Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

CITEViz: interactively classify cell populations in CITE-Seq via a flow cytometry-like gating workflow using R-Shiny.

BACKGROUND: The rapid advancement of new genomic sequencing technology has enabled the development of multi-omic single-cell sequencing assays. These assays profile multiple modalities in the same cell and can often yield new insights not revealed with a single modality. For example, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) simultaneously profiles the RNA transcriptome and the surface protein expression. The surface protein markers in CITE-Seq can be used to identify cell populations similar to the iterative filtration process in flow cytometry, also called "gating", and is an essential step for downstream analyses and data interpretation. While several packages allow users to interactively gate cells, they often do not process multi-omic sequencing datasets and may require writing redundant code to specify gate boundaries. To streamline the gating process, we developed CITEViz which allows users to interactively gate cells in Seurat-processed CITE-Seq data. CITEViz can also visualize basic quality control (QC) metrics allowing for a rapid and holistic evaluation of CITE-Seq data. RESULTS: We applied CITEViz to a peripheral blood mononuclear cell CITE-Seq dataset and gated for several major blood cell populations (CD14 monocytes, CD4 T cells, CD8 T cells, NK cells, B cells, and platelets) using canonical surface protein markers. The visualization features of CITEViz were used to investigate cellular heterogeneity in CD14 and CD16-expressing monocytes and to detect differential numbers of detected antibodies per patient donor. These results highlight the utility of CITEViz to enable the robust classification of single cell populations. CONCLUSIONS: CITEViz is an R-Shiny app that standardizes the gating workflow in CITE-Seq data for efficient classification of cell populations. Its secondary function is to generate basic feature plots and QC figures specific to multi-omic data. The user interface and internal workflow of CITEViz uniquely work together to produce an organized workflow and sensible data structures for easy data retrieval. This package leverages the strengths of biologists and computational scientists to assess and analyze multi-omic single-cell datasets. In conclusion, CITEViz streamlines the flow cytometry gating workflow in CITE-Seq data to help facilitate novel hypothesis generation.

Seek and ye shall find: The utility of perioperative VTE screening in high-risk oncologic patients undergoing abdominopelvic surgery.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the prevalence and risk factors associated with perioperative venous thromboembolism (VTE) in patients undergoing major oncologic surgery using an epidural catheter (EC) for postoperative analgesia with mechanical prophylaxis and without chemoprophylaxis. METHODS: Six hundred and twenty-six patients undergoing major oncologic surgery from 2009 to 2023 were evaluated. VTE was defined as deep vein thrombosis above the level of the knee. Lower extremity venous duplexes (LEVDs) were done preoperatively and postoperatively after the EC was removed. All patients received mechanical thromboprophylaxis, but not chemical prophylaxis, while the EC was in place. A generalized linear multivariable model was constructed to identify risk factors that predict pre and postoperative VTE. RESULTS: 29/626 patients (4.6%) were found to have preoperative VTE. 16/626 (2.6%) were found to have a postoperative VTE when their preoperative LEVD was negative. In comparison to patients without preoperative VTE, those with VTE were more likely to be male, anticoagulated, and have a history of coronary artery disease. Patients in the postoperative VTE group were older, male, anticoagulated, and had a history of VTE. On multivariable analysis, previous history of VTE was the risk factor most strongly associated with both pre and postoperative VTE. CONCLUSION: Oncologic patients undergoing elective abdominopelvic surgery with epidural analgesia should be screened in the perioperative setting with LEVD to identify VTE and possibly prevent PE.

Mental health stigma and health-seeking behaviors amongst pregnant women in Vietnam: a mixed-method realist study.

