshtein Noa , Cardona Francesco , Hedderly Tammy , Heyman Isobel , Huyser Chaim , Morer Astrid , Mueller Norbert , Munchau Alexander , Plessen Kerstin J , Porcelli Cesare , Walitza Susanne , Schrag Anette , Martino Davide ,   The EMTICS collaborative group, Dietrich Andrea ,   The TS-EUROGRAIN Network, Mathews Carol A. , Scharf Jeremiah M. , Hoekstra Pieter J. , Davis Lea K. , Paschou Peristera
Published: September 2022

Tourette Syndrome (TS) is a complex neurodevelopmental disorder characterized by vocal and motor tics lasting more than a year. It is highly polygenic in nature with both rare and common previously associated variants. Epidemiological studies have shown TS to be correlated with other phenotypes, but large-scale phenome wide analyses in biobank level data have not been performed to date. In this study, we used the summary statistics from the latest meta-analysis of TS to calculate the polygenic risk score (PRS) of individuals in the UK Biobank data and applied a Phenome Wide Association Study (PheWAS) approach to determine the association of disease risk with a wide range of phenotypes. A total of 57 traits were found to be significantly associated with TS polygenic risk, including multiple psychosocial factors and mental health conditions such as anxiety disorder and depression. Additional associations were observed with complex non-psychiatric disorders such as Type 2 diabetes, heart palpitations, and respiratory conditions. Cross-disorder comparisons of phenotypic associations with genetic risk for other childhood-onset disorders (e.g.: attention deficit hyperactivity disorder [ADHD], autism spectrum disorder [ASD], and obsessive-compulsive disorder [OCD]) indicated an overlap in associations between TS and these disorders. ADHD and ASD had a similar direction of effect with TS while OCD had an opposite direction of effect for all traits except mental health factors. Sex-specific PheWAS analysis identified differences in the associations with TS genetic risk between males and females. Type 2 diabetes and heart palpitations were significantly associated with TS risk in males but not in females, whereas diseases of the respiratory system were associated with TS risk in females but not in males. This analysis provides further evidence of shared genetic and phenotypic architecture of different complex disorders.
Human blood vessel organoids reveal a critical role for CTGF in maintaining microvascular integrity
Authors: Romeo Sara G , Secco Ilaria , Schneider Edoardo , Reumiller Christina M , Santos Celio XC , Pooni Aman , Yin Xiaoke , Theofilatos Konstantinos , Trevelin Silvia Cellone , Zeng Lingfang , Mann Giovanni E , Margariti Andriana , Mayr Manuel , Shah Ajay M , Giacca Mauro , Zampetaki Anna
Published: September 2022

The microvasculature plays a key role in tissue perfusion, transport of mediators, and exchange of gases and metabolites to and from tissues. Microvascular dysfunction has emerged as an important contributor to cardiovascular diseases. In this study we used human blood vessel organoids (BVOs) as a model of the microvasculature to delineate the mechanisms of microvascular dysfunction caused by metabolic rewiring. BVOs fully recapitulated key features of the normal human microvasculature, including reliance of mature endothelial cells (ECs) on glycolytic metabolism, as concluded from metabolic flux assays using13C-glucose labelling and mass spectrometry-based metabolomics. Treatment of BVOs with PFK15, a pharmacological inhibitor of glycolysis, resulted in rapid tissue restructuring, vessel regression with reduced pericyte coverage and alterations in tight junction morphology. Proteomic analysis of the BVO secretome revealed remodelling of the extracellular matrix and differential expression of paracrine mediators such as CTGF. Treatment with recombinant CTGF recovered tight junction formation and increased pericyte coverage in microvessels. Our metabolic and proteomics findings demonstrate that BVOs rapidly undergo restructuring in response to metabolic changes and identify CTGF as a critical paracrine regulator of microvascular integrity.
20E-dependent tyrosine phosphorylation of phospholipase C gamma underpins egg development in the malaria vectorAnopheles gambiae
Authors: Ferracchiato Simona , Catteruccia Flaminia , Peirce Matthew J.
Published: September 2022

