CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.

Ageing impairs the ability of neural stem cells (NSCs) to transition from quiescence to proliferation in the adult mammalian brain. Functional decline of NSCs results in the decreased production of new neurons and defective regeneration following injury during ageing1-4. Several genetic interventions have been found to ameliorate old brain function5-8, but systematic functional testing of genes in old NSCs-and more generally in old cells-has not been done. Here we develop in vitro and in vivo high-throughput CRISPR-Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screens in primary cultures of young and old NSCs uncovered more than 300 gene knockouts that specifically restore the activation of old NSCs. The top gene knockouts are involved in cilium organization and glucose import. We also establish a scalable CRISPR-Cas9 screening platform in vivo, which identified 24 gene knockouts that boost NSC activation and the production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes the GLUT4 glucose transporter, is a top intervention that improves the function of old NSCs. Glucose uptake increases in NSCs during ageing, and transient glucose starvation restores the ability of old NSCs to activate. Thus, an increase in glucose uptake may contribute to the decline in NSC activation with age. Our work provides scalable platforms to systematically identify genetic interventions that boost the function of old NSCs, including in vivo, with important implications for countering regenerative decline during ageing.

The germline coordinates mitokine signaling.

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C. elegans, neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the Wnt ligand EGL-20, which activate the mitochondrial unfolded protein response (UPRMT) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-periphery UPRMT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, Wnt and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPRMT activation. We also find that the germline tissue itself is essential for UPRMT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

The extracellular matrix integrates mitochondrial homeostasis.

Cellular homeostasis is intricately influenced by stimuli from the microenvironment, including signaling molecules, metabolites, and pathogens. Functioning as a signaling hub within the cell, mitochondria integrate information from various intracellular compartments to regulate cellular signaling and metabolism. Multiple studies have shown that mitochondria may respond to various extracellular signaling events. However, it is less clear how changes in the extracellular matrix (ECM) can impact mitochondrial homeostasis to regulate animal physiology. We find that ECM remodeling alters mitochondrial homeostasis in an evolutionarily conserved manner. Mechanistically, ECM remodeling triggers a TGF-ß response to induce mitochondrial fission and the unfolded protein response of the mitochondria (UPRMT). At the organismal level, ECM remodeling promotes defense of animals against pathogens through enhanced mitochondrial stress responses. We postulate that this ECM-mitochondria crosstalk represents an ancient immune pathway, which detects infection- or mechanical-stress-induced ECM damage, thereby initiating adaptive mitochondria-based immune and metabolic responses.

CRISPR screens and lectin microarrays identify novel high mannose N-glycan regulators.

Glycans play critical roles in cellular signaling and function. Unlike proteins, glycan structures are not templated from genes but the concerted activity of many genes, making them historically challenging to study. Here, we present a strategy that utilizes pooled CRISPR screens and lectin microarrays to uncover and characterize regulators of cell surface glycosylation. We applied this approach to study the regulation of high mannose glycans - the starting structure of all asparagine(N)-linked-glycans. We used CRISPR screens to uncover the expanded network of genes controlling high mannose surface levels, followed by lectin microarrays to fully measure the complex effect of select regulators on glycosylation globally. Through this, we elucidated how two novel high mannose regulators - TM9SF3 and the CCC complex - control complex N-glycosylation via regulating Golgi morphology and function. Notably, this method allowed us to interrogate Golgi function in-depth and reveal that similar disruption to Golgi morphology can lead to drastically different glycosylation outcomes. Collectively, this work demonstrates a generalizable approach for systematically dissecting the regulatory network underlying glycosylation.

The germline coordinates mitokine signaling.

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C. elegans , neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the WNT ligand EGL-20, which activate the mitochondrial unfolded protein response (UPR MT ) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-peripheral UPR MT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, like WNT and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPR MT activation. We also find that the germline tissue itself is essential in UPR MT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

Lipid homeostasis is essential for a maximal ER stress response.

Changes in lipid metabolism are associated with aging and age-related diseases, including proteopathies. The endoplasmic reticulum (ER) is uniquely a major hub for protein and lipid synthesis, making its function essential for both protein and lipid homeostasis. However, it is less clear how lipid metabolism and protein quality may impact each other. Here, we identified let-767, a putative hydroxysteroid dehydrogenase in Caenorhabditis elegans, as an essential gene for both lipid and ER protein homeostasis. Knockdown of let-767 reduces lipid stores, alters ER morphology in a lipid-dependent manner, and blocks induction of the Unfolded Protein Response of the ER (UPRER). Interestingly, a global reduction in lipogenic pathways restores UPRER induction in animals with reduced let-767. Specifically, we find that supplementation of 3-oxoacyl, the predicted metabolite directly upstream of let-767, is sufficient to block induction of the UPRER. This study highlights a novel interaction through which changes in lipid metabolism can alter a cell's response to protein-induced stress.

Large-scale genetic screens identify BET-1 as a cytoskeleton regulator promoting actin function and life span.

The actin cytoskeleton is a three-dimensional scaffold of proteins that is a regulatory, energyconsuming network with dynamic properties to shape the structure and function of the cell. Proper actin function is required for many cellular pathways, including cell division, autophagy, chaperone function, endocytosis, