bluuebunny/crossref_metadata_2025 huggingface dataset:
Responding to Discriminatory Patient Requests
Authors: Moore Rachel , Loe Irene M. , Whitgob Emily , Cowden John D. , Nyp Sarah S.
Published: May 2021

CASE:

Julia is a 13-year-old White adolescent girl who was referred for psychological counseling given concerns related to mood, nonadherence, and adjustment secondary to her new diagnosis of type 1 diabetes. The family lives in a rural town located several hours from the academic medical center where she was diagnosed. After several months on a waitlist, the family was contacted to schedule a telehealth appointment with a predoctoral psychology trainee. When the scheduler informed the mother that her daughter would be scheduled with Ms. Huang, the mother abruptly stopped the conversation stating, “I do not want to waste everyone's time” and initially declined the appointment offered. When the scheduler asked about her hesitance, the mother disclosed previous interactions with doctors at the hospital who were “not born in the United States” that she felt were “textbook” (e.g., smiling even when discussing a new chronic medical condition) and “hard to understand” (i.e., because of different dialect/accent). The mother shared that she found these experiences to be stressful and felt the interactions had negatively affected Julia's care. When informed about the length of the waitlist for another clinician, the mother agreed to initiate services with the trainee.

The supervising psychologist shared the mother's concerns and comments with Ms. Huang. After discussion, Ms. Huang agreed to provide intervention services, “as long as the family was willing.” During the initial telehealth sessions, Ms. Huang primarily focused on building rapport and strengthening the therapeutic alliance with the family. During this time, Julia's mother was reluctant to incorporate suggested parent management strategies at home. Julia also made minimal improvement in her medical management (i.e., A1c levels remained high), had difficulty using behavioral coping strategies, and experienced ongoing mood symptoms (i.e., significant irritability, sleep difficulties, and depressive symptoms). Ms. Huang began to wonder whether the family's resistance and inability to implement recommendations were in some part because of the family's initial concerns and reluctance to engage in therapy with her as a clinician.

Should Ms. Huang address the previously identified concerns with the patient and her family? What should be considered when determining how to approach this situation to ensure provision of both the best care for this patient and support for this trainee?
An Actinomyces Long-Bone Osteomyelitis Case Report and Review
Authors: Bush Larry M. , Viggiano Vico , Tayon Matthew , Sama Nicholas
Published: May 2021

Historically, anaerobic bacteria as monomicrobial pathogens or components of polymicrobial infections have been found to play a minor role in osteomyelitis. Various Actinomyces species reside as constituents of the normal human microbial flora found in the oropharynx and in the most people may also be present as commensal microorganisms in the gastrointestinal, genitourinary, and, to a lesser degree, the upper respiratory tracts. Bone infections due to Actinomyces species are relatively uncommon and usually associated with concomitant cervicofacial disease. To date, only a sparse amount of cases of long bone osteomyelitis due to Actinomyces bacteria have been published in the medical literature. We report a case of monomicrobial subacute late-onset tibia bone osteomyelitis involving Actinomyces presenting 10 years after an open reduction and internal fixation surgical procedure performed to stabilize a traumatic fracture and review actinomycotic osteomyelitis including the rare association of Actinomyces odontolyticus with long bone infections.
Clinical Outcomes for Telavancin for Salvage Therapy in Methicillin-resistant Staphylococcus Aureus Bacteremia
Authors: Garcia Brandon , Gonzalez Jimmy , Chaudhry Saira
Published: May 2021

Background

Little evidence exists for the use of telavancin in methicillin-resistant Staphylococcus aureus (MRSA) bacteremia as salvage therapy. This study aims to evaluate the efficacy and safety of telavancin as salvage therapy in the treatment of MRSA bacteremia.

Methods

Electronic medical charts of patients who received telavancin for the treatment of MRSA bacteremia at an academic, tertiary care medical center in the United States between January 1, 2015, and May 31, 2019, were reviewed for clinical cure, reinfection, prior antibiotic use, source of infection, microbiological culture clearance, patient mortality, and adverse effects. The primary outcome was clinical cure.

Results

Thirty-eight charts were identified, and 11 met inclusion criteria for this analysis. Vancomycin or daptomycin was given for an average of 5.4 and 2.6 days, respectively, before the initiation of telavancin. The average telavancin dose used was 7.5 mg/kg. All patients achieved clinical cure, and there were no instances of 60-day mortality. Two patients experienced reinfection: 1 patient after 268 days and another after 467 days. Four patients experienced either QTc prolongation or acute renal failure.

Conclusions

The use of telavancin in MRSA bacteremia demonstrated microbiological cure, clinical cure, and minimal reinfection in the reviewed patient population. Telavancin may be a potential treatment option for patients with MRSA bacteremia as salvage therapy.
Absenteeism-Related Wage Loss Associated With Multimorbidity Among Employed Adults in the United States
Authors: Mohamed Rowida , Patel Jayeshkumar , Shaikh Nazneen Fatima , Sambamoorthi Usha
Published: May 2021

Objective:

To estimate the incremental absenteeism-related wage loss associated with multimorbidity and examine the influence of paid sick leave policy (PSLP) on multimorbidity-associated absenteeism wage loss.

Methods:

We used the Medical Expenditure Panel Survey (MEPS) 2015 data. Two-part generalized linear models (GLM) were employed, using binomial distribution and gamma distribution with a log link.

Results:

Nationally, multimorbidity was associated with a $9 billion incremental absenteeism-related wage loss annually among working adults. Absenteeism-related wage loss was higher among those with multimorbidity than those without multimorbidity. The incremental annual absenteeism-related wage loss associated with multimorbidity was lower in settings that offered paid sick leave than that did not offer paid sick leaves.

Conclusion:

Multimorbidity is associated with higher absenteeism-related wage loss. Paid sick leave policies can reduce the impact of multimorbidity on absenteeism-related wage loss.
Resolution of large vessel stenosis in giant cell arteritis (GCA) by tocilizumab
Authors: Matsumoto Haruki , Fujita Yuya , Asano Tomoyuki , Suzuki Erina , Kiko Yuichiro , Suzuki Osamu , Matsuda Mitsuko , Sato Shuzo , Temmoku Jumpei , Yashiro-Furuya Makiko , Matsuoka Naoki , Suzuki Eiji , Watanabe Hiroshi , Migita Kiyoshi
Published: May 2021

Introduction:

Progressive large vessel involvements including stenosis are commonly associated with giant cell arteritis (GCA). Glucocorticoids (GCs) are the mainstay of therapy for GCA stenosis, however, adverse effects including cardiovascular events are frequent.

Patients concerns:

We describe an 87-years old male patient with a medical history of diabetes mellitus and old cerebral infarction who presented with right temporal headache.

Diagnosis:

Color duplex sonography revealed the thickening of arterial wall and halo sign in the affected temporal arteries. In laboratory data, the elevated levels of acute phase reactants, including C-reactive protein and serum amyloid A were noted. Temporal artery biopsied specimens showed the lymphocytic infiltration and granulomatous inflammation within the arterial media, which are typical findings associated with GCA.

Intervention:

The patient was treated with subcutaneous injections of tocilizumab (TCZ, 162 mg) once weekly. Initially, glucocorticoids were withheld since the patient was diabetic and had a history of cerebral infarction. However, low dose prednisolone was combined with the reduced doses of TCZ (162 mg biweekly) injections due to the TCZ-related leukopenia.

Outcomes:

This treatment strategy resulted in the improvement of the patient's temporal headache and normalized levels of C-reactive protein and serum amyloid A. Follow-up imaging by color duplex sonography revealed the resolution of the temporal artery stenosis after 5 months of TCZ treatment.

Conclusions:

Patients with GCA often require long duration of steroid therapy and commonly suffer steroid-related complications. TCZ monotherapy, could induce early resolution of progressive vascular inflammation and stenosis in untreated GCA cases.
Papillary thyroid carcinoma arising in struma ovarii
Authors: Ioannidis Aristidis , Kourtidis Lazaros , Zatagias Apostolos , Chorti Angeliki , Gourvas Viktoras , Efthimiadis Christoforos , Michalopoulos Antonios
Published: May 2021

Introduction:

Struma Ovarii is one type of mature teratoma in which thyroid tissue represents more than 50% of the ovarian tumor. Diagnosis is very difficult due to lack of typical symptoms. In majority of these patients, diagnosis is incidental.

Patient concerns:

A 65- year-old female presented to our hospital with complaints of uterine bleeding and abdominal pain.

Diagnosis:

Ultrasonography showed no lesions in the uterus and left ovary, while the right ovary was enlarged, comprising a dense mass of 4 cm in diameter. Computed tomography scan revealed a mass, with fatty density and absence of fluid in the Douglas space. The lymph nodes in the posterior peritoneal space showed no enlargement. Additional Magnetic Resonance Imaging confirmed the presence of the mass with clear margins, high T1 signal and T2 sequence indicating dermal cysts. Blood tumor markers were normal. However, beta-human chorionic gonadotropin levels were slightly high.

Interventions:

Total hysterectomy was performed along with bilateral salpingo-oophorectomy.

Outcomes:

The biopsy of the mass revealed features of papillary thyroid carcinoma. The lesion was found to be benign, and the patient showed no recurrence so far.

Conclusion:

Struma Ovarii is a rare condition, found in only 1% of all ovarian tumors; it is benign in 95% of the cases. The clinical presentation can vary in terms of symptoms and signs. Ultrasonography can reveal the differences between a benign struma ovarii and a malignant one. Complete hysterectomy and bilateral salpingo-oophorectomy are the treatment of choice. Specific guidelines for pre- and postoperative management of the patients are yet to be elucidated.
After 800 Mtp Events, Mortality Due to Hemorrhagic Shock Remains High and Unchanged Despite Several In-Hospital Hemorrhage Control Advancements
Authors: Duchesne Juan , Taghavi Sharven , Ninokawa Scott , Harris Charles , Schroll Rebecca , McGrew Patrick , McGinness Clifton , Reily Robert , Guidry Chrissy , Tatum Danielle
Published: May 2021

Background:

Numerous advancements in hemorrhage control and volume replacement that comprise damage control resuscitation (DCR) have been implemented in the last decade to reduce deaths from bleeding. We sought to determine the impact of DCR interventions on mortality over 12 years in a massive transfusion protocol (MTP) population. We hypothesized that mortality would be decreased in later years, which would have used more DCR interventions.

