Deciphering Combinatorial Genetics.

High-order interactions among components of interconnected genetic networks regulate complex functions in biological systems, but deciphering these interactions is challenging. New strategies are emerging to decode these combinatorial genetic interactions across a wide range of organisms. Here, we review advances in multiplexed and combinatorial genetic perturbation technologies and high-throughput profiling platforms that are enabling the systematic dissection of complex genetic networks. These rapidly evolving technologies are being harnessed to probe combinatorial gene functions in functional genomics studies and have the potential to advance our understanding of how genetic networks regulate sophisticated biological phenotypes, to generate novel therapeutic strategies, and to enable the engineering of complex artificial gene networks.

Regulation of mast cells by overlapping but distinct protein interactions of Siglec-6 and Siglec-8.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood. METHODS: Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec-6 and Siglec-8. For functional characterization, each receptor was evaluated biochemically and in ex vivo and in vivo inhibition models of IgE and non-IgE-mediated MC activation in Siglec-6- or Siglec-8-expressing transgenic mice. RESULTS: Siglec-6 and Siglec-8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec-6 and Siglec-8 interacted with a large cluster of proteins involved in IgE and non-IgE-mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL-4 and IL-33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec-6 and Siglec-8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec-6. Indeed, Siglec-6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti-Siglec-6 antibody significantly inhibited SCF-mediated MC activation more in comparison to targeting Siglec-8. CONCLUSION: These data demonstrate a central role for Siglec-6 and Siglec-8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec-6 has a role distinct from that of Siglec-8 in regulating MC function and represents a distinct potential therapeutic target in mast cell-driven diseases.

Cardiovascular manifestations of Erdheim-Chester disease: A narrative review with two cases.

Erdheim-Chester disease (ECD) is a rare 'L' (Langerhans) group histiocytic neoplasm that affects a multitude of organ systems, causing osteosclerotic bone lesions, periaortic encasement ('coated' aorta), retroperitoneal fibrosis involving kidneys and ureters ('hairy kidney'), and infiltration of the central nervous system. Cardiovascular involvement can occur in up to 70% of patients and is usually found during computed tomography/magnetic resonance imaging evaluation. When present, cardiovascular symptoms can have wide variability in presentation from asymptomatic to pericarditis, fatal cardiac tamponade, myocardial infarction, conduction abnormalities, heart failure, renal artery stenosis, and claudication. Cardiac involvement found on imaging includes right atrial pseudotumor, right atrioventricular groove infiltration, and pericardial effusions. ECD can involve the large- and medium-sized arteries, often seen as periarterial thickening (commonly coating the aorta) with stenosis/occlusion. Although more cardiovascular ECD cases have begun to be published in the literature, more data are needed on the outcomes of these patients, as well as how cardiovascular manifestations respond to treatment of ECD.

High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9's DNA- and guide RNA- interacting residues to optimize its editing fidelity.

Parallels between wound healing, epimorphic regeneration and solid tumors.

Striking similarities between wound healing, epimorphic regeneration and the progression of solid tumors have been uncovered by recent studies. In this Review, we discuss systemic effects of tumorigenesis that are now being appreciated in epimorphic regeneration, including genetic, cellular and metabolic heterogeneity, changes in circulating factors, and the complex roles of immune cells and immune modulation at systemic and local levels. We suggest that certain mechanisms enabling regeneration may be co-opted by cancer to promote growth at primary and metastatic sites. Finally, we advocate that working with a unified approach could complement research in both fields.

A roadmap to high-resolution standard microcoil MAS NMR spectroscopy for metabolomics.

Current microcoil probe technology has emerged as a significant advancement in NMR applications to biofluids research. It has continued to excel as a hyphenated tool with other prominent microdevices, opening many new possibilities in multiple omics fields. However, this does not hold for biological samples such as intact tissue or organisms, due to the considerable challenges of incorporating the microcoil in a magic-angle spinning (MAS) probe without relinquishing the high-resolution spectral data. Not until 2012 did a microcoil MAS probe show promise in profiling the metabolome in a submilligram tissue biopsy with spectral resolution on par with conventional high-resolution MAS (HR-MAS) NMR. This result subsequently triggered a great interest in the possibility of NMR analysis with microgram tissues and striving toward the probe development of "high-resolution" capable microcoil MAS NMR spectroscopy. This review gives an overview of the issues and challenges in the probe development and summarizes the advancements toward metabolomics.

Guideline-based and restricted fluid resuscitation strategy in sepsis patients with heart failure: A systematic review and meta-analysis.

