bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential ion dehydration energetics explains selectivity in the non-canonical lysosomal K+ channel TMEM175
Authors: Oh SeCheol , Marinelli Fabrizio , Zhou Wenchang , Lee Jooyeon , Choi Ho Jeong , Kim Min , Faraldo-Gómez José D. , Hite Richard K.
Published: November 2021

Structures of the human lysosomal K+ channel TMEM175 in open and closed states revealed a novel architecture lacking the canonical K+ selectivity filter motif present in previously known K+ channel structures. A hydrophobic constriction composed of four isoleucine residues was resolved in the pore and proposed to serve as the gate in the closed state, and to confer ion selectivity in the open state. Here, we achieve higher-resolution structures of the open and closed states and employ molecular dynamics simulations to analyze the conducting properties of the putative open state, demonstrating that it is capable of permeating K+ ions at the expected rate. Monovalent cations must dehydrate significantly to penetrate the narrow hydrophobic constriction, but ion flow is assisted by a favorable electrostatic field generated by the protein that spans the length of the pore. The balance of these opposing energetic factors explains why permeation is feasible, and why TMEM175 is selective for K+ over Na+, despite the absence of the canonical selectivity filter. Accordingly, mutagenesis experiments reveal an exquisite sensitivity of the channel to perturbations that mitigate the constriction. Together, these data reveal a novel mechanism for selective permeation of ions by TMEM175 that is unlike that of other K+ channels.
Transcriptional regulation of sphingolipid metabolism in budding yeast
Authors: Komatsu Nao , Ishino Yuko , Shirai Rina , Sakata Ken-taro , Tani Motohiro , Maeda Tatsuya , Tanaka Naotaka , Tabuchi Mitsuaki
Published: November 2021

Global control for the synthesis of lipids constituting a bilayer of cell membranes is known to be with a small number of transcription factors called master transcriptional regulators, which target a wide range of genes encoding lipid metabolism enzymes and/or their regulators. Although master transcriptional regulators of glycerophospholipids and sterols have been identified in both yeast and mammals, this aspect of sphingolipid metabolism is not yet understood. In the present study, we identified the C2H2-type zinc finger transcription factor, Com2, as a master transcriptional regulator of sphingolipid metabolism in the budding yeast, Saccharomyces cerevisiae. The target of rapamycin complex 2 (TORC2)-activated protein kinase Ypk1 is known to regulate sphingolipid metabolism. Activated Ypk1 stimulates the activity of serine palmitoyl transferase (SPT), the first-step enzyme in sphingolipid biosynthesis, by phosphorylating and inhibiting Orm1/2, a negative regulator of SPT. This regulation of SPT activity is thought to be a major pathway in the regulation of sphingolipid metabolism. In the present study, we found that inhibition of sphingolipid synthesis upregulates the expression of Com2, which in turn leads to the concomitant expression of Ypk1. The upregulation of Ypk1 expression was found to be dependent on a putative Com2-binding site in the YPK1 promoter. Our results also suggested that Com2 senses intracellular sphingolipid levels through a pathway independent of TORC2-Ypk1-mediated sensing of sphingolipids. Our results revealed an additional layer of mechanistic regulation that allows cells to maintain appropriate levels of sphingolipid biosynthesis and to rapidly induce this process in response to environmental stresses.

Significance Statement

One of the major regulatory mechanisms involved in the control of lipid metabolism in bilayers of biological membranes is regulation at the transcriptional level by master transcriptional regulators that control the transcription of genes encoding lipid metabolism enzymes and/or their regulators. In the present study, we identified the C2H2-type zinc finger transcription factor Com2 as a master transcriptional regulator in sphingolipid metabolism. We found that Com2 regulates sphingolipid metabolism by transcriptionally controlling the expression of Ypk1, which regulates Orm1/2, a negative regulator of serine palmitoyl transferase, the first-step enzyme in sphingolipid biosynthesis, through phosphorylation. Our study revealed a new layer of regulation that allows the maintenance of an appropriate level of sphingolipid biosynthesis for a rapid response to environmental stresses.
Aggregation controlled by condensate rheology
Authors: Pönisch Wolfram , Michaels Thomas C.T. , Weber Christoph A.
Published: November 2021

Biomolecular condensates in living cells can exhibit a complex rheology including viscoelastic and glassy behaviour. This rheological behavior of condensates was suggested to regulate polymerisation of cytoskeletal filaments and aggregation of amyloid fibrils. Here, we theoretically investigate how the rheological properties of condensates can control the formation of linear aggregates. To this end, we propose a kinetic theory for linear aggregation in coexisting phases, which accounts for the aggregate size distribution and the exchange of aggregates between inside and outside of condensates. The rheology of condensates is accounted in our model via aggregate mobilities that depend on aggregate size. We show that condensate rheology determines whether aggregates of all sizes or dominantly small aggregates are exchanged between condensate inside and outside on the time-scale of aggregation. As a result, the ratio of aggregate numbers inside to outside of condensates differs significantly. Strikingly, we also find that weak variations in the rheological properties of condensates can lead to a switch-like change of the number of aggregates. These results suggest a possible physical mechanism for how living cells could control linear aggregation in a switch-like fashion through variations in condensate rheology.