Study Design:

This was a retrospective review of all MTP patients treated at a large regional Level I trauma center from 2008 to 2019. Interventions by year of implementation examined included MTP 1:1 ratio (2009), liquid plasma (2010), tranexamic acid (2012), prehospital tourniquets (2013), REBOA/TEG (2017), satellite blood station (2018), and whole blood transfusion (2019). Relative risk and odds of mortality for DCR interventions were examined.

Results:

There were 824 MTP patients included. The cohort was primarily male (80.6%) injured by penetrating mechanism (68.1%) with median (interquartile range) age 31 years (23–44) and New Injury Severity Score 25 (16–34). Overall mortality was unchanged [(38.3%–56.6%); P = 0.26]. Tourniquets (P = 0.02) and whole blood (WB) (P = 0.03) were associated with lower unadjusted mortality; only tourniquets remained significant after adjustment (OR: 0.39; 95% CI: 0.17–0.89; P = 0.03).

Conclusions:

Despite lower mortality with use of tourniquets and WB, mortality rates due to hemorrhage have not improved at our high MTP volume institution, suggesting implementation of new in-hospital strategies is insufficient to reduce mortality. Future efforts should be directed toward moving hemorrhage control and effective resuscitation interventions to the injury scene.
Phase separation on cell surface: a mechanism of basic fibroblast growth factor signal transduction with heparan sulphate
Authors: Xue Song , Zhou Fan , Zhao Tian , Zhao Huimin , Wang Xuewei , Chen Long , Li Jin-ping , Tan Tianwei , Luo Shi-Zhong
Published: May 2021

Liquid-liquid phase separation (LLPS) driven by weak, multivalent interactions among biomolecules is an important means of cellular compartmentation and plays a central role in cellular processes including stress resistance, RNA processing and other cellular activities. Coordination of the condensates and inner membrane was recently revealed, mediating intracellular processes like cell signalling and cargo trafficking. Intracellular LLPS has been observed extensively in vivo, whereas LLPS in extracellular compartments has not been reported under physiological conditions. Here we show, for the first time, that basic fibroblast growth factor (bFGF) undergoes LLPS on the cell surface by interacting with heparan sulphate proteoglycans (HSPG) and the phase transition is required for effective downstream signalling. The condensation is driven by multivalent interactions between bFGF and sulpho-groups on heparan sulphate (HS), and dimerization and oligomerization of bFGF promote the LLPS process. Compared with free bFGF, phase separated bFGF with HS showed higher thermo stability, providing a potential mechanism for the preservation of bFGF activity. Furthermore, we have found that downstream signalling is triggered by phase separation of a ternary complex formed by bFGF, HSPGs and FGFR on cell surface. Our results revealed a molecular mechanism that HS can serve as a platform to promote extracellular proteins like bFGF to condensate on outer membrane, consequently coordinating the signal transduction activities. This novel finding expands the horizons of phase separation in vivo, providing a new dimension on how HSPG may regulate extracellular protein behaviour and cell signalling.
Youth susceptibility to tobacco use: Is it general or specific?
Authors: Cheng Hui G. , Lizhnyak Pavel N. , Knight Natasha A. , Vansickel Andrea R. , Largo Edward G.
Published: May 2021

Importance

Susceptibility to tobacco use can help identify youth that are at risk for tobacco use.

Objective

To estimate the extent of overlap in susceptibilities across various tobacco products, investigate correlates with susceptibilities, and examine whether the relationship linking susceptibility with the onset of use is product specific or is accounted for by a general susceptibility-onset relationship.

Design

Prospective cohort study.

Setting

Analysis of data from the longitudinal Population Assessment of Tobacco and Health study wave 4 (December 2016 to January 2018) and wave 4.5 youth surveys (December 2017 to November 2018).

Participants

A nationally representative sample of non-institutionalized youth 12-17 years old who had never used a tobacco product at baseline assessment.

Main variable of interest

Susceptibility to the use of each type of tobacco product assessed at wave 4.

Main outcomes

Onset of use of various tobacco products defined as the first use occurring between waves 4 and 4.5 assessments.

Results

Cigarettes and e-cigarettes were the most common (∼25%), while snus was the least common (<5%), tobacco product to which youth were susceptible. There was a high degree of overlap in susceptibilities across tobacco products (65% of tobacco-susceptible youth were susceptible to more than one tobacco product). Tobacco-susceptible youth were more likely to have used cannabis or consumed alcohol in the past 30 days or to have tobacco-using peers. Susceptibility to use predicted the onset of use (incidence ratio = 3.2 to 12.9). Estimates for the product-specific path were null, except for e-cigarettes (β=0.08, 95% CI=0.04 to 0.13) and filtered cigars (β= -0.09, 95% CI= -0.13 to -0.05), after accounting for the general susceptibility-to-tobacco-onset relationship (β=0.50, 95% CI=0.42 to 0.58).

Conclusions and Relevance

Youth susceptibility to tobacco use overlaps widely across different tobacco products and other risky behaviors. Public health efforts may benefit from a holistic approach to risk behavior prevention planning.

Key Points

Question

Is susceptibility to tobacco use product specific, or does it represent a general openness to tobacco use?

Findings

There was a large degree of overlap in the susceptibility to tobacco use across various product categories. Associations linking susceptibility and the onset of use of a tobacco product were not statistically robust once the general level of susceptibility to tobacco use was considered.

Meaning

Susceptibility to tobacco use is better conceived as a general openness to tobacco use rather than product specific.
Prenatal Alcohol and THC E-Cigarette Exposure Effects on Motor Development
Authors: Breit Kristen R. , Rodriguez Cristina , Lei Annie , Hussain Samirah , Thomas Jennifer D.
Published: May 2021

It has been well established that prenatal alcohol exposure can lead to a wide range of neurological and behavioral deficits, including alterations in motor domains. However, much less is known about the effects of prenatal cannabis exposure on motor development, despite the fact that cannabis is the most commonly consumed illicit drug among women. Cannabis use among pregnant women has become increasingly popular given the wide-spread perception that consumption is safe during pregnancy. Moreover, alcohol and cannabis are commonly used together, even among pregnant women. Yet, few studies have explored the potential consequences of combined prenatal exposure on behavioral domains. Using our previously established model, pregnant Sprague-Dawley rats were exposed to vaporized alcohol, THC via e-cigarettes, the combination, or a vehicle from gestational days 5-20. Following birth, offspring were tested on early sensorimotor development, adolescent motor coordination, and adolescent activity levels. Prenatal e-cigarette THC exposure delayed sensorimotor development early in life and impaired motor coordination later in adolescence. However, combined prenatal alcohol and THC exposure produced hyperactivity among male offspring. These data suggest that prenatal cannabis exposure may lead to impaired motor skills throughout early development, and that combined exposure with alcohol during gestation may also lead to hyperactivity in adolescence. These findings have important implications for pregnant women and public policy.
Loose ends in cancer genome structure
Authors: Behr Julie M. , Yao Xiaotong , Hadi Kevin , Tian Huasong , Deshpande Aditya , Rosiene Joel , Lange Titia de , Imieliński Marcin
Published: May 2021

Recent pan-cancer studies have delineated patterns of structural genomic variation across thousands of tumor whole genome sequences. It is not known to what extent the shortcomings of short read (≤ 150 bp) whole genome sequencing (WGS) used for structural variant analysis has limited our understanding of cancer genome structure. To formally address this, we introduce the concept of “loose ends” - copy number alterations that cannot be mapped to a rearrangement by WGS but can be indirectly detected through the analysis of junction-balanced genome graphs. Analyzing 2,319 pan-cancer WGS cases across 31 tumor types, we found loose ends were enriched in reference repeats and fusions of the mappable genome to repetitive or foreign sequences. Among these we found genomic footprints of neotelomeres, which were surprisingly enriched in cancers with low telomerase expression and alternate lengthening of telomeres phenotype. Our results also provide a rigorous upper bound on the role of non-allelic homologous recombination (NAHR) in large-scale cancer structural variation, while nominatingINO80,FANCA, andARID1Aas positive modulators of somatic NAHR. Taken together, we estimate that short read WGS maps >97% of all large-scale (>10 kbp) cancer structural variation; the rest represent loose ends that require long molecule profiling to unambiguously resolve. Our results have broad relevance for future research and clinical applications of short read WGS and delineate precise directions where long molecule studies might provide transformative insight into cancer genome structure.
Somatic reversion impacts MDS/AML evolution in the short telomere syndromes
Authors: Schratz Kristen E. , Gaysinskaya Valeriya , Cosner Zoe L. , DeBoy Emily A. , Xiang Zhimin , Kasch-Semenza Laura , Florea Liliana , Shah Pali D. , Armanios Mary
Published: May 2021

Inherited mutations in telomerase and other telomere maintenance genes manifest in the premature aging short telomere syndromes. Myelodysplastic syndromes and acute myeloid leukemia (MDS/AML) account for 75% of associated malignancies, but how these cancers overcome the germline telomere defect is unknown. We used targeted ultra-deep sequencing to detect candidate somatic reversion mutations hypothesizing they may promote MDS/AML evolution. While no controls carried somatic mutations in telomere maintenance genes (0 of 28), 29% of adults with germline telomere maintenance defects carried at least one (16 of 56, P<0.001). In addition to TERT promoter mutations which were present in 19%, we identified POT1 and TERF2IP mutations in 13%. POT1 mutations impaired telomere binding and in some cases were identical to those found in familial melanoma associated with longer telomere length. Exclusively in patients with germline defects in telomerase RNA (TR), we identified somatic mutations in nuclear RNA exosome genes, RBM7, SKIV2L2, and DIS3, where loss-of-function upregulates mature TR levels. Paradoxically, somatic reversion events were more prevalent in patients who were MDS/AML-free (P=0.01, RR 5.0, 95% CI 1.4-18.9), and no MDS/AML patient had more than one mutant clone (P=0.048). Our data identify diverse somatic adaptive mechanisms in the short telomere syndromes, and raise the possibility that their presence alleviates the telomere crisis that promotes transformation to MDS/AML.
Synaptic density and neuronal metabolic function measured by PET in the unilateral 6-OHDA rat model of Parkinson’s disease
Authors: Raval Nakul Ravi , Gudmundsen Frederik , Juhl Morten , Andersen Ida Vang , Speth Nikolaj Raahauge , Videbæk Annesofie , Petersen Ida Nymann , Mikkelsen Jens Damsgaard , Fisher Patrick MacDonald , Herth Matthias Manfred , Plavén-Sigray Pontus , Knudsen Gitte Moos , Palner Mikael
Published: May 2021