The efficient development of eggs following a blood meal is central to the vector capacity ofAnopheles gambiaefemales. The ecdysteroid hormone 20-hydroxyecdysone (20E) plays a pivotal role in this process, yet the signaling mechanisms by which 20E exerts its effects remain incompletely understood. Here we show blood feeding is associated with increased tyrosine phosphorylation of an array of proteins coincident with the increase in titers of 20E that follows a blood meal. Injection of genistein, a tyrosine kinase inhibitor, reduces both the appearance of phosphotyrosine-containing proteins and fecundity, linking tyrosine phosphorylation to egg development. We identify one of the proteins phosphorylated after a blood meal as phospholipase C gamma (PLCγ) and show that its blood feeding-induced phosphorylation is dependent on endogenously synthesized 20E via the ecdysone receptor (EcR). Interruption of Src-like tyrosine kinase signaling inhibits phosphorylation of PLCγ as well as egg development after blood feeding, implicating Src-like kinases in the phosphorylation of PLCγ. Taken together, these data suggest that the effects of 20E onAn. gambiaeegg development are at least partially mediated through a tyrosine phosphorylation-dependent signaling cascade involving Src family tyrosine kinases and PLCγ.
A new strategy for identifying polysialylated proteins reveals they are secreted from cancer cells as soluble proteins and as part of extracellular vesicles
Authors: Hunter Carmanah , Derksen Tahlia , Karathra Julieanna , Baker Kristi , Nitz Mark , Willis Lisa M.
Published: September 2022

Polysialic acid (polySia) is a long homopolymer consisting of α2,8-linked sialic acid with tightly regulated expression in humans. In healthy adults, it occurs on cell surface glycoproteins in neuronal, reproductive, and immune tissues; however, it is aberrantly present in many cancers and its overexpression correlates with significantly increased metastasis and poor prognosis. Prompted by the observation that the MCF-7 breast cancer cell line contains only intracellular polySia, we investigated the secretion of polySia from MCF-7 cells. PolySia was found predominantly on soluble proteins in MCF-7 conditioned media, but also on extracellular vesicles (EVs), secreted from the cells. Since MCF-7 cells do not express known polysialylated proteins, we developed a robust method for purifying polysialylated proteins that uses a metabolic labelling strategy to introduce a bioorthogonal functionality into polySia. Using this method we identified three previously unknown polysialylated proteins, and found that two of these proteins - AGR2 and QSOX2 – were secreted from MCF-7 cells. We confirmed that QSOX2 found in EV-depleted MCF-7 cell conditioned media was polysialylated. Herein we report the secretion of polysialic acid on both soluble and EV-associated proteins from MCF-7 cancer cells and introduce a new method to efficiently identify polysialylated proteins. These findings have exciting implications for understanding the roles of polySia in cancer progression and metastasis and for identifying new cancer biomarkers.
Developmental shift in testosterone influence on prefrontal emotion control
Authors: Tyborowska Anna , Volman Inge , Niermann Hannah C. M. , Dapprich Anna L. , Smeekens Sanny , Cillessen Antonius H. N. , Toni Ivan , Roelofs Karin
Published: September 2022

A paradox of testosterone effects is seen in adolescents vs. adults in social emotional approach-avoidance behavior. During adolescence, high testosterone levels are associated with increased anterior prefrontal (aPFC) involvement in emotion control, whereas during adulthood this neuro-endocrine relation is reversed. Rodent work shows that, during puberty, testosterone transitions from a neuro-developmental to a social-sexual activating hormone. In this study, we explored whether this functional transition is also present in human adolescents and young adults. Using a prospective longitudinal design, we investigated the role of testosterone on neural control of social emotional behavior during the transitions from middle to late adolescence and into young adulthood. Seventy-one individuals (tested at ages 14, 17, and 20 years) performed an fMRI-adapted approach-avoidance (AA) task involving automatic and controlled actions in response to social emotional stimuli. In line with predictions from animal models, the effect of testosterone on aPFC engagement decreased between middle and late adolescence, and shifted into an activational role by young adulthood - impeding neural control of emotions. This change in testosterone function was accompanied by increased testosterone-modulated amygdala reactivity. These findings qualify the testosterone-dependent maturation of the prefrontal-amygdala circuit supporting emotion control during the transition from middle adolescence into young adulthood.
Conceptual associations generate sensory predictions
Authors: Yan Chuyao , de Lange Floris P. , Richter David
Published: September 2022

A crucial ability of the human brain is to learn and exploit probabilistic associations between stimuli to facilitate perception and behavior by predicting future events. While studies have shown how perceptual relationships are used to predict sensory inputs, relational knowledge is often between concepts rather than percepts (e.g., we learned to associate cats with dogs, rather than specific images of cats and dogs). Here we asked if and how sensory responses to visual input may be modulated by predictions derived from conceptual associations. To this end we exposed participants to arbitrary word-word pairs (e.g., car – dog) repeatedly, creating an expectation of the second word, conditional on the occurrence of the first. In a subsequent session, we exposed participants to novel word-picture pairs, while measuring fMRI BOLD responses. All word-picture pairs were equally likely, but half of the pairs conformed to the previously formed conceptual (word-word) associations, whereas the other half violated this association. Results showed suppressed sensory responses throughout the ventral visual stream, including early visual cortex, to pictures that corresponded to the previously expected words compared to unexpected words. This suggests that the learned conceptual associations were used to generate sensory predictions that modulated processing of the picture stimuli. Moreover, these modulations were tuning-specific, selectively suppressing neural populations tuned towards the expected input. Combined, our results suggest that recently acquired conceptual priors are generalized across domains and used by the sensory brain to generate feature specific predictions, facilitating processing of expected visual input.
Activity-dependent glassy cell mechanics I : Mechanical properties measured with active microrheology
Authors: Ebata H. , Umeda K. , Nishizawa K. , Nagao W. , Inokuchi S. , Sugino Y. , Miyamoto T. , Mizuno D.
Published: September 2022