SIGNIFICANCE

The intracellular space can be organized through phase-separated condensates that often exhibit rheological properties reminiscent of complex fluids. These condensates can affect biochemical processes such as the formation of linear aggregates, in particular biofilaments or amyloids. Here, we propose a theoretical model for how condensate rheology can control the irreversible formation of linear aggregates. A key finding is that size and number of aggregates change in a switch-like fashion upon weak changes in condensate rheology. Our model paves the way to unravel the physiochemical mechanisms of how the rheology of condensates can control aberrant protein aggregation. Such mechanisms may explain how rheological changes, such as ageing or the transition to dormancy, give rise to diseases related to protein aggregation.
Probabilistic Genotyping of Single Cell Replicates from Complex DNA Mixtures Recovers Higher Contributor LRs than Standard Analysis
Authors: Huffman Kaitlin , Hanson Erin , Ballantyne Jack
Published: November 2021

DNA mixtures are a common source of crime scene evidence and are often one of the more difficult sources of biological evidence to interpret. With the implementation of probabilistic genotyping (PG), mixture analysis has been revolutionized allowing previously unresolvable mixed profiles to be analyzed and probative genotype information from contributors to be recovered. However, due to allele overlap, artifacts, or low-level minor contributors, genotype information loss inevitably occurs. In order to reduce the potential loss of significant DNA information from donors in complex mixtures, an alternative approach is to physically separate individual cells from mixtures prior to performing DNA typing thus obtaining single source profiles from contributors. In the present work, a simplified micromanipulation technique combined with enhanced single-cell DNA typing was used to collect one or few cells, referred to as direct single-cell subsampling (DSCS). Using this approach, single and 2-cell subsamples were collected from 2-6 person mixtures. Single-cell subsamples resulted in single source DNA profiles while the 2-cell subsamples returned either single source DNA profiles or new mini-mixtures that are less complex than the original mixture due to the presence of fewer contributors. PG (STRmix™) was implemented, after appropriate validation, to analyze the original bulk mixtures, single source cell subsamples, and the 2-cell mini mixture subsamples from the original 2-6-person mixtures. PG further allowed replicate analysis to be employed which, in many instances, resulted in a significant gain of genotype information such that the returned donor likelihood ratios (LRs) were comparable to that seen in their single source reference profiles (i.e., the reciprocal of their random match probabilities). In every mixture, the DSCS approach gave improved results for each donor compared to standard bulk mixture analysis. With the 5- and 6- person complex mixtures, DSCS recovered highly probative LRs (> 1020) from donors that had returned non-probative LRs (<103) by standard methods.
Molecular evolution of classic Hodgkin lymphoma revealed through whole genome sequencing of Hodgkin and Reed Sternberg cells
Authors: Maura Francesco , Ziccheddu Bachisio , Xiang Jenny Z. , Bhinder Bhavneet , Abascal Federico , Maclachlan Kylee H. , Eng Kenneth Wha , Uppal Manik , He Feng , Zhang Wei , Gao Qi , Yellapantula Venkata , Park Sunita , Oberley Matthew , Ruckdeschel Elizabeth , Lim Megan S. , Wertheim Gerald , Barth Matthew , Horton Terzah M. , Forlenza Christopher , Zhang Yanming , Landgren Ola , Moskowitz Craig H , Cesarman Ethel , Imielinski Marcin , Elemento Olivier , Roshal Mikhail , Giulino-Roth Lisa
Published: November 2021

The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells within a classic Hodgkin lymphoma (cHL) biopsy limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Here we leveraged this method to report the first whole genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events prior to the clinical diagnosis of cHL. We identified alterations in driver genes not previously described in cHL, a high activity of the APOBEC mutational signature, and the presence complex structural variants including chromothripsis. We found that the high ploidy observed in cHL is often acquired through multiple, independent large chromosomal gain events including whole genome duplication. The first of these likely occurs several years prior to the diagnosis of cHL, and the last gains typically occur very close to the time of diagnosis. Evolutionary timing analyses revealed that driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID driven mutagenesis usually preceded large chromosomal gains. The study provides the first temporal reconstruction of cHL pathogenesis and suggests a relatively long time course between the first pathogenic event and the clinical diagnosis.
Structure and Function of the Retina of Low-density Lipoprotein Receptor-related Protein 5 (Lrp5)-deficient Rats
Authors: Ubels John L. , Lin Cheng-Mao , Antonetti David A. , Diaz-Coranguez Monica , Diegel Cassandra R. , Williams Bart O.
Published: November 2021

Loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), result in familial exudative vitreoretinopathy (FEVR), osteoporosis-pseudoglioma syndrome (OPPG), and Norrie disease. CRISPR/Cas9 gene editing was used to produce rat strains deficient in Lrp5. The purpose of this study was to validate this rat model for studies of hypovascular, exudative retinopathies. The retinal vasculature of wildtype and Lrp5 knockout rats was stained with Giffonia simplifolia isolectin B4 and imaged by fluorescence microscopy. Effects on retinal structure were investigated by histology. The integrity of the blood-retina barrier was analyzed by staining for claudin-5 and measurement of permeability to Evans blue dye. Retinas were imaged by fundus photography and SD-OCT, and electroretinograms were recorded. Lrp5 gene deletion led to sparse superficial retinal capillaries and loss of the deep and intermediate plexuses. Autofluorescent exudates were observed, correlated with absence of claudin-5 expression in superficial vessels and increased Evans blue permeability. OCT images show pathology similar to OCT of humans with FEVR, and retinal thickness is reduced by 50% compared to wild-type rats. Histology and OCT reveal that photoreceptor and outer plexiform layers are absent. The retina failed to demonstrate an ERG response. CRISPR/Cas9 gene-editing produced a predictable rat Lrp5 knockout model with extensive defects in the retinal vascular and neural structure and function. This rat model should be useful for studies of exudative retinal vascular diseases involving the Wnt and norrin pathways.
Construction of lux-based promoter-reporter platforms in Mycobacterium bovis BCG for screening of drug repurposing small-molecule compounds as new anti-tuberculosis drugs
Authors: Zhu Li , Lee Annie Wing-Tung , Wu Kelvin Ka-Lok , Gao Peng , Tam Kingsley King-Gee , Rajwani Rahim , Chaburte Galata Chala , Ng Timothy Ting-Leung , Chan Chloe Toi-Mei , Lao Hiu Yin , Yam Wing Cheong , Kao Richard Yi-Tsun , Siu Gilman Kit Hang
Published: November 2021

The emergence of multidrug-resistant strains and hyper-virulent strains of Mycobacterium tuberculosis are big therapeutic challenges for tuberculosis (TB) control. Repurposing bioactive small-molecule compounds has recently become a new therapeutic approach against TB. This study aimed to construct a rapid screening system to identify novel anti-TB agents from a library of small-molecule compounds.

In this study, a total of 320 small-molecule compounds were used to screen for their ability to suppress the expression of a key virulence gene, phoP, of M. tuberculosis complex using luminescence (lux)-based promoter-reporter platforms. The minimum inhibitory and bactericidal concentrations on drug-resistant M. tuberculosis and cytotoxicity to human macrophage were determined. RNA-sequencing (RNA-seq) was conducted to determine the drug mechanisms of the selected compounds as novel antibiotics or anti-virulent agents against the M. tuberculosis complex.

Six compounds displayed bactericidal activity against M. bovis BCG, in which Ebselen demonstrated the lowest cytotoxicity to macrophage and was considered as a potential antibiotic for TB. Another ten compounds did not inhibit the in vitro growth of the M. tuberculosis complex but down-regulated the expression of phoP specifically. Of them, ST-193 and ST-193 (hydrochloride) showed low cytotoxicity and could dysregulate the entire phoP-associated gene network, and thus identified as potential anti-virulence agents for M. tuberculosis. This study provides a rapid screening platform coupled with a systematic validation and eventually suggested one potential antibiotic and two anti-virulence agents for M. tuberculosis infections.
Whole-genome sequencing of Chinese native goat offers biological insights into cashmere fiber formation
Authors: Han Hu , Yang Man-Man , Dan Jiang , Zhang Xing-Ju , Wei Qiang , Chen Tao , Wang Qi-Ju , Yang Cheng-Ye , Wulan Bater , Zhang Ting-Ting , Gen Gang , Mengkedala  , Li Bin , Deng Wei-Dong , Miao Ze-Pu , Wang Ran , Zhang Qing-Feng , Li Lin , Chao Sheng-Yu , Fang Ming , Li Yong
Published: November 2021

Cashmere evolved naturally in the goat, and almost all breeds of goat can produce more or less cashmere fibers. However, the genetic alterations underlying cashmere trait selection are still unclear.We sequenced 120 Chinese native goat including two cashmere goat breeds (Ujumain, Chaidamu) and six ordinary goat breeds (Jining Gray, Matou, Guizhou Black, Jintang Black, Yunnan Black Bone, Chengdu Brown). The genome-wide selective sweep of cashmere goat and ordinary goat revealed a novel set of candidate genes as well as pathways, such as Nuclear factor kappa-B and Wnt Signaling pathways. Of them, theLHX2gene regulating hair follicle development, was evident from the strongest selection signal when comparing the Uhumqin cashmere goat and ordinary goat. Interestingly, we identified a 582bp deletion at 367 kb upstream ofLHX2with higher frequency in cashmere goats and their ancient relatives. This mutation probably rises along the breeding procedures, and is putatively responsible for cashmere production and diameter, as revealed by association studies. Luciferase assay shows that the deletion, which acts as an insulator, restrains the expression ofLHX2by interfering its upstream enhancers.Our study discovers a novel insulator of theLHX2involved in regulating cashmere production and diameter, which would be beneficial to understanding hair follicle development and regeneration. Our findings also provide new insights into the genetic formation of cashmere, and facilitate subsequent molecular breeding for cashmere goat improvement.
The role of Imp and Syp RBPs in precise neuronal elimination by apoptosis through the regulation of TFs
Authors: Guan Wenyue , Nie Ziyan , Laurençon Anne , Bouchet Mathilde , Godin Christophe , Chérif Kabir , Darnas Aurélien , Enriquez Jonathan
Published: November 2021

Neuronal stem cells generate a limited and consistent number of neuronal progenies, each possessing distinct morphologies and functions. The precise production of neurons with distinct identities must be meticulously regulated throughout development to ensure optimal brain function. In our study, we focused on a neuroblast lineage in Drosophila known as Lin A/15, which gives rise to motoneurons (MNs) and glia. Interestingly, the Lin A/15 neuroblast dedicates 40% of its time to producing immature MNs that are subsequently eliminated through apoptosis. Two RNA-binding proteins, Imp and Syp, play crucial roles in this process of neuronal elimination. We found that Imp+ MNs survive, while Imp-, Syp+ MNs undergo apoptosis. Our results indicate that Imp promotes survival, whereas Syp promotes cell death in immature MNs. Furthermore, our investigations revealed that late-born motoneurons face elimination due to their failure to express a functional code of transcription factors that control their morphological fate (mTFs).

Late-born MNs possess a unique and distinct set of TFs compared to early-born MNs. By manipulating the expression of Imp and Syp in late-born motoneurons, we observed a shift in the TF code of late MNs towards that of early-born MNs, resulting in their survival. Additionally, introducing the TF code of early MNs into late-born MNs also promoted their survival. These findings demonstrate that the differential expression of Imp and Syp in immature MNs establishes a connection between generating a precise number of MNs and producing MNs with distinct identities through the regulation of mTFs.

Importantly, both Imp and Syp are conserved in vertebrates, suggesting that they play a central role in determining the number of neurons produced during development. The Drosophila model, along with its genetic tools, provides a unique opportunity to further explore and decipher the functions of these RNA-binding proteins in neural stem cells versus immature neurons. The insights gained from these studies could shed light on the broader mechanisms of neurogenesis and neuronal identity determination in more complex organisms.
Malaria transmission relies on concavin-mediated maintenance ofPlasmodiumsporozoite cell shape
Authors: Kehrer Jessica , Formaglio Pauline , Muthinja Julianne Mendi , Weber Sebastian , Baltissen Danny , Lance Christopher , Ripp Johanna , Grech Janessa , Meissner Markus , Funaya Charlotta , Amino Rogerio , Frischknecht Friedrich
Published: November 2021

During transmission of malaria-causing parasites from mosquitoes to mammals,Plasmodiumsporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here we report on a newly identified protein, concavin, that is important for maintenance of the sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin-GFP localized at the cytoplasmic periphery of sporozoites andconcavin(−)sporozoites progressively rounded up upon entry of salivary glands. These roundedconcavin(−)sporozoites failed to pass through the narrow salivary ducts and were hence rarely ejected by mosquitoes. However, normally shapedconcavin(−)sporozoites could be transmitted and migrated in the skin or skin like environments. Strikingly, motileconcavin(−)sporozoites could disintegrate while migrating through narrow strictures in the skin leading to parasite arrest or death and decreased transmission efficiency. We suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex.

SIGNIFICANCE

Malaria parasites are transmitted byAnophelesmosquitoes and rely on rapid migration for establishing an infection. We identified and characterized a protein, named concavin, essential for maintaining the shape of the sporozoite. Concavin is a membrane associated protein facing the cytoplasm suggesting that it contributes to riveting the plasma membrane to the subtending inner membrane complex. Sporozoites lacking concavin can round up in the salivary glands, are less well transmitted to mice and disintegrate while migrating in the skin. Hence, concavin is essential for parasite transmission and infectivity.

Highlights

- A membrane associated protein is essential forPlasmodiumshape maintenance

-Migrating parasites disintegrate in the absence of concavin

-First protein essential for cellular integrity ofPlasmodiumsporozoites

-Thickened and deformedPlasmodiumsporozoites fail to be transmitted by mosquitoes
The IPF fibroblastic focus is an active collagen biosynthesis factory embedded in a distinct extracellular matrix
Authors: Herrera Jeremy A. , Dingle Lewis , Montero M. Angeles , Shah Rajesh , Venkateswaran Rajamiyer V , Blaikley John F. , Lawless Craig , Schwartz Martin A.
Published: November 2021

Background

The Fibroblastic Focus (FF) is the signature lesion of Idiopathic Pulmonary Fibrosis (IPF) where myofibroblasts accumulate and extracellular matrix (ECM) is produced. However, the molecular composition and function of the FF and surrounding tissue remain undefined.

Methods

Utilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 IPF specimens plus 6 non-fibrotic alveolar specimens as controls. The data were subject to qualitative and quantitative analysis, and validation by immunohistochemistry.

Results

We found that the protein signature of IPF alveoli is defined by immune deregulation as the strongest category. The IPF mature scar was classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared to non-fibrotic control.