Parkinson’s disease (PD) is caused by progressive neurodegeneration and characterised by motor dysfunction. Neurodegeneration of dopaminergic neurons also causes aberrations within the cortico-striato-thalamo-cortical (CSTC) circuit, which has been hypothesised to lead to non-motor symptoms such as depression. Individuals with PD have both lower synaptic density and changes in neuronal metabolic function in the basal ganglia, as measured using [11C]UCB-J and [18F]FDG positron emission tomography (PET), respectively. However, the two radioligands have not been directly compared in the same PD subject or in neurodegeneration animal models. Here, we investigate [11C]UCB-J binding and [18F]FDG uptake in the CSTC circuit following a unilateral dopaminergic lesion in rats and compare it to sham lesioned rats.

Rats received either a unilateral injection of 6-hydroxydopamine (6-OHDA) or saline in the medial forebrain bundle and rostral substantia nigra (n=4/group). After three weeks, all rats underwent two PET scans using [18F]FDG, followed by [11C]UCB-J on a separate day. [18F]FDG uptake and [11C]UCB-J binding were both lower in the ipsilateral striatal regions compared to the contralateral regions. Using [11C]UCB-J, we could detect an 8.7% decrease in the ipsilateral ventral midbrain, compared to a 2.9% decrease in ventral midbrain using [18F]FDG. Differential changes between hemispheres for [11C]UCB-J and [18F]FDG outcomes were also evident in the CSTC circuit’s cortical regions, especially in the orbitofrontal cortex and medial prefrontal cortex where higher synaptic density yet lower neuronal metabolic function was observed, following lesioning.

In conclusion, [11C]UCB-J and [18F]FDG PET can detect divergent changes following a dopaminergic lesion in rats, especially in cortical regions that are not directly affected by the neurotoxin. These results suggest that combined [11C]UCB-J and [18F]FDG scans could yield a better picture of the heterogeneous cerebral changes in neurodegenerative disorders.
Surveillance of in situ tumor arrays reveals early environmental control of cancer immunity
Authors: Ortiz-Muñoz Guadalupe , Brown Markus , Carbone Catherine B. , Pechuan-Jorge Joaquin , Ritter Alex T. , Orlova Darya , Mantri Shreya R. , Yang Angela , Doerr Jonas , Mariathasan Sanjeev , Wang Yulei , Turley Shannon , Gomez-Roca Carlos , de Andrea Carlos Eduardo , Spigel David , Wu Thomas , Modrusan Zora , Price Richard , Mellman Ira , Moussion Christine
Published: May 2021

The immune phenotype of a tumor is a key predictor of its response to immunotherapy1–4. Patients who respond to immune checkpoint blockade generally present with tumors that are infiltrated by activated T cells, a tumor-immune phenotype referred to as ‘immune inflamed’5–7. However, not all immune inflamed tumors respond to therapy, and in addition the majority of patients presents with tumors that lack T cells (‘immune desert’) or that exclude T cells in the periphery of the tumor islet (‘immune excluded’)8. Despite the importance of these tumor-immune phenotypes in patients, little is known about their development, heterogeneity or dynamics due to an inability to model these features pre-clinically.

Here, we describe an approach designated STAMP (skin tumor array by micro-poration), which combines in vivo noninvasive, high-throughput time-lapse imaging with excisional biopsies and next generation sequencing to characterize the establishment of the immunological niche and follow its evolution during immunotherapy. STAMP involves the seeding of dozens to hundreds clonal tumors in the superficial dermis of a single mouse ear that can be visualized in situ over weeks to months. Using this approach, we found that genetically identical tumors could display surprisingly different immune phenotypes. Although individual tumors of the same array were populated by the same T cell clonotypes, regression or progression of individual tumors were associated with distinct patterns of spatial organization of the T cells. In situ imaging of 14K tumors revealed that immune phenotypes were not static over-time but could rather evolve with tumor growth and response to treatment. Therapy-induced or spontaneous early conversion to the immune inflamed phenotype correlated with tumor regression and enhanced cytotoxic T cell activity. Therefore, STAMP provides a flexible approach to study the relationship between tumor evolution, immune cell dynamics, and tumor microenvironment with therapeutic response.
Heligmosomoides bakeriandToxoplasma gondiico-infection leads to increased mortality associated with intestinal pathology
Authors: Szabo Edina K. , Bowhay Christina , Forrester Emma , Liu Holly , Dong Beverly , Coria Aralia Leon , Perera Shashini , Fung Beatrice , Badawadagi Namratha , Gaio Camila , Bailey Kayla , Ritz Manfred , Bowron Joel , Ariyaratne Anupama , Finney Constance A. M.
Published: May 2021

Co-infections are a common reality but understanding how the immune system responds in this context is complex and can be unpredictable.Heligmosomoides bakeri(parasitic roundworm, previouslyHeligmosomoides polygyrus) andToxoplasma gondii(protozoan parasite) are well studied organisms that stimulate a characteristic Th2 and Th1 response, respectively. Several studies have demonstrated reduced inflammatory cytokine responses in animals co-infected with such organisms. However, while general cytokine signatures have been examined, the impact of the different cytokine producing lymphocytes on parasite control/clearance is not fully understood. We investigated five different lymphocyte populations (NK, NKT, γδ T, CD4+T and CD8+T cells), five organs (small intestine, Peyer’s patches, mesenteric lymph nodes, spleen and liver), and 4 cytokines (IFN γ, IL-4, IL-10 and IL-13) at two different time points (days 5 and 10 postT. gondiiinfection). We found that co-infected animals had significantly higher mortality than either single infection. This was accompanied by transient and local changes in parasite loads and cytokine profiles. Despite the early changes in lymphocyte and cytokine profiles, severe intestinal pathology in co-infected mice likely contributed to early mortality due to significant damage by both parasites in the small intestine. Our work demonstrates the importance of taking a broad view during infection research, studying multiple cell types, organs/tissues and time points to link and/or uncouple immunological from pathological findings. Our results provide insights into how co-infection with parasites stimulating different arms of the immune system can lead to drastic changes in infection dynamics.
ColFeatures: Automated data extraction and classification of bacterial colonies
Authors: García-Soriano Daniela A. , Andersen Frederikke Dybdahl , Nygaard Jens Vinge , Tørring Thomas
Published: May 2021

Examining microbial colonies on agar plates have been at the core of microbiology for many decades. It is usually done manually, and therefore subject to bias besides requiring a considerable amount of time and effort. In order to optimize and standardize the identification of bacterial colonies growing on agar plates, we have developed an open access tool available on GitHub: ColFeatures. The software allows automated identification of bacterial colonies, extracts key morphological data and generate labels that ensure tracking of temporal development. We included machine learning algorithms that provide sorting of environmental isolates by using cluster methodologies. Furthermore, we show how cluster performance is evaluated using index scores (Silhouette, Calinski-Harabasz, Davies-Bouldin) to ensure the outcome of colony classification. As automation becomes more prominent in microbiology, tools as ColFeatures will assist identification of bacterial colonies on agar plates, bypassing human bias and complementing sequencing or mass spectrometry information that often comes attached with a considerable price tag.
Estradiol Influences Adenosinergic Signaling and NREM Sleep Need in Adult Female Rats
Authors: Smith Philip C. , Phillips Derrick J. , Pocivavsek Ana , Byrd Carissa A. , Viechweg Shaun S. , Hampton Brian , Mong Jessica A.
Published: May 2021

Studies report estradiol (E2) suppresses sleep in females; however, the mechanisms of E2 action remain largely undetermined. Our previous findings suggest that the median preoptic nucleus (MnPO) is a key nexus for E2 action on sleep. Here, using behavioral, neurochemical and pharmacological approaches, we investigated whether E2 influenced the sleep homeostat as well as adenosinergic signaling in the MnPO of adult female rats. During the Light Phase, where rats accumulate the majority of sleep, E2 markedly reduced NREM-SWA (a measure of the homeostatic sleep need). Following 6-hours of sleep deprivation, levels of NREM-SWA were significantly increased compared to baseline sleep. However, the NREM-SWA levels were not different between E2 and control treatment despite a significant increase in wake at the expense of NREM sleep. Analysis of NREM-SWA differences between baseline and recovery sleep following sleep deprivation demonstrated that E2 induced a 2-fold increase in delta power compared to controls suggesting that E2 significantly expanded the dynamic range for the sleep homeostat. Correlated with E2-induced changes in physiological markers of homeostatic sleep was a marked increase in extracellular adenosine (a molecular marker of homeostatic sleep need) during unrestricted and recovery sleep following a 6-hour deprivation. Additionally, E2 blocked the ability of an adenosine A2A receptor agonist (CGS-21680) to increase NREM sleep compared to controls. Thus, taken together, the findings that E2 increased extracellular adenosine content, while blocking A2A signaling in the MnPO suggests a potential mechanism for how estrogens impact sleep in the female brain.