BACKGROUND: Approximately 15% of women in low-and middle-income countries experience common perinatal mental disorders. Yet, many women, even if diagnosed with mental health conditions, are untreated due to poor quality care, limited accessibility, limited knowledge, and stigma. This paper describes how mental health-related stigma influences pregnant women's decisions not to disclose their conditions and to seek treatment in Vietnam, all of which exacerbate inequitable access to maternal mental healthcare. METHODS: A mixed-method realist study was conducted, comprising 22 in-depth interviews, four focus group discussions (total participants n = 44), and a self-administered questionnaire completed by 639 pregnant women. A parallel convergent model for mixed methods analysis was employed. Data were analyzed using the realist logic of analysis, an iterative process aimed at refining identified theories. Survey data underwent analysis using SPSS 22 and descriptive analysis. Qualitative data were analyzed using configurations of context, mechanisms, and outcomes to elucidate causal links and provide explanations for complexity. RESULTS: Nearly half of pregnant women (43.5%) would try to hide their mental health issues and 38.3% avoid having help from a mental health professional, highlighting the substantial extent of stigma affecting health-seeking and accessing care. Four key areas highlight the role of stigma in maternal mental health: fear and stigmatizing language contribute to the concealment of mental illness, rendering it unnoticed; unconsciousness, normalization, and low literacy of maternal mental health; shame, household structure and gender roles during pregnancy; and the interplay of regulations, referral pathways, and access to mental health support services further compounds the challenges. CONCLUSION: Addressing mental health-related stigma could influence the decision of disclosure and health-seeking behaviors, which could in turn improve responsiveness of the local health system to the needs of pregnant women with mental health needs, by offering prompt attention, a wide range of choices, and improved communication. Potential interventions to decrease stigma and improve access to mental healthcare for pregnant women in Vietnam should target structural and organizational levels and may include improvements in screening and referrals for perinatal mental care screening, thus preventing complications.

Signatures of immune dysfunction in HIV and HCV infection share features with chronic inflammation in aging and persist after viral reduction or elimination.

Chronic inflammation is thought to be a major cause of morbidity and mortality in aging, but whether similar mechanisms underlie dysfunction in infection-associated chronic inflammation is unclear. Here, we profiled the immune proteome, and cellular composition and signaling states in a cohort of aging individuals versus a set of HIV patients on long-term antiretroviral therapy therapy or hepatitis C virus (HCV) patients before and after sofosbuvir treatment. We found shared alterations in aging-associated and infection-associated chronic inflammation including T cell memory inflation, up-regulation of intracellular signaling pathways of inflammation, and diminished sensitivity to cytokines in lymphocytes and myeloid cells. In the HIV cohort, these dysregulations were evident despite viral suppression for over 10 y. Viral clearance in the HCV cohort partially restored cellular sensitivity to interferon-α, but many immune system alterations persisted for at least 1 y posttreatment. Our findings indicate that in the HIV and HCV cohorts, a broad remodeling and degradation of the immune system can persist for a year or more, even after the removal or drastic reduction of the pathogen load and that this shares some features of chronic inflammation in aging.

Community Pharmacy Chlamydia and Gonorrhea Test-To-Treat Program: Development of an Implementation Toolkit.

BACKGROUND: Sexually transmitted infection (STI) surveillance showed more than 2.5 million cases of chlamydia, gonorrhea, and syphilis nationally in the United States in 2022. Individuals often seek out non-emergency medical care at pharmacies. This makes community pharmacies well-positioned to address rising STI rates by offering services to screen and treat common STIs. A local health department, an independent pharmacy, and a school of pharmacy in Pennsylvania partnered to implement a test-to-treat service for chlamydia and gonorrhea within a pharmacy. This pilot program utilized: (1) patient self-collected test kits for chlamydia and gonorrhea screening and; (2) standing orders for treatment at the pharmacy. One goal of this pilot was to develop resources others can use to implement similar pharmacy-based chlamydia and gonorrhea testing and treatment services. OBJECTIVE: Develop an expert-informed implementation toolkit for a chlamydia and gonorrhea test-to-treat program at a community pharmacy. METHODS: The "How to Build an Implementation Toolkit from Start to Finish" framework from the University of California at Berkeley was used to design the initial toolkit outline. Toolkit content was triangulated from three sources: (1) comprehensive literature review; (2) pilot program implementation team meetings; and (3) feedback from public health and other experts. Pilot program partners met regularly to review and edit the toolkit. The draft toolkit was then reviewed by outside experts and potential end-users . RESULTS: An 11-item toolkit was developed. Toolkit contents were reviewed by 11 outside experts and potential end-users. Toolkit resources included STI training resources for pharmacy teams, testing and treatment standing orders, pharmacy treatment screening form, marketing strategies, patient education materials, sample workflow, essential supply list, and other key resources. CONCLUSION: Pharmacies may need additional resources for STI testing and treatment program implementation. Toolkit resources developed from this pilot program may help pharmacies overcome implementation barriers for similar programs.

Development of a Pharmacy Point-of-Dispensing Toolkit for Anthrax Post-Exposure Prophylaxis for Allegheny County Postal Workers.

CONTEXT: The Centers for Disease Control and Prevention (CDC) and the US Postal Service (USPS) consider anthrax to be a potential threat to USPS workers. A county health department-owned pharmacy supports local USPS response in the event of an exposure. The pharmacy team identified the need to review and update the local anthrax response plan. PROGRAM/POLICY: A Pharmacy Point-of-Dispensing Toolkit and response plan for initial 10-day post-exposure antibiotic prophylaxis was developed for use by a local health department in the event of a mass anthrax exposure at a US Post Office sorting facility. The pharmacist's role in medical countermeasures planning for anthrax exposure is also discussed to illustrate how pharmacists' medication expertise can be utilized. EVALUATION: The CDC's Public Health Preparedness Capabilities: National Standards for State and Local Planning framework and inputs from an interprofessional stakeholder team were used to develop a Medical Countermeasures Response Plan and Implementation Toolkit for mass point-of-dispensing (POD) in the event of an anthrax exposure. IMPLEMENTATION AND DISSEMINATION: Stakeholders attended a USPS Community Partner Training event where additional revisions to the toolkit were made. The toolkit and standing order are now implemented at the local health department to be reviewed and updated on a yearly basis by health department leadership. DISCUSSION: Pharmacists can use their medication expertise and experience with patient education to design emergency response plans focused on increasing patient safety and medication adherence. Pharmacists should be involved in emergency response and medical countermeasures planning that involve medications.

Photon-counting versus Dual-Source CT of Congenital Heart Defects in Neonates and Infants: Initial Experience.

Background Photon-counting CT (PCCT) has been shown to improve cardiovascular CT imaging in adults. Data in neonates, infants, and young children under the age of 3 years are missing. Purpose To compare image quality and radiation dose of ultrahigh-pitch PCCT with that of ultrahigh-pitch dual-source CT (DSCT) in children suspected of having congenital heart defects. Materials and Methods This is a prospective analysis of existing clinical CT studies in children suspected of having congenital heart defects who underwent contrast-enhanced PCCT or DSCT in the heart and thoracic aorta between January 2019 and October 2022. CT dose index and dose-length product were used to calculate effective radiation dose. Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated by standardized region-of-interest analysis. SNR and CNR dose ratios were calculated. Visual image quality was assessed by four independent readers on a five-point scale: 5, excellent or absent; 4, good or minimal; 3, moderate; 2, limited or substantial; and 1, poor or massive. Results Contrast-enhanced PCCT (n = 30) or DSCT (n = 84) was performed in 113 children (55 female and 58 male participants; median age, 66 days [IQR, 15-270]; median height, 56 cm [IQR, 52-67]; and median weight, 4.5 kg [IQR, 3.4-7.1]). A diagnostic image quality score of at least 3 was obtained in 29 of 30 (97%) with PCCT versus 65 of 84 (77%) with DSCT. Mean overall image quality ratings were higher for PCCT versus DSCT (4.17 vs 3.16, respectively; P < .001). SNR and CNR were higher for PCCT versus DSCT with SNR (46.3 ± 16.3 vs 29.9 ± 15.3, respectively; P = .007) and CNR (62.0 ± 50.3 vs 37.2 ± 20.8, respectively; P = .001). Mean effective radiation doses were similar for PCCT and DSCT (0.50 mSv vs 0.52 mSv; P = .47). Conclusion At a similar radiation dose, PCCT offers a higher SNR and CNR and thus better cardiovascular imaging quality than DSCT in children suspected of having cardiac heart defects. © RSNA, 2023.

Predicting the Strength of Cohesive and Adhesive Interparticle Interactions for Dry Powder Inhalation Blends of Terbutaline Sulfate with α-Lactose Monohydrate.

Grid-based systematic search methods are used to investigate molecule-molecule, molecule-surface, and surface-surface contributions to interparticle interactions in order to identify the crystal faces that most strongly affect particle behavior during powder blend formulation and delivery processes. The model system comprises terbutaline sulfate (TBS) as an active pharmaceutical ingredient (API) and α-form lactose monohydrate (LMH). A combination of systematic molecular modeling and X-ray computed tomography (XCT) is used to determine not only the adhesive and cohesive interparticle energies but, also the agglomeration behavior during manufacturing and de-agglomeration behavior during delivery after inhalation. This is achieved through a detailed examination of the balance between the adhesive and cohesive energies with the XCT results confirming the blend segregation tendencies, through the particle-particle de-agglomeration process. The results reveal that the cohesive interaction energies of TBS-TBS are higher than the adhesive energies between TBS and LMH, but that the cohesive energies of LMH-LMH are the smallest between molecule and molecule, molecule and surface, and surface and surface. This shows how systematic grid-search molecular modeling along with XCT can guide the digital formulation design of inhalation powders in order to achieve optimum aerosolization and efficacy for inhaled medicines. This will lead to faster pharmaceutical design with less variability, higher quality, and enhanced performance.

A qualitative study identifying challenges resulting from complex evidence on lifestyle factors and cancer: perspectives from Black and Latina cancer survivors and healthcare providers.

PURPOSE: Most breast cancer survivors have challenges with adopting healthy lifestyle behaviors. This may be due to contextual challenges that result from the complex nature of the evidence. To address this gap, we explored the experiences of breast cancer survivors of color and oncology healthcare providers. METHODS: Content analysis with inductive and deductive approaches was used for semi-structured interviews with 26 female breast cancer survivors and 10 oncology healthcare providers from Greater New Haven, Connecticut. RESULTS: Survivors identified substantial confusion on the evidence regarding lifestyle behaviors and breast cancer, stemming from inadequate healthcare provider counseling and an overreliance on informal sources of information. Providers identified lack of evidence-based knowledge as a barrier to counseling on these topics. There was a mixed perspective regarding the consistency of evidence, stemming from a combination of gaps in the available evidence and accessing evidence-based knowledge from a wide range of professional resources. Some providers perceived the guidelines as consistent; others felt guidelines were constantly changing, impacting how and on what they counseled. Therefore, many healthcare providers in oncology care relied on generic messaging on lifestyle behaviors after a cancer diagnosis. CONCLUSIONS: Inconsistent information sources, the rapidly changing evidence, and gaps in the current evidence contribute to generic messaging about lifestyle behaviors and may inhibit a survivor's ability to engage in behavior change. Consistent and uniform healthy lifestyle guidelines for cancer outcomes may address both provider and patient level barriers to knowledge.

Gender differences and cooperation in medical authorships - an analysis of the recent ten years in five key medical disciplines.

BACKGROUND: Career prospects in academic medicine are strongly linked to scientific authorship and this marker has been widely used as an indicator of gender equity in academia. However, direct comparisons of medical disciplines regarding their proportion of female physicians (FP) in different countries are missing. This study examines the gender parity and gender cooperation using first authorships (FA) and senior authorships (SA) of scientific publications in five medical disciplines and six different OECD countries over a 10-year time-trend. METHODS: Articles from three high-impact journals in each of the medical discipline radiology, urology, surgery, gynecology, and pediatrics from the years 2007/8 and 2017/18 were retrospectively reviewed. The gender and affiliation location of the FA and SA of original research articles and reviews were assigned and compared with the proportion of in each discipline for the United States of America, Canada, United Kingdom, France, Germany, and Japan. Mantel-Haenszel test and multinomial logistic regression models were used to calculate differences in proportions of women authors and FP and to assess trends and proportions of FA and SA. RESULTS: 30,803 articles were evaluated. Equally, with rising proportions of FP in all disciplines, the number of women authors increased across years. The shares of women FAs were either significantly higher (urology/surgery/gynecology) or balanced (pediatrics/radiology) compared to the proportion of FP. In contrast, the shares of women SA were balanced only in disciplines with a low proportion of FP (urology and surgery) and otherwise reduced. Women same-gender cooperation was as common as men same-gender cooperation and preferred over a women-led mixed gender cooperation in disciplines where this seemed to be practicable due to the high proportions of FP. CONCLUSION: In contrast to FA, a significant disparity persists in SA, particularly in disciplines with a high proportion of FP. The discrepancy between FA and SA may reflect, among others, dropout from an academic career in early or mid-academic levels, for example, due to structural inequality; together with the findings on gender preference in authorship collaborations, this may inform future strategies for promoting equal career advancement for women physicians.

Influence of minimal invasive extracorporeal circuits on dialysis dependent patients undergoing cardiac surgery.

INTRODUCTION: Cardiac surgery in patients on chronic renal dialysis is associated with significant morbidity and mortality. Minimally invasive extracorporeal circuits (MiECC) have shown a positive impact on patient outcome in different high-risk populations. This retrospective study compares the outcome of these high-risk patients undergoing heart surgery either with a MiECC or a conventional extracorporeal circulation (CECC). METHODS: This is a single-center experience including 131 consecutive dialysis dependent patients undergoing cardiac surgery between January 2006 and December 2016. A propensity score matching was employed leaving 30 matched cases in each group. RESULTS: After propensity score matching the 30-day mortality was significantly lower in the MiECC group (n = 3 (10%) vs n = 10 (33%) in the CECC group, p = .028). Further, intraoperative transfused units of packed red blood cells were lower in the MiECC group (1.4 ± 1.8 units vs 2.8 ± 1.7, p < .001). CONCLUSIONS: There are evident advantages to using MiECC in dialysis dependent patients, especially regarding mortality. These findings necessitate additional research in MiECC usage in high-risk populations.

A Depth-Based Hybrid Approach for Safe Flight Corridor Generation in Memoryless Planning.

This paper presents a depth-based hybrid method to generate safe flight corridors for a memoryless local navigation planner. It is first proposed to use raw depth images as inputs in the learning-based object-detection engine with no requirement for map fusion. We then employ an object-detection network to directly predict the base of polyhedral safe corridors in a new raw depth image. Furthermore, we apply a verification procedure to eliminate any false predictions so that the resulting collision-free corridors are guaranteed. More importantly, the proposed mechanism helps produce separate safe corridors with minimal overlap that are suitable to be used as space boundaries for path planning. The average intersection of union (IoU) of corridors obtained by the proposed algorithm is less than 2%. To evaluate the effectiveness of our method, we incorporated it into a memoryless planner with a straight-line path-planning algorithm. We then tested the entire system in both synthetic and real-world obstacle-dense environments. The obtained results with very high success rates demonstrate that the proposed approach is highly capable of producing safe corridors for memoryless local planning.

Radiological risk assessment of outdoor222Rn and220Rn around rare earth element and uranium mines from northern Vietnam.