Active microrheology was conducted in living cells by applying an optical-trapping force to vigorously-fluctuating tracer beads with feedback-tracking technology. The complex shear viscoelastic modulusG(ω) =G′(ω) –iG″(ω) was measured in HeLa cells in an epithelial-like confluent monolayer. We found thatG(ω) ∝ (−iω)1/2over a wide range of frequencies (1 Hz <ω/2π<10 kHz). Actin disruption and cell-cycle progression from G1 to S and G2 phases only had a limited effect onG(ω) in living cells. On the other hand,G(ω) was found to be dependent on cell metabolism; ATP-depleted cells showed an increased elastic modulusG′(ω) at low frequencies, giving rise to a constant plateau such thatG(ω) =G0+A(−iω)1/2. Both the plateau and the additional frequency dependency ∝ (−iω)1/2of ATP-depleted cells are consistent with a rheological response typical of colloidal jamming. On the other hand, the plateauG0disappeared in ordinary metabolically active cells, implying that living cells fluidize their internal states such that they approach the critical jamming point.

Statement of Significance

Intracellular mechanical properties were measured using optical-trap-based microrheology. Despite expectations to the contrary, shear viscoelasticity was hardly affected by reorganization of cytoskeletal structures during cell-cycle progression (G1 to S and G2 phases), nor by artificial disruption of the actin cytoskeleton induced by chemical inhibitors. Rather, the mechanics of cell interiors is governed by the glassy cytoplasm. Cells depleted of ATP solidified, whereas living cells that maintained metabolic activities were more fluid-like. Instead of a completely fluid response, however, we observed a characteristic power-law viscoelasticityG(ω) ∝ (−iω)1/2over the whole range of frequencies measured. Based on our current understanding of jamming rheology, we discuss how cells fluidize their internal state in a way that pushes the system towards the critical jamming transition.
Hyperaerated metalation calculator for E. coli strain JM109 (DE3) grown in LB media
Authors: Clough Sophie E. , Osman Deenah , Young Tessa R. , Robinson Nigel J.
Published: September 2022

We recently produced three web-based calculators that predict in vivo metal occupancies of proteins, based on the metal affinities of a protein of interest along with estimates of the availabilities of the labile buffered pools of metals inside a cell. Metal availabilities were calculated from the calibrated responses of DNA-binding, metal-sensing, transcriptional regulators. The availability of intracellular Fe(II) was estimated to be similar in E. coli grown under anaerobic conditions compared to cells grown aerobically in LB medium. The purpose of this article is to archive the background data that underpins the release of a new calculator for hyperaerated cells grown in flasks with baffles, with relatively low culture volumes plus high shaking speeds to give elevated oxygenation. The intracellular availability of Fe(II) calculated from the responses of the intracellular Fe(II) sensor Fur was estimated to be significantly lower in these hyperaerated cells than either of the previous values determined for anaerobic or aerobic cultures. The total number of atoms of Mn(II) per cell increased in hyperaerated cells albeit with only modest change in intracellular Mn(II) availability as estimated from the responses of the Mn(II) sensor MntR. Accurate determination of intracellular Ni(II) availability will require further calibration of the magnitude of the responses of the Ni(II) sensor NikR in hyperaerated cells to take account of the state of Fnr. The hyperaerated metalation calculator is made available online and as a spreadsheet, for use by others.
Attenuation of polysialic acid biosynthesis in cells by the small molecule inhibitor 8-keto-sialic acid
Authors: Hunter Carmanah , Gao Zhizeng , Chen Hong-Ming , Thompson Nicole , Wakarchuk Warren , Nitz Mark , Withers Stephen G. , Willis Lisa M.
Published: September 2022