Statement of Significance

While gonadal steroids and gender are implicated as risk factors for sleep disruptions and insomnia, the relationship between ovarian steroids and sleep is poorly understood. Understanding the mechanisms through which estradiol (E2) is working to influence sleep-wake behavior is a critical first step toward a better understanding of the role of estrogens in sleep pathologies. Using a rodent model, the current study presents novel findings suggesting that estradiol (E2) is influencing adenosinergic actions in the MnPO. The ability of E2 to attenuate the local effects of the A2A receptors in the MnPO suggests that E2 modulation of A2A receptor signaling may underlie estrogenic suppression of sleep behavior as well as changes in homeostatic sleep need.
Chop-n-drop: in silico assessment of a novel single-molecule protein fingerprinting method employing fragmentation and nanopore detection
Authors: de Lannoy Carlos , Maglia Giovanni , de Ridder Dick
Published: May 2021

The identification of proteins at the single-molecule level would open exciting new venues in biological research and disease diagnostics. Previously we proposed a nanopore-based method for protein identification called chop-n-drop fingerprinting, in which the fragmentation pattern induced and measured by a proteasome-nanopore construct is used to identify single proteins. However whether such fragmentation patterns are sufficiently characteristic of proteins to identify them in complex samples remained unclear. In the simulation study presented here, we show that 97.9% of human proteome constituents are uniquely identified under close to ideal measuring circumstances, using a simple alignment-based classification method. We show that our method is robust against experimental error, as 78.8% can still be identified if the resolution is twice as low as currently attainable and 10% of proteasome restriction sites and protein fragments are randomly ignored. Based on these results and our experimental proof-of-concept, we argue that chop-n-drop fingerprinting has the potential to make cost-effective single-molecule protein identification feasible in the near future.
InferringMycobacterium bovistransmission between cattle and badgers using isolates from the Randomised Badger Culling Trial
Authors: van Tonder Andries J. , Thornton Mark , Conlan Andrew J.K. , Jolley Keith A. , Goolding Lee , Mitchell Andrew P. , Dale James , Palkopoulou Eleftheria , Hogarth Philip J. , Glyn Hewinson R. , Wood James L.N. , Parkhill Julian
Published: May 2021

Mycobacterium bovis(M. bovis)is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial,M. bovisisolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission ofM. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger.
Parameterization of regulatory nodes for engineering broad host range heterologous gene expression
Authors: Nikel Pablo I. , Benedetti Ilaria , de Lorenzo Victor , Calles Belen
Published: May 2021

By building on the SEVA (Standard European Vector Architecture) format we have refactored a number of regulatory nodes recruited from both Gram-negative and Gram-positive bacteria for rigorously comparing and parameterizing five expression devices that respond to diverse and unrelated chemical inducers, i.e. LacIq-Ptrc, XylS-Pm, AlkS-PalkB, CprK-PDB3and ChnR-PchnB. These were assembled as cargoes following the SEVA standard within exactly the same vector backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance in anEscherichia colistrain of reference was then analyzed through the readout a fluorescence reporter gene that contained strictly identical translation signal elements in all cases and in the same DNA context. This study allowed us to describe and compare the cognate expression systems with unprecedented quantitative detail. The systems under scrutiny diverged considerably in their capacity, expression noise, inducibility and OFF/ON ratios. These features, along with the absence of physiological effects caused by the inducers and the lack of cross-regulation offer a panoply of choices to potential users and help interoperability of the specific constructs.
A distributed residue network permits conformational binding specificity in a conserved family of actin remodelers
Authors: Hwang Theresa , Parker Sara S , Hill Samantha M. , Ilunga Meucci W. , Grant Robert A. , Mouneimne Ghassan , Keating Amy E.
Published: May 2021

Metazoan proteomes contain many paralogous proteins that have evolved distinct functions. The Ena/VASP family of actin regulators consists of three members that share an EVH1 interaction domain with a 100% conserved binding site. A proteome-wide screen revealed ciliary protein PCARE as a high-affinity ligand for ENAH EVH1. Here we report the surprising observation that PCARE is ~100-fold specific for ENAH over paralogs VASP and EVL and can selectively bind and inhibit ENAH-dependent adhesion in cells. Specificity arises from a mechanism whereby PCARE stabilizes a conformation of the ENAH EVH1 domain that is inaccessible to family members VASP and EVL. Structure-based modeling rapidly identified seven residues distributed throughout EVL that are sufficient to differentiate binding by ENAH vs. EVL. By exploiting the ENAH-specific conformation, we rationally designed the tightest and most selective ENAH binder to date. Our work uncovers a conformational mechanism of interaction specificity that distinguishes highly similar paralogs and establishes tools for dissecting specific Ena/VASP functions in processes including cancer cell invasion.
Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability
Authors: Dahal Sumedha , Siddiqua Humaira , Sharma Shivangi , Babu Ravi K. , Manjunath Meghana , Sharma Sheetal , Choudhary Bibha , Raghavan Sathees C.
Published: May 2021

Having its own genome makes mitochondria a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is double-stranded closed circular molecule of about 16 kb coding 37 genes. Mutations, including deletions in the mitochondrial genome can culminate in different human diseases. Mapping of the deletion junctions suggests that the breakpoints are generally seen at hotspots. ‘9-bp deletion’ (8271-8281), seen in the intergenic region of cytochrome c oxidase II/tRNALys, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with ‘9-bp deletion’, when it is present on a plasmid or in the mitochondrial genome. Through a series of in vitro and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Besides, we reconstitute the whole process of ‘9-bp deletion’ using purified Endonuclease G to induce breaks in mtDNA, followed by mitochondrial extract-mediated DSB repair and establish that microhomology-mediated end joining is responsible for the generation of mtDNA deletion. Finally, we show that the whole process is regulated by different stress conditions, which may modulate release of Endonuclease G to the mitochondrial matrix. Therefore, we uncover a new role for Endonuclease G in generating deletions, which is dependent on the formation of G4 DNA within the mitochondrial genome. Thus, in this study we identify a novel property of Endonuclease G, besides its role in apoptosis, and the recently described ‘elimination of paternal mitochondria during fertilization.
Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA
Authors: Hormeno S , Wilkinson OJ , Aicart-Ramos C , Kuppa S , Antony E , Dillingham MS , Moreno-Herrero F
Published: May 2021