OBJECTIVES: To examine whether a fluid resuscitation strategy based on guidelines (at least 30 mL/kg IV crystalloids) vs. a restrictive approach with <30 mL/kg within three hours affects in-hospital mortality in patients with sepsis and a history of heart failure (HF). DATA SOURCES: On 03/07/2023, we searched Embase, PubMed, and Scopus for peer-reviewed papers and abstracts using the PRISMA guidelines. STUDY SELECTION: The language was limited to English. Studies published since 2016 included if they had sepsis patients with a history of HF, or a subgroup of patients with HF, and in-hospital mortality data on these patients that did or did not meet the 30 mL/kg by 3 h (30 × 3) goal. Duplicate studies, studies that focused on a broader period than 3 h from the diagnosis of sepsis or without mortality breakdown for HF patients or with unrelated title/abstract, or without an IRB approval were excluded. DATA EXTRACTION: In-hospital mortality data was taken from the final studies for HF patients with sepsis who did or did not meet the 30 × 3 goal. DATA SYNTHESIS: The meta-analysis was performed using the Review Manager 5.4 program with ORs as the effect measure. The ProMeta program version 3.0 was used to evaluate the publication bias. Egger's linear regression and Berg and Mazumdar's rank correlation was used to evaluate the publication bias. The result was visually represented by a funnel plot. To estimate the proportion of variance attributable to heterogeneity, the I2 statistic was calculated. RESULTS: The search yielded 26,069 records, which were narrowed down to 4 studies. Compared to those who met the 30 × 3 goal, the <30 × 3 group had a significantly higher risk of in-hospital mortality (OR = 1.81, 95% CI = 1.13-2.89, P = 0.01). CONCLUSIONS: Restrictive fluid resuscitation increased the risk of in-hospital mortality in HF patients with sepsis. More rigorous research is required to determine the optimal fluid resuscitation strategy for this population.

The effect of task on object processing revealed by EEG decoding.

Recent studies showed that task demand affects object representations in higher-level visual areas and beyond but not so much in earlier areas. There are, however, limitations in those studies including the relatively weak manipulation of task due to the use of familiar real-life objects, the low temporal resolution in functional magnetic resonance imaging (fMRI) and the emphasis on the amount and not the source of information carried by brain activations. In the current study, observers categorised images of artificial objects in one of two orthogonal dimensions, shape and texture, while their brain activity was recorded with electroencephalogram (EEG). Results showed that object processing along the texture dimension was affected by task demand starting from a relatively late time (320- to 370-ms time window) after image onset. The findings are consistent with the view that task exerts an effect on the later phases of object processing.

Publisher Correction: Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.

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

Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.

The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.

Characterizing extracellular diffusion properties using diffusion-weighted MRS of sucrose injected in mouse brain.

Brain water and some critically important energy metabolites, such as lactate or glucose, are present in both intracellular and extracellular spaces (ICS/ECS) at significant levels. This ubiquitous nature makes diffusion MRI/MRS data sometimes difficult to interpret and model. While it is possible to glean information on the diffusion properties in ICS by measuring the diffusion of purely intracellular endogenous metabolites (such as NAA), the absence of endogenous markers specific to ECS hampers similar analyses in this compartment. In past experiments, exogenous probes have therefore been injected into the brain to assess their apparent diffusion coefficient (ADC) and thus estimate tortuosity in ECS. Here, we use a similar approach in mice by injecting sucrose, a well-known ECS marker, in either the lateral ventricles or directly in the prefrontal cortex. For the first time, we propose a thorough characterization of ECS diffusion properties encompassing (1) short-range restriction by looking at signal attenuation at high b values, (2) tortuosity and long-range restriction by measuring ADC time-dependence at long diffusion times and (3) microscopic anisotropy by performing double diffusion encoding (DDE) measurements. Overall, sucrose diffusion behavior is strikingly different from that of intracellular metabolites. Acquisitions at high b values not only reveal faster sucrose diffusion but also some sensitivity to restriction, suggesting that the diffusion in ECS is not fully Gaussian at high b. The time evolution of the ADC at long diffusion times shows that the tortuosity regime is not reached yet in the case of sucrose, while DDE experiments suggest that it is not trapped in elongated structures. No major difference in sucrose diffusion properties is reported between the two investigated routes of injection and brain regions. These original experimental insights should be useful to better interpret and model the diffusion signal of molecules that are distributed between ICS and ECS compartments.

Metabolic NMR mapping with microgram tissue biopsy.