Conclusion

Spatial proteomics demonstrated distinct protein compositions in the histologically defined regions of IPF tissue. These data revealed that the FF is the main site of collagen biosynthesis and that the alveoli adjacent to the FF are abnormal. This new and essential information will inform future mechanistic studies on mechanisms of IPF progression.
A Prospective Cohort Study Towards Improving Enhanced Recovery After Cesarean (ERAC) Pathways
Authors: Cojocaru Liviu , Pahlavan Autusa , Alton Suzanne , Coghlan Martha , Seung Hyunuk , Trilling Ariel , Kodali Bhavani S , Crimmins Sarah , Goetzinger Katherine
Published: November 2021

Objective

To evaluate whether Enhanced Recovery After Cesarean (ERAC) pathways reduces inpatient and outpatient opioid use, pain scores and improves the indicators of postoperative recovery.

Study design

This is a prospective cohort study of all patients older than 18 undergoing an uncomplicated cesarean delivery (CD) at an academic medical center. We excluded complicated CD, patients with chronic pain disorders, chronic opioid use, acute postpartum depression, or mothers whose neonate demised before their discharge. Lastly, we excluded non-English and non-Spanish speaking patients. Our study compared the outcomes in patients before (pre-ERAC) and after (post-ERAC) implementation of an ERAC pathways. Primary outcomes were inpatient morphine milligram equivalent (MME) use and the patient’s delta pain scores. Secondary outcomes were outpatient MME prescriptions as well as indicators of postoperative recovery.

Results

Of 308 patients undergoing CD from October 2019 to September 2020, 196 were enrolled in the pre-ERAC cohort and 112 in the post-ERAC cohort. Patients in the post-ERAC cohort were less likely to require opioids in the postoperative period compared to the pre-ERAC cohort (35.7% vs. 18.4%, p<0.001). In addition, there was a significant reduction in the MME per stay in this cohort [16.8 MME (11.2-33.9) vs. 30 MME (20-49), p<0.001]. In the post-ERAC cohort, there was also a reduction in the number of patients who required prescribed opioids at the time of discharge (86.6 vs. 98%, p<0.001) as well as in the amount of MMEs prescribed [150 MME (112-150) vs. 150 MME (150-225), p<0.001; different shape of distribution]. Patients in the post-ERAC cohort had lower delta pain scores [2.2 (1.3-3.7) vs. 3.3 (2.3-4.7), p<0.001].

Conclusion

Our study has illustrated that our ERAC pathways reduced inpatient and outpatient opioid use as well as patient-reported pain scores while improving indicators of postoperative recovery.
Construction of a bioluminescence-based assay for bitter taste receptors (TAS2Rs)
Authors: Tan Shi Min , Seetoh Wei-Guang
Published: November 2021

In humans, a family of 25 bitter taste receptors (TAS2Rs) mediates bitter taste perception. A common approach to characterize bitter causative agents involves expressing TAS2Rs and the appropriate signal transducers in heterologous cell systems, and monitoring changes in the intracellular free calcium levels upon ligand exposure using a fluorescence-based modality, which typically suffers from a low signal window, and is susceptible to interference by autofluorescence, therefore prohibiting its application to screening of plant or food extracts, which are likely to contain autofluorescent compounds. Here, we report the development and validation of a bioluminescence-based intracellular calcium release assay for TAS2Rs that has a better assay performance than a fluorescence-based assay. Furthermore, the bioluminescence-based assay enables the evaluation of TAS2R agonists within an autofluorescent matrix, highlighting its potential utility in the assessment of the bitterness-inducing properties of plant or food fractions by the food industry. Additionally, improvement to the bioluminescence-based assay for some TAS2Rs was achieved by altering their N-terminal signal sequences, leading to signal window enhancement. Altogether, the bioluminescence-based TAS2R assay can be used to perform functional studies of TAS2Rs, evaluate TAS2R-modulating properties of autofluorescent samples, and facilitate the discovery of compounds that can function as promising bitter taste modulators.
Probing the modulation of enzyme kinetics by multi-temperature, time-resolved serial crystallography
Authors: Schulz Eike C. , Prester Andreas , von Stetten David , Gore Gargi , Hatton Caitlin E. , Bartels Kim , Leimkohl Jan-Philipp , Schikora Hendrik , Ginn Helen M. , Tellkamp Friedjof , Mehrabi Pedram
Published: November 2021

We present an environmental enclosure for fixed-target serial crystallography, enabling X-ray diffraction experiments in a temperature window from below 10 °C to above 70 °C - a universal parameter of protein function. Via 5D-SSX time-resolved experiments can now be carried out at physiological temperatures, providing fundamentally new insights into protein function. We show temperature-dependent modulation of turnover kinetics for the mesophilicβ-lactamase CTX-M-14 and for the hyperthermophilic enzyme xylose isomerase.
Cortical and autonomic responses during staged Taoist meditation: two distinct meditation strategies
Authors: Volodina Maria , Smetanin Nikolai , Lebedev Mikhail , Ossadtchi Alexei
Published: November 2021

Meditation is a consciousness state associated with specific physiological and neural correlates. Numerous investigations of these correlates reported controversial results which prevented a consistent depiction of the underlying neurophysiological processes. Here we investigated the dynamics of multiple neurophysiological indicators during a staged meditation session. We measured the physiological changes at rest and during the guided Taoist meditation in experienced meditators and naive subjects. We recorded EEG, respiration, galvanic skin response, and photoplethysmography. All subjects followed the same instructions split into 16 stages. In the experienced meditators group we identified two subgroups with different physiological markers dynamics. One subgroup showed several signs of general relaxation evident from the changes in heart rate variability, respiratory rate, and EEG rhythmic activity. The other subgroup exhibited mind concentration patterns primarily noticeable in the EEG recordings while no autonomic responses occurred. The duration and type of previous meditation experience or any baseline indicators we measured did not explain the segregation of the meditators into these two groups. These results suggest that two distinct meditation strategies could be used by experienced meditators, which partly explains the inconsistent results reported in the earlier studies evaluating meditation effects. Our findings are also relevant to the development of the high-end biofeedback systems.
Exploiting convergent evolution to derive a pan-cancer cisplatin response gene expression signature
Authors: Scarborough Jessica A. , Eschrich Steven A. , Torres-Roca Javier , Dhawan Andrew , Scott Jacob G.
Published: November 2021

Precision medicine offers remarkable potential for the treatment of cancer, but is largely focused on tumors that harbor actionable mutations. Gene expression signatures can expand the scope of precision medicine by predicting response to traditional (cytotoxic) chemotherapy agents without relying on changes in mutational status. We present a novel signature extraction method, inspired by the principle of convergent evolution, which states that tumors with disparate genetic backgrounds may evolve similar phenotypes independently. This evolutionary-informed method can be utilized to produce signatures predictive of response to over 200 chemotherapeutic drugs found in the Genomics of Drug Sensitivity in Cancer Database. Here, we demonstrate its use by extracting the Cisplatin Response Signature, CisSig, for use in predicting a common trait (sensitivity to cisplatin) across disparate tumor subtypes (epithelial-origin tumors). CisSig is predictive of cisplatin response within the cell lines and clinical trends in independent datasets of tumor samples. Finally, we demonstrate preliminary validation of CisSig for use in muscle-invasive cancer, predicting overall survival in patients who undergo cisplatin-containing chemotherapy. This novel methodology can be used to produce robust signatures for the prediction of traditional chemotherapeutic response, dramatically increasing the reach of personalized medicine in cancer.

Translational Relevance

Most precision medicine research focuses on using targeted drugs on patients with known driver mutations, yet the majority of patients don’t have actionable mutations. Using a novel signature extraction method, we produce the Cisplatin Response Signature (CisSig) to predict how well patients with epithelial-origin tumors will respond to cisplatin, a common cytotoxic chemotherapy. We show that expression of CisSig is correlated to clinical trends of cisplatin use in treatment guidelines using independent tumor databases. Then, we look at preliminary validation of CisSig for use in patients with muscle-invasive bladder cancer. Using two independent cohorts of pre-treatment tumor samples, we show that a CisSig-trained model is predictive of overall survival in patients who did receive cisplatin, but this signal is lost in patients who did not receive cisplatin–indicating that the model is predictive of therapeutic response, not simply prognosis.
A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair
Authors: Lechuga Susana , Cartagena-Rivera Alexander X. , Khan Afshin , Crawford Bert I. , Narayanan Vani , Conway Daniel E. , Lehtimäki Jaakko , Lappalainen Pekka , Rieder Florian , Longworth Michelle S. , Ivanov Andrei I.
Published: November 2021

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. While biochemical mechanisms that regulate activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC-45A were decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.
Lysosome-targeted lipid probes reveal sterol transporters NPC1 and LIMP-2 as sphingosine transporters
Authors: Altuzar Janathan , Notbohm Judith , Stein Frank , Haberkant Per , Heybrock Saskia , Worsch Jutta , Saftig Paul , Höglinger Doris
Published: November 2021

Lysosomes are central catabolic organelles involved in lipid homeostasis and their dysfunction is associated with pathologies ranging from lysosomal storage disorders to common neurodegenerative diseases. The mechanism of lipid efflux from lysosomes is well understood for cholesterol, while the export of other lipids, particularly sphingosine, is less well studied. To overcome this knowledge gap, we have developed functionalized sphingosine and cholesterol probes that allow us to follow their metabolism, protein interactions as well as their subcellular localization. These probes feature a modified cage group for lysosomal targeting and controlled release of the active lipids with high temporal precision. An additional photo-crosslinkable group allowed for the discovery of lysosomal interactors for both sphingosine and cholesterol. In this way, we found that two lysosomal cholesterol transporters, NPC1 and LIMP-2/SCARB2, also directly bind to sphingosine. In addition, we showed that absence of either protein leads to lysosomal sphingosine accumulation which suggests a sphingosine transport role of both proteins. Furthermore, artificial elevation of lysosomal sphingosine levels impaired cholesterol efflux, consistent with sphingosine and cholesterol sharing a common export mechanism.
The evolution of metazoan shelterin
Authors: Myler Logan R. , Kinzig Charles G. , Sasi Nanda K. , Zakusilo George , Cai Sarah W. , de Lange Titia
Published: November 2021

The mammalian telomeric shelterin complex—comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1—blocks the DNA damage response at chromosome ends and interacts with telomerase and the CST complex to regulate telomere length. The evolutionary origins of shelterin are unclear, partly because unicellular organisms have distinct telomeric proteins. Here, we describe the evolution of metazoan shelterin, showing that TRF1 emerged in vertebrates upon duplication of a TRF2-like ancestor. TRF1 and TRF2 diverged rapidly during vertebrate evolution through the acquisition of new domains and interacting factors. Vertebrate shelterin is also distinguished by the presence of an HJRL domain in the split C-terminal OB fold of POT1, whereas invertebrate POT1s carry inserts of variable nature. Importantly, the data reveal that, apart from the primate and rodent POT1 orthologs, all metazoan POT1s are predicted to have a fourth OB fold at their N termini. Therefore, we propose that POT1 arose from a four-OB-fold ancestor, most likely an RPA70-like protein. This analysis provides insights into the biology of shelterin and its evolution from ancestral telomeric DNA-binding proteins.
DrosophilaMushroom Bodies Are Dispensable for Visual, Tactile, and Motor Learning
Authors: Wolf Reinhard , Wittig Tobias , Liu Li , Wustmann Gerold , Eyding Dirk , Heisenberg Martin
Published: November 2021

A total of 18 associative learning/memory tests have been applied toDrosophila melanogasterflies lacking mushroom bodies. Only in paradigms involving chemosensory cues as conditioned stimuli have flies been found to be compromised by a block in the mushroom body pathway. Among the learning tasks not requiring these structures are a case of motor learning (yaw torque/heat), a test of the fly’s spatial orientation in total darkness, conditioned courtship suppression by mated females, and nine different examples of visual learning. The latter used the reinforcers of heat, visual oscillations, mechanical shaking, or sucrose, and as conditioned stimuli, color, intensity contrast, as well as stationary and moving visual patterns. No forms of consolidated memory have been tested in mushroom body-less flies. With respect to short-term memory the mushroom bodies ofDrosophilaare specially required for chemosensory learning tasks, but not for associative learning and memory in general.
Hyperactivity and Learning Deficits in Transgenic Mice Bearing a Human Mutant Thyroid Hormone β1 Receptor Gene
Authors: McDonald Michael P. , Wong Rosemary , Goldstein Gregory , Weintraub Bruce , Cheng Sheue-yann , Crawley Jacqueline N.
Published: November 2021

Resistance to thyroid hormone (RTH) is a human syndrome mapped to thethyroid receptor β(TRβ) gene on chromosome 3, representing a mutation of the ligandbinding domain of theTRβgene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutantTRβgene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant humanTRβgene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD.
α7 Nicotinic Receptor Subunits Are Not Necessary for Hippocampal-Dependent Learning or Sensorimotor Gating: A Behavioral Characterization of Acra7-Deficient Mice
Authors: Paylor Richard , Nguyen Michelle , Crawley Jacqueline N. , Patrick James , Beaudet Arthur , Orr-Urtreger Avi
Published: November 2021

The α7 nicotinic acetylcholine receptor (nAChR) subunit is abundantly expressed in the hippocampus and contributes to hippocampal cholinergic synaptic transmission suggesting that it may contribute to learning and memory. There is also evidence for an association between levels of α7 nAChR and in sensorimotor gating impairments. To examine the role of α7 nAChRs in learning and memory and sensorimotor gating, Acra7 homozygous mutant mice and their wild-type littermates were tested in a Pavlovian conditioned fear test, for spatial learning in the Morris water task, and in the prepulse inhibition paradigm. Exploratory activity, motor coordination, and startle habituation were also evaluated. Acra7 mutant mice displayed the same levels of contextual and auditory-cue condition fear as wild-type mice. Similarly, there were no differences in spatial learning performance between mutant and wild-type mice. Finally,Acra7 mutant and wild-type mice displayed similar levels of prepulse inhibition. Other behavioral responses in Acra7 mutant mice were also normal, except for an anxiety-related behavior in the open-field test. The results of this study show that the absence of α7 nAChRs has little impact on normal, base-line behavioral responses. Future studies will examine the contribution of α7 nAChR to the enhancement of learning and sensorimotor gating following nicotine treatments.
On the Respective Roles of Nitric Oxide and Carbon Monoxide in Long-Term Potentiation in the Hippocampus
Authors: Zhuo Min , Laitinen Jarmo T. , Li Xiao-Ching , Hawkins Robert D.
Published: November 2021

Perfusion of hippocampal slices with an inhibitor of nitric oxide (NO) synthase-blocked induction of long-term potentiation (LTP) produced by a one-train tetanus and significantly reduced LTP by a two-train tetanus, but only slightly reduced LTP by a four-train tetanus. Inhibitors of heme oxygenase, the synthetic enzyme for carbon monoxide (CO), significantly reduced LTP by either a two-train or four-train tetanus. These results suggest that NO and CO are both involved in LTP but may play somewhat different roles. One possibility is that NO serves a phasic, signaling role, whereas CO provides tonic, background stimulation. Another possibility is that NO and CO are phasically activated under somewhat different circumstances, perhaps involving different receptors and second messengers. Because NO is known to be activated by stimulation of NMDA receptors during tetanus, we investigated the possibility that CO might be activated by stimulation of metabotropic glutamate receptors (mGluRs). Consistent with this idea, long-lasting potentiation by the mGluR agonist tACPD was blocked by inhibitors of heme oxygenase but not NO synthase. Potentiation by tACPD was also blocked by inhibitors of soluble guanylyl cyclase (a target of both NO and CO) or cGMP-dependent protein kinase, and guanylyl cyclase was activated by tACPD in hippocampal slices. However, biochemical assays indicate that whereas heme oxygenase is constitutively active in hippocampus, it does not appear to be stimulated by either tetanus or tACPD. These results are most consistent with the possibility that constitutive (tonic) rather than stimulated (phasic) heme oxygenase activity is necessary for potentiation by tetanus or tACPD, and suggest that mGluR activation stimulates guanylyl cyclase phasically through some other pathway.
Landmark Discrimination Learning in the Dog
Authors: Milgram Norton W. , Adams Beth , Callahan Heather , Head Elizabeth , Mackay Bill , Thirlwell Celeste , Cotman Carl W.
Published: November 2021

Allocentric spatial memory was studied in dogs of varying ages and sources using a landmark discrimination task. The primary goal of this study was to develop a protocol to test landmark discrimination learning in the dog. Using a modified version of a landmark test developed for use in monkeys, we successfully trained dogs to make a spatial discrimination on the basis of the position of a visual landmark relative to two identical discriminanda. Task performance decreased, however, as the distance between the landmark and the “discriminandum” was increased. A subgroup of these dogs was also tested on a delayed nonmatching to position spatial memory task (DNMP), which relies on egocentric spatial cues. These findings suggest that dogs can acquire both allocentric and egocentric spatial tasks. These data provide a useful tool for evaluating the ability of canines to use allocentric cues in spatial learning.
Mapping of the Anatomical Circuit of CaM Kinase-Dependent Courtship Conditioning in Drosophila
Authors: Joiner Mei-ling A. , Griffith Leslie C.
Published: November 2021

Globally inhibiting CaM kinase activity in Drosophila, using a variety of genetic techniques, disrupts associative memory yet leaves visual and chemosensory perception intact. These studies implicate CaM kinase in the plastic processes underlying learning and memory but do not identify the neural circuitry that specifies the behavior. In this study, we use the GAL4/UAS binary expression system to define areas of the brain that require CaM kinase for modulation of courtship conditioning. The CaM kinase-dependent neurons that determine the response to the mated female during conditioning and those involved in formation and expression of memory were found to be located in distinct areas of the brain. This supports the idea that courtship conditioning results in two independent behavioral modifications: a decrement in courtship during the conditioning period and an associative memory of conditioning. This study has allowed us for the first time to genetically determine the circuit of information flow for a memory process in Drosophila. The map we have generated dissects the behavior into multiple components and will provide tools that allow both molecular and electrophysiological access to this circuit.
Signaling Mechanisms Mediating BDNF Modulation of Synaptic Plasticity in the Hippocampus
Authors: Gottschalk Wolfram A. , Jiang Hao , Tartaglia Nicole , Feng Linyin , Figurov Alexander , Lu Bai
Published: November 2021

Although recent studies indicate that brain-derived neurotrophic factor (BDNF) plays an important role in hippocampal synaptic plasticity, the underlying signaling mechanisms remain largely unknown. Here, we have characterized the signaling events that mediate the BDNF modulation of high-frequency synaptic transmission. Mitogen-associated protein kinase (MAPK), phosphotidylinositol-3 kinase (PI3K), and phospholipase C-γ (PLC-γ) are the three signaling pathways known to mediate neurotrophin signaling in other systems. In neonatal hippocampal slices, application of BDNF rapidly activated MAPK and PI3K but not PLC-γ. BDNF greatly attenuated synaptic fatigue at CA1 synapses induced by a train of high-frequency, tetanic stimulation (HFS). Inhibition of the MAPK and PI3K, but not PLC-γ, prevented the BDNF modulation of high-frequency synaptic transmission. Neurotrophin-3 (NT-3), a close relative of BDNF, did not activate MAPK or PI3K and had no effect on synaptic fatigue in the neonatal hippocampus. Neither forskolin, which activated MAPK but not PI3 kinase, nor ciliary neurotrophic factor (CNTF), which activated PI3K but not MAPK, affected HFS-induced synaptic fatigue. Treatment of the slices with forskolin together with CNTF still had no effect on synaptic fatigue. Thus, although the activation of MAPK and PI3K is required, the two together are not sufficient to mediate the BDNF effect. Inhibition of new protein synthesis by anisomycin or cycloheximide did not prevent the BDNF effect. These data suggest that BDNF modulation of high-frequency transmission is independent of protein synthesis but requires MAPK and PI3K and yet another signaling pathway to act together in the hippocampus.