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of ∼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′-to-3′ direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.
Deciphering signatures of natural selection via deep learning
Authors: Qin Xinghu , Chiang Charleston W. K. , Gaggiotti Oscar E.
Published: May 2021

Identifying genomic regions influenced by natural selection provides fundamental insights into the genetic basis of local adaptation. We propose a deep learning-based framework, DeepGenomeScan, that can detect signatures of local adaptation. We demonstrate that DeepGenomeScan outperformed PCA and RDA-based genome scans in identifying loci underlying quantitative traits subject to complex spatial patterns of selection. Noticeably, DeepGenomeScan increases statistical power by up to 47.25% under non-linear environmental selection patterns. We applied DeepGenomeScan to a European human genetic dataset and identified some well-known genes under selection and a substantial number of clinically important genes that were not identified using existing methods.
Spatial-transcriptomics reveals unique defining molecular features of fluorescence-sorted 5-aminolevulinic acid+ infiltrative tumor cells associated with glioblastoma recurrence and poor survival
Authors: Andrieux Geoffroy , Das Tonmoy , Griffin Michaela , Smith Stuart J. , Rahman Ruman , Chakraborty Sajib
Published: May 2021

Spatiotemporal-heterogeneity of glioblastoma (GBM) originating from the genomic and transcriptional variation in spatially distinct intra-tumor sites, may contribute to subtype switching in GBM prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) has enabled the isolation of infiltrative margin tumor cells (5ALA+ cells) from a background of non-neoplastic cells. We have explored the spatial-transcriptomic (ST) landscape to interrogate molecular signatures unique to infiltrating 5ALA+ cells in comparison to GBM core, rim, and invasive margin non-neoplastic cells. ST analysis reveals that GBM molecular subtype plasticity is not restricted to recurrence, but manifests regionally in a cell-type-specific manner. Whilst GBM core and rim are highly enriched with Classical and Proneural subtypes, the unique enrichment of the Mesenchymal subtype (MES) in 5ALA+ cells supports the hypothesis that MES 5ALA+ cells may drive GBM recurrence. Upregulation of the wound response pathway in 5ALA+ cells signifies the possibility of hijacking the wound healing pathway of neural cells to promote tumor growth. Exon-intron split analysis revealed an upregulation of exonic counts for MES and wound-response genes in 5ALA+ cells, implying that these genes are under active post-transcriptional control. Network analysis suggests that wound response genes, including chemokineCCL2that recruits regulatory T-cells and monocytic myeloid-derived suppressor cells, are controlled by anIRF8-mediated transcriptional program in 5ALA+ cells. A higher stemness signature both in 5ALA+ cells and non-neoplastic cells of the invasive margin emphasizes the role of this microenvironment in stemness acquisition and defines 5ALA+ cells as a rare sub-population of GBM stem cells. Finally, we establish a link between the unique molecular signatures of 5ALA+ cells and poor survival and GBM recurrence. Characterization of the 5ALA+ infiltrative sub-population offers an opportunity to develop more effective GBM treatments and urges focus away from the GBM proliferative core, upon which failed targeted therapies have been predicated.
A comprehensive mass spectral library for human thyroid tissues
Authors: Sun Yaoting , Li Lu , Ge Weigang , Dong Zhen , Liu Wei , Chen Hao , Xiao Qi , Cai Xue , Zhang Fangfei , Xiao Junhong , Wang Guangzhi , He Yi , Kon Oi Lian , Iyer N. Gopalakrishna , Zhao Yongfu , Guo Tiannan
Published: May 2021

Thyroid nodules occur in about 60% of the population. Current diagnostic strategies, however, often fail at distinguishing malignant nodules before surgery, thus leading to unnecessary, invasive treatments. As proteins are involved in all physio/pathological processes, a proteome investigation of biopsied nodules may help correctly classify and identify malignant nodules and discover therapeutic targets. Quantitative mass spectrometry data-independent acquisition (DIA) enables highly reproducible and rapid throughput investigation of proteomes. An exhaustive spectral library of thyroid nodules is essential for DIA yet still unavailable. This study presents a comprehensive thyroid spectral library covering five types of thyroid tissue: multinodular goiter, follicular adenoma, follicular and papillary thyroid carcinoma, and normal thyroid tissue. Our library includes 925,330 transition groups, 157,548 peptide precursors, 121,960 peptides, 9941 protein groups, and 9826 proteins from proteotypic peptides. This library resource was evaluated using three papillary thyroid carcinoma samples and their corresponding adjacent normal thyroid tissue, leading to effective quantification of up to 7863 proteins from biopsy-level thyroid tissues.