The outdoor222Rn and220Rn concentrations at 320 sampling points at 1 m above the ground in different sites surrounding rare earth element (REE) and uranium mines from northern Vietnam were measured using the RAD7. Results showed that222Rn concentrations were always higher than220Rn concentrations with large variation ranges from 25.7 to 573 Bq m-3and from 18.5 to 385 Bq m-3, respectively. The high correlation between220Rn and228Ra concentrations in surface soil of the studied sites were observed. The highest220Rn and222Rn concentrations are found at the sampling points of the REE NX-Lai Chau site. The220Rn and222Rn activities surrounding the REE mines were found to be higher than those surrounding the uranium mines. The average annual committed effective doses originated from the inhalation of220Rn and222Rn outdoor concentrations is about five times higher than the worldwide average value.

Semi-supervised adversarial discriminative domain adaptation.

Domain adaptation is a potential method to train a powerful deep neural network across various datasets. More precisely, domain adaptation methods train the model on training data and test that model on a completely separate dataset. The adversarial-based adaptation method became popular among other domain adaptation methods. Relying on the idea of GAN, the adversarial-based domain adaptation tries to minimize the distribution between the training and testing dataset based on the adversarial learning process. We observe that the semi-supervised learning approach can combine with the adversarial-based method to solve the domain adaptation problem. In this paper, we propose an improved adversarial domain adaptation method called Semi-Supervised Adversarial Discriminative Domain Adaptation (SADDA), which can outperform other prior domain adaptation methods. We also show that SADDA has a wide range of applications and illustrate the promise of our method for image classification and sentiment classification problems.

Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells.

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.

Mutations in LNPK, Encoding the Endoplasmic Reticulum Junction Stabilizer Lunapark, Cause a Recessive Neurodevelopmental Syndrome.

The dynamic shape of the endoplasmic reticulum (ER) is a reflection of its wide variety of critical cell biological functions. Consequently, perturbation of ER-shaping proteins can cause a range of human phenotypes. Here, we describe three affected children (from two consanguineous families) who carry homozygous loss-of-function mutations in LNPK (previously known as KIAA1715); this gene encodes lunapark, which is proposed to serve as a curvature-stabilizing protein within tubular three-way junctions of the ER. All individuals presented with severe psychomotor delay, intellectual disability, hypotonia, epilepsy, and corpus callosum hypoplasia, and two of three showed mild cerebellar hypoplasia and atrophy. Consistent with a proposed role in neurodevelopmental disease, LNPK was expressed during brain development in humans and mice and was present in neurite-like processes in differentiating human neural progenitor cells. Affected cells showed the absence of full-length lunapark, aberrant ER structures, and increased luminal mass density. Together, our results implicate the ER junction stabilizer lunapark in establishing the corpus callosum.

Reducing phenotypic instabilities of a microbial population during continuous cultivation based on cell switching dynamics.

Predicting the fate of individual cells among a microbial population (i.e., growth and gene expression) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation. Indeed, the dynamic nature of a continuous cultivation process implies the potential diversification of the microbial population resulting in genotypic and phenotypic heterogeneity. The present work focused on the induction of the arabinose operon in Escherichia coli as a model system to study this diversification process in continuous cultivations. As a preliminary step, the green fluorescent protein (GFP) level triggered by an arabinose-inducible ParaBAD promoter was tracked by flow cytometry in chemostat cultivations with glucose-arabinose co-feeding. For a wide range of glucose-arabinose co-feeding concentrations in the chemostats, the simultaneous occurrence of GFP positive and negative subpopulation was observed. In the second set of experiments, continuous cultivation was performed by adding glucose continuously and arabinose based on the capability of individual cells to switch from low GFP to high GFP expression states, performed with a technology setup called segregostat. In the segregostat cultivation mode, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the phenotypic switching profiles of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to prolonged maintenance of the induction level with a limited increase in the phenotypic diversity for more than 60 generations. The results suggest that the steady forcing of individual cells into a given phenotypic trajectory may not be the best strategy for controlling cell populations. Instead, allowing individual cells to switch periodically around a predefined threshold seems to be a more robust strategy leading to oscillations, but within a predictable cell population behavior range.