This study explores the potential of profiling a microgram-scale soft tissue biopsy by NMR spectroscopy. The important elements of high resolution and high sensitivity for the spectral data are achieved through a unique probe, HR-µMAS, which allowed comprehensive profiling to be performed on microgram tissue for the first time under MAS conditions. Thorough spatially resolved metabolic maps were acquired across a coronal brain slice of rat C6 gliomas, which rendered the delineation of the tumor lesion. The results present a unique ex vivo NMR possibility to analyze tissue pathology that cannot be fully explored by the conventional approach, HR-MAS and in vivo MRS. Aside from the capability of analyzing a small localized region to track its specific metabolism, it could also offer the possibility to carry out longitudinal investigations on live animals due to the feasibility of minimally invasive tissue excision.

Operando NMR characterization of a metal-air battery using a double-compartment cell design.

Applying operando investigations is becoming essential for acquiring fundamental insights into the reaction mechanisms and phenomena at stake in batteries currently under development. The capability of a real-time characterization of the charge/discharge electrochemical pathways and the reactivity of the electrolyte is critical to decipher the underlying chemistries and improve the battery performance. Yet, adapting operando techniques for new chemistries such as metal-oxygen (i.e. metal-air) batteries introduces challenges in the cell design due notably to the requirements of an oxygen gas supply at the cathode. Herein a simple operando cell is presented with a two-compartment cylindrical cell design for NMR spectroscopy. The design is discussed and evaluated. Operando7Li static NMR characterization on a Li-O2 battery was performed as a proof-of-concept. The productions of Li2O2, mossy Li/Li dendrites and other irreversible parasitic lithium compounds were captured in the charge/discharge processes, demonstrating the capability of tracking the evolution of the anodic and cathodic chemistry in metal-oxygen batteries.

Perspectives of fast magic-angle spinning 87 Rb NMR of organic solids at high magnetic fields.

We report solid-state 87 Rb NMR spectra from two Rb-ionophore complexes obtained with fast magic-angle spinning (MAS) (up to 60 kHz) at 21.1 T. These Rb-ionophore complexes containing macrocycles such as benzo-15-crown-5 and cryptand [2.2.2] are typical of organic Rb salts that exhibit very large 87 Rb quadrupole coupling constants (close to 20 MHz). We have also obtained static 87 Rb NMR spectra for these two compounds and determined both 87 Rb quadrupole coupling and chemical shift tensors. The experimental 87 Rb NMR tensor parameters are compared with those obtained by quantum chemical computations. Our results demonstrate that the combination of fast MAS (60 kHz or higher) and a high magnetic field (21.1 T or higher) is sufficient to produce high-quality solid-state 87 Rb NMR spectra for organic Rb solids at the natural abundance level. We anticipate that, with additional 87 Rb isotope enrichment (up to 99%), the sensitivity of solid-state 87 Rb NMR will be 400 times higher than 39 K NMR, which makes the former an attractive surrogate probe for studying K+ ion binding in biological systems.

A reliable and valid tool for measuring visual recognition ability with musical notation.

Recognizing musical notation is an important skill to a full participation of Western classical music, but remains a largely under-researched topic in the psychology of music. One plausible reason of such omission is that, in the past, the research field has heavily relied on self-report of music reading ability, which was subjective and highly variable. This paper presents a reliable and valid tool for objectively measuring individual abilities in visual recognition of musical notation. The visual fluency task measures how fast one can accurately recognize a sequence of musical notation at a desired accuracy level using the adaptive psychometric method QUEST. We checked the reliability of this task in over 200 participants in terms of Guttman's λ-2 and Cronbach's alpha. Also, we evaluated the construct validity of this task by considering the convergent validity of this task with multiple external real-world measures of one's musical training background, with numerous experimental measures of visual tendencies of musical notation recognition and with sight-reading performance. Overall, the visual fluency task achieved satisfactory reliability and validity for measuring abilities in recognizing musical notation. This opens the door for characterizing the cognitive mechanisms, development, and individual differences in musical notation recognition, for understanding music learning and music psychology and for understanding of visual perceptual expertise in general.

Ectopic expression of recipient CD47 inhibits mouse macrophage-mediated immune rejection against human stem cell transplants.

Like conventional transplants, immunosuppression is required to facilitate survival and function of human embryonic stem cell (hESC) derivatives after implantation into xenogeneic recipients. We have previously reported that T cells alone are sufficient to reject allogeneic murine ESC derivatives; and strategies that inhibit T-cell activation, including coreceptor and costimulation blockade, prevent hESC derivatives from being rejected. This study aimed to investigate, in addition to T cells, whether macrophages contribute to transplant rejection of hESC xenografts with nonobese diabetic (NOD)/SCID mice that lack functional T and B cells but have macrophages. We show that acute rejection against hESC-derived endothelial cells (hESC-ECs) was mediated, to some degree, by infiltrating macrophages that phagocytosed them. Transgenic expression of murine CD47 on cell surface of hESC-ECs mitigates macrophage-mediated phagocytosis and improves their survival after transplantation. Our results highlight that innate immune cells, such as macrophages, can reject hESC derivatives, raising concern against the use of NOD/SCID as transplant recipients for testing in vivo function of hESC-derived tissues. Augmenting CD47 signaling promotes survival and function of hESC derivatives after xenogeneic transplantation.-Leung, C. S., Li, J., Xu, F., Wong, A. S. L., Lui, K. O. Ectopic expression of recipient CD47 inhibits mouse macrophage-mediated immune rejection against human stem cell transplants.

