The fasciola cinereum of the hippocampal tail as an interventional target in epilepsy.

Targeted tissue ablation involving the anterior hippocampus is the standard of care for patients with drug-resistant mesial temporal lobe epilepsy. However, a substantial proportion continues to suffer from seizures even after surgery. We identified the fasciola cinereum (FC) neurons of the posterior hippocampal tail as an important seizure node in both mice and humans with epilepsy. Genetically defined FC neurons were highly active during spontaneous seizures in epileptic mice, and closed-loop optogenetic inhibition of these neurons potently reduced seizure duration. Furthermore, we specifically targeted and found the prominent involvement of FC during seizures in a cohort of six patients with epilepsy. In particular, targeted lesioning of the FC in a patient reduced the seizure burden present after ablation of anterior mesial temporal structures. Thus, the FC may be a promising interventional target in epilepsy.

Secuencia anemia-policitemia en la gestación monocorial. Manejo prenatal y resultados perinatales / Twin anaemia polycythemia polycythemia sequence in monochorionic gestation. Prenatal management and perinatal outcomes

Introducción: El objetivo de este trabajo consiste en describir y analizar los resultados perinatales de una serie de casos de anemia-policitemia (TAPS) seguidos y tratados en un centro de referencia en Medicina Fetal, en términos del tipo TAPS y la conducta prenatal aplicada. Métodos: Análisis estadístico descriptivo e inferencial con el programa IBM SPSS Statistics. Resultados: Se diagnosticaron 13 casos de TAPS en un total de 510 gestaciones monocoriales. La mayoría de TAPS espontáneos se diagnosticaron en estadios precoces (5 casos), mientras que, en TAPS posláser, 3/6 casos se diagnosticaron en estadios avanzados. Cuatro fetos (4/26; 15,4%) presentaron alteraciones en la neurosonografía, 3 (3/14; 21,4%) en TAPS espontáneos y uno (1/12, 8,3%) en TAPS posláser (p = 0,308). Se detectó hipertrofia miocárdica en 7 fetos, 5 (71,4%) de los cuales correspondían a TAPS en estadios avanzados. Cuatro eran TAPS espontáneos (4/14; 28,6%) y 3 TAPS posláser (3/12; 25%) (p = 0,797). Ocho casos (8/13; 62,5%) precisaron algún procedimiento de terapia fetal. Se produjeron 3 muertes fetales anteparto y una pareja decidió interrupción legal del embarazo (19,23%). Las 5 pérdidas se produjeron en estadio IV (p=0,008).La supervivencia global fue del 80,8% (21/26 recién nacidos vivos). No hubo diferencias significativas en función del tipo TAPS (p = 0,159) y la conducta prenatal adoptada (p = 0,746). Conclusiones: El TAPS espontáneo es, por tanto, una entidad clínica con un impacto en los resultados perinatales similar al posláser. Parece que la conducta expectante y el láser consiguen mayor edad gestacional al nacimiento.(AU)

Introduction: The aim of this paper is to describe and analyze the perinatal outcomes of a series of TAPS cases followed and treated in a Fetal Medicine referral center, in terms of the type of TAPS and the prenatal behavior applied. Methods: Descriptive and inferential statistical analysis with IBM SPSS Statistics software. Results: Thirteen cases of TAPS were diagnosed in a total of 510 monocorial gestations. Most of the spontaneous TAPS were diagnosed at early stages (5 cases), whereas, in post-laser TAPS, 3/6 cases were diagnosed at advanced stages. Four fetuses (4/26; 15.4%) presented alterations in neurosonography, 3 (3/14; 21.4%) in spontaneous TAPS and one (1/12, 8.3%) in post-laser TAPS (P=0.308). Myocardial hypertrophy was detected in 7 fetuses, 5 (71.4%) of which corresponded to advanced stage TAPS. Four were spontaneous TAPS (4/14; 28.6%) and 3 were post-laser TAPS (3/12; 25%) (P=0.797). Eight cases (8/13; 62.5%) required some fetal therapy procedure. There were 3 antepartum fetal deaths and one couple decided to legally terminate the pregnancy (19.23%). All 5 losses were stage IV (P=0.008). Overall survival was 80.8% (21/26 live newborns). There were no significant differences according to the type of TAPS (P= 0.159) and the prenatal behavior adopted (P=0.746).Conclusions: Spontaneous TAPS is therefore a clinical entity with an impact on perinatal outcomes similar to post-laser. It seems that expectant management and laser achieve higher gestational age at birth.(AU)

Comparison of maternal, fetal, obstetric and neonatal outcomes for 234 triplet pregnancies conceived in vivo versus IVF and ICSI conceptions.

RESEARCH QUESTION: Is there a difference in maternal, fetal, obstetric and neonatal outcomes for triplet pregnancies when comparing in vivo conceptions with those conceived by assisted reproductive technology (ART)? DESIGN: This single-centre, retrospective cohort study included all triplet pregnancies followed up at La Paz University Hospital, Madrid between 2000 and 2022. The characteristics of the pregnant women, and maternal, fetal, obstetric and perinatal outcomes were examined. Univariate and multivariate statistical analyses were performed. RESULTS: In total, 234 triplet pregnancies were analysed: 92 in the natural and assisted insemination conception group (in-vivo conception) and 142 in the in vitro fertilization and intracytoplasmic sperm injection conception group (ART conception). ART triplet pregnancies were more common between 2000 and 2010 (P = 0.003). The percentage of monochorionic triamniotic pregnancies was significantly higher (P = 0.02) in the in-vivo conception group, and the percentage of dichorionic triamniotic pregnancies was significantly higher (P = 0.003) in the ART conception group. After adjusting for confounders, intrauterine growth restriction (IUGR) remained significantly more common in the ART conception group (adjusted odds ratio 8.65, 95% CI 1.66-45.03; P = 0.01). Differences in maternal age (P = 0.61), threatened preterm labour (P = 0.10), Apgar score ≤5 at 5 min (P = 0.99), umbilical cord pH <7.20 (P = 0.99) and fetal death (P = 0.99) disappeared after adjustment for confounders. CONCLUSION: ART triplet pregnancies had a higher rate of IUGR than in vivo triplet pregnancies. This could be related to higher maternal age, and higher rates of Apgar score ≤5 at 5 min and umbilical cord pH <7.20 in these pregnancies. In these cases, placental examination could provide valuable information.

Synthetic yeast chromosome XI design provides a testbed for the study of extrachromosomal circular DNA dynamics.

We describe construction of the synthetic yeast chromosome XI (synXI) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design.

Serum anti-GM2 and anti-GalNAc-GD1a IgG antibodies are biomarkers for acute canine polyradiculoneuritis.

OBJECTIVES: A previous single-country pilot study indicated serum anti-GM2 and anti-GA1 anti-glycolipid antibodies as potential biomarkers for acute canine polyradiculoneuritis. This study aims to validate these findings in a large geographically heterogenous cohort. MATERIALS AND METHODS: Sera from 175 dogs clinically diagnosed with acute canine polyradiculoneuritis, 112 dogs with other peripheral nerve, cranial nerve or neuromuscular disorders and 226 neurologically normal dogs were screened for anti-glycolipid antibodies against 11 common glycolipid targets to determine the immunoglobulin G anti-glycolipid antibodies with the highest combined sensitivity and specificity for acute canine polyradiculoneuritis. RESULTS: Anti-GM2 anti-glycolipid antibodies reached the highest combined sensitivity and specificity (sensitivity: 65.1%, 95% confidence interval 57.6 to 72.2%; specificity: 90.2%, 95% confidence interval 83.1 to 95.0%), followed by anti-GalNAc-GD1a anti-glycolipid antibodies (sensitivity: 61.7%, 95% confidence interval 54.1 to 68.9%; specificity: 89.3%, 95% confidence interval 82.0 to 94.3%) and these anti-glycolipid antibodies were frequently present concomitantly. Anti-GA1 anti-glycolipid antibodies were detected in both acute canine polyradiculoneuritis and control animals. Both for anti-GM2 and anti-GalNAc-GD1a anti-glycolipid antibodies, sex was found a significantly associated factor with a female to male odds ratio of 2.55 (1.27 to 5.31) and 3.00 (1.22 to 7.89), respectively. Anti-GalNAc-GD1a anti-glycolipid antibodies were more commonly observed in dogs unable to walk (OR 4.56, 1.56 to 14.87). CLINICAL SIGNIFICANCE: Anti-GM2 and anti-GalNAc-GD1a immunoglobulin G anti-glycolipid antibodies represent serum biomarkers for acute canine polyradiculoneuritis.