Sialic acids are key mediators of cell function, particularly with regards to cellular interactions with the surrounding environment. Reagents that modulate the display of specific sialyl glycoforms at the cell surface would be useful biochemical tools and potentially allow for therapeutic intervention in numerous challenging chronic diseases. While multiple strategies are being explored for the control of cell surface sialosides, none that shows high selectivity between sialyltransferases or that targets a specific sialyl glycoform has yet to emerge. Here we describe a strategy to block the formation of α2,8-linked sialic acid chains (oligo- and polysialic acid) through the use of 8-keto-sialic acid as a chain-terminating metabolic inhibitor that, if incorporated, can not be elongated. 8-Keto-sialic acid is non-toxic at effective concentrations and serves to block polysialic acid synthesis in cancer cell lines and primary immune cells, with minimal effects on other sialyl glycoforms.
A role for BCL2L13 and autophagy in germline purifying selection of mtDNA
Authors: Kremer Laura S. , Bozhilova Lyuba V. , Rubalcava-Gracia Diana , Filograna Roberta , Upadhyay Mamta , Koolmeister Camilla , Chinnery Patrick F. , Larsson Nils-Göran
Published: September 2022

Mammalian mitochondrial DNA (mtDNA) is inherited uniparentally through the female germline without undergoing recombination. This poses a major problem as deleterious mtDNA mutations must be eliminated to avoid a mutational meltdown over generations. At least two mechanisms that can decrease the mutation load during maternal transmission are operational: a stochastic bottleneck for mtDNA transmission from mother to child, and a directed purifying selection against transmission of deleterious mtDNA mutations. However, the molecular mechanisms controlling these processes remain unknown. In this study, we systematically tested whether decreased autophagy contributes to purifying selection by crossing the C5024T mouse model harbouring a single pathogenic heteroplasmic mutation in the tRNAAla gene of the mtDNA with different autophagy-deficient mouse models, including knockouts of Parkin, Bcl2l13, Ulk1, and Ulk2. Our study reveals a robust effect of knockout of Bcl2l13 on the selection process, and weaker evidence for the effect of Ulk1 and potentially Ulk2, while no statistically significant impact is seen for knockout of Parkin. This points at distinctive roles of these players in germline purifying selection. Overall, our approach provides a framework for investigating the roles of other important factors involved in the enigmatic process of purifying selection and guides further investigations for the role of BCL2L13 in the elimination of non-synonymous mutations in protein-coding genes.
Global profiling of the RNA and protein complexes ofEscherichia coliby size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq)
Authors: Chihara Kotaro , Gerovac Milan , Hör Jens , Vogel Jörg
Published: September 2022

New methods for the global identification of RNA-protein interactions have led to greater recognition of the abundance and importance of RNA-binding proteins (RBPs) in bacteria. Here, we expand this tool kit by developing SEC-seq, a method based on a similar concept as the established Grad-seq approach. In Grad-seq, cellular RNA and protein complexes of a bacterium of interest are separated in a glycerol gradient, followed by high-throughput RNA-sequencing and mass spectrometry analyses of individual gradient fractions. New RNA-protein complexes are predicted based on the similarity of their elution profiles. In SEC-seq, we have replaced the glycerol gradient with separation by size exclusion chromatography, which shortens operation times and offers greater potential for automation. Applying SEC-seq toEscherichia coli, we find that the method provides a higher resolution than Grad-seq in the lower molecular weight range up to ∼500 kDa. This is illustrated by the ability of SEC-seq to resolve two distinct, but similarly sized complexes of the global translational repressor CsrA with either of its antagonistic small RNAs, CsrB and CsrC. We also characterized changes in the SEC-seq profiles of the small RNA MicA upon deletion of its RNA chaperones Hfq and ProQ and investigated the redistribution of these two proteins upon RNase treatment. Overall, we demonstrate that SEC-seq is a tractable and reproducible method for the global profiling of bacterial RNA-protein complexes that offers the potential to discover yet-unrecognized associations between bacterial RNAs and proteins.
Improved Genome Editing by an Engineered CRISPR-Cas12a
Authors: Ma Enbo , Chen Kai , Shi Honglue , Stahl Elizabeth C. , Adler Ben , Liu Junjie , Zhou Kaihong , Ye Jinjuan , Doudna Jennifer A.
Published: September 2022

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a (LbCas12a) that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2-18-fold depending on the genomic locus. Finally, we found that a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy of coupled rational engineering and directed evolution establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The availability of experimental and predicted structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome editing proteins.
The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins
Authors: Miller Alexandria N. , Houlihan Patrick R. , Matamala Ella , Cabezas-Bratesco Deny , Lee Gi Young , Cristofori-Armstrong Ben , Dilan Tanya L. , Sanchez-Martinez Silvia , Matthies Doreen , Yan Rui , Yu Zhiheng , Ren Dejian , Brauchi Sebastian E. , Clapham David E.
Published: September 2022

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.