Emerging drugs for the treatment of hidradenitis suppurativa.

INTRODUCTION: Hidradenitis suppurativa (HS) is a severe, chronic inflammatory disorder that causes recurrent occlusion of hair follicles in the intertriginous regions of the skin. Mild to moderate HS has been successfully treated with oral antibiotics, topical therapy, and lifestyle modifications. However, moderate to severe HS is known to be refractory to conventional treatments. Wide excision surgery is a treatment option for severe HS, but often leads to functional impairments. Additionally, recurrence is common. The proper management of moderate to severe HS continues to be a challenge to practitioners. AREAS COVERED: A comprehensive literature search was conducted to identify published HS treatments using PubMed databases, in addition, ongoing studies were sought in clinicaltrials.gov. Search terms included 'hidradenitis suppurativa,' 'treatment,' and 'management.' EXPERT OPINION: Although adalimumab is currently the only biologic approved by the United States Food and Drug Administration for treatment of HS, there are many studies underway involving the development of drugs with a variety of immunological targets. Those potential HS therapies in either Phase II or Phase III trials show much promise. Since HS is a complicated disease that involves both pathological and environmental factors, treating HS continues to involve a multidisciplinary approach and monotherapy tends to not be efficacious.

Intact NMR spectroscopy: slow high-resolution magic angle spinning chemical shift imaging.

High-Resolution Magic-Angle Spinning Chemical Shift Imaging (HR-MAS CSI) has recently been explored for nuclear magnetic resonance (NMR) metabolomics and shows considerable promise in organism research. This is due to its ability to offer a supplemental dimension - spatial metabolic distribution - for profiling. However, HR-MAS CSI suffers from the large centrifugal stress exerted on the sample, which inevitably hinders the metabolic assessment. Herein, a slow sample spinning strategy was implemented and evaluated. The results demonstrate its potential as a highly informative profiling approach for intact specimens, with high quality data and feasibility.

Multiple processing limitations underlie multitasking costs.

Human multitasking is typically defined as the practice of performing more than one task at the same time (dual task) or rapidly alternating between multiple tasks (task switching). The majority of research in multitasking has been focusing on individual paradigms, with surprisingly little effort in understanding their relationships. We adopted an individual-difference approach to reveal the limitations underlying multitasking costs measured in different paradigms. Exploratory factor analyses revealed not a general multitasking factor but instead three different processing limitations associated with response selection, retrieval and maintenance of task information, and task-set reconfiguration. The three factors were only weakly correlated with and thus not reducible to common measures of processing speed, working memory capacity and fluid intelligence. Males and females excelled in different aspects of multitasking, demonstrating the benefit of using a multifaceted view of multitasking competency in group comparison. Findings of the current study help resolve conflicting results between studies using different paradigms, and form the basis of more comprehensive measurement tools and training protocols covering different aspects of multitasking limitations. The study will also help future integration of multitasking abilities into the theoretical framework of executive function.

Beauty and the beast: Promotion concerns and the pursuit of physically attractive mates.

OBJECTIVE: In the mating market, individuals differ in their aspirations to pursue opposite-sex mates who have a relatively higher (vs. similar) level of physical attractiveness. Few studies have explored how motivational concerns outside the mating domain can account for these individual differences in romantic aspiration. Based on regulatory focus theory, this research tested how broad concerns for promotion and prevention influence the aspiration and dating outcome. METHOD: Four studies tested whether promotion concerns increase romantic aspiration and the chance to mate with a more physically attractive partner. The first three studies tested how promotion concerns, either measured (Studies 1a and 2) or manipulated (Study 1b), can influence romantic aspiration. Study 3 further tested how one's chronic promotion concerns are related to the physical attractiveness of the current partner (as rated by observers). RESULTS: The first three studies supported the prediction that promotion concerns increase aspiration to pursue more physically attractive mates. The last study also found that, controlling for their own physical attractiveness, individuals with stronger promotion concerns tend to mate with physically attractive partners. CONCLUSIONS: The results highlight the significant roles of broad motivational concerns in determining both aspiration and chance to date a more physically attractive partner.