An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens.

The trafficking of specific protein cohorts to correct subcellular locations at correct times is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or fluorescence-activated cell sorting (FACS). To empower the study of protein trafficking processes with gene perturbation, we developed a genetically encoded molecular tool named HiLITR (High-throughput Localization Indicator with Transcriptional Readout). HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identified genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in mislocalization and destabilization of many mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.

Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease.

Molecular integrators, in contrast to real-time indicators, convert transient cellular events into stable signals that can be exploited for imaging, selection, molecular characterization, or cellular manipulation. Many integrators, however, are designed as complex multicomponent circuits that have limited robustness, especially at high, low, or nonstoichiometric protein expression levels. Here, we report a simplified design of the calcium and light dual integrator FLARE. Single-chain FLARE (scFLARE) is a single polypeptide chain that incorporates a transcription factor, a LOV domain-caged protease cleavage site, and a calcium-activated TEV protease that we designed through structure-guided mutagenesis and screening. We show that scFLARE has greater dynamic range and robustness than first-generation FLARE and can be used in culture as well as in vivo to record patterns of neuronal activation with 10-min temporal resolution.

A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving Aversion.

The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here, we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCRE's modular design, we expressed an optogenetic channel selectively in this cell type and showed that direct recruitment of this otherwise genetically inaccessible population elicits behavioral aversion. The specificity and minute resolution of FLiCRE enables molecularly informed characterization, manipulation, and reprogramming of activated cellular ensembles.

The prognostic value of the Koret CT score in dogs following traumatic brain injury.

Traumatic brain injury (TBI) is a common condition in veterinary medicine with relatively high mortality rate. Recently, a study that correlated abnormal computed tomography (CT) findings with outcome in dogs with head trauma established a prognostic scoring system termed Koret CT score (KCTS). The purpose of this study was to evaluate the accuracy of the KCTS in making short- and long-term prognosis in dogs presented within 72 h of TBI. Thirty-five dogs that were admitted to a hospital during 2010-2019 with TBI and were CT-scanned within 72 h of injury were included in the study. Retrospectively collected data included signalment, modified Glasgow Coma Scale score (MGCS), CT findings, and outcome, i.e. short-term (defined as 10 days) and long-term (6 months) survival. CT images were reviewed and the KCTS was calculated for all dogs. Association between KCTS and outcome was examined. A significant negative association was found between KCTS and both short- and long-term survival. The area under receiver operating characteristic curve for KCTS for short- and long-term survival was 0.9 and 0.87, respectively. Furthermore, the probability of survival in the short term was predicated by the KCTS in an almost linear fashion and a score of 3 points or less on the KCTS was associated with survival with 85% sensitivity and 100% specificity. These results validate the prognostic value of the KCTS in dogs with TBI and provide a complementary tool for serial clinical and neurological evaluation.

Correction: Novel broad-spectrum activity-based probes to profile malarial cysteine proteases.

[This corrects the article DOI: 10.1371/journal.pone.0227341.].

MitoBlue as a tool to analyze the mitochondria-lysosome communication.

MitoBlue is a fluorescent bisamidine that can be used to easily monitor the changes in mitochondrial degradation processes in different cells and cellular conditions. MitoBlue staining pattern is exceptional among mitochondrial dyes and recombinant fluorescent probes, allowing the dynamic study of mitochondrial recycling in a variety of situations in living cells. MitoBlue is a unique tool for the study of these processes that will allow the detailed characterization of communication between mitochondria and lysosomes.

Publisher Correction: Directed evolution improves the catalytic efficiency of TEV protease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Novel broad-spectrum activity-based probes to profile malarial cysteine proteases.

Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability.

Directed evolution improves the catalytic efficiency of TEV protease.

Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV's cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our TEV-S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-s temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed-evolution platform used to generate them could be beneficial across a wide range of applications.

Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.

Dipeptidyl aminopeptidases (DPAPs) are cysteine proteases that cleave dipeptides from the N-terminus of protein substrates and have been shown to play important roles in many pathologies including parasitic diseases such as malaria, toxoplasmosis and Chagas's disease. Inhibitors of the mammalian homologue cathepsin C have been used in clinical trials as potential drugs to treat chronic inflammatory disorders, thus proving that these enzymes are druggable. In Plasmodium species, DPAPs play important functions at different stages of parasite development, thus making them potential antimalarial targets. Most DPAP inhibitors developed to date are peptide-based or peptidomimetic competitive inhibitors. Here, we used a high throughput screening approach to identify novel inhibitor scaffolds that block the activity of Plasmodium falciparum DPAP1. Most of the hits identified in this screen also inhibit Plasmodium falciparum DPAP3, cathepsin C, and to a lesser extent other malarial clan CA proteases, indicating that these might be general DPAP inhibitors. Interestingly, our mechanism of inhibition studies indicate that most hits are allosteric inhibitors, which opens a completely new strategy to inhibit these enzymes, study their biological function, and potentially develop new inhibitors as starting points for drug development.

Canonical DNA minor groove insertion of bisbenzamidine-Ru(ii) complexes with chiral selectivity.

We report the first Ru(ii) coordination compounds that interact with DNA through a canonical minor groove insertion mode and with selectivity for A/T rich sites. This was made possible by integrating a bis-benzamidine minor groove DNA-binding agent with a ruthenium(ii) complex. Importantly, one of the enantiomers (Δ-[Ru(bpy)2 b4bpy]2+, Δ-4Ru) shows a considerably higher DNA affinity than the parent organic ligand and the other enantiomer, particularly for the AATT sequence, while the other enantiomer preferentially targets long AAATTT sites with overall lower affinity. Finally, we demonstrate that the photophysical properties of these new binders can be exploited for DNA cleavage using visible light.

Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies differences in amino acid preferences between peptide-based substrates and covalent inhibitors.

Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes, it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of nonoptimal peptide substrates. Finally, our homology modelling and docking studies provide potential structural explanations of the differences in specificity between DPAP1, DPAP3, and CatC, and between substrates and inhibitors in the case of DPAP3. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds.

Plasmodium falciparum dipeptidyl aminopeptidase 3 activity is important for efficient erythrocyte invasion by the malaria parasite.

Parasite egress from infected erythrocytes and invasion of new red blood cells are essential processes for the exponential asexual replication of the malaria parasite. These two tightly coordinated events take place in less than a minute and are in part regulated and mediated by proteases. Dipeptidyl aminopeptidases (DPAPs) are papain-fold cysteine proteases that cleave dipeptides from the N-terminus of protein substrates. DPAP3 was previously suggested to play an essential role in parasite egress. However, little is known about its enzymatic activity, intracellular localization, or biological function. In this study, we recombinantly expressed DPAP3 and demonstrate that it has indeed dipeptidyl aminopeptidase activity, but contrary to previously studied DPAPs, removal of its internal prodomain is not required for activation. By combining super resolution microscopy, time-lapse fluorescence microscopy, and immunoelectron microscopy, we show that Plasmodium falciparum DPAP3 localizes to apical organelles that are closely associated with the neck of the rhoptries, and from which DPAP3 is secreted immediately before parasite egress. Using a conditional knockout approach coupled to complementation studies with wild type or mutant DPAP3, we show that DPAP3 activity is important for parasite proliferation and critical for efficient red blood cell invasion. We also demonstrate that DPAP3 does not play a role in parasite egress, and that the block in egress phenotype previously reported for DPAP3 inhibitors is due to off target or toxicity effects. Finally, using a flow cytometry assay to differentiate intracellular parasites from extracellular parasites attached to the erythrocyte surface, we show that DPAP3 is involved in the initial attachment of parasites to the red blood cell surface. Overall, this study establishes the presence of a DPAP3-dependent invasion pathway in malaria parasites.

Time-gated detection of protein-protein interactions with transcriptional readout.

Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery.

A light- and calcium-gated transcription factor for imaging and manipulating activated neurons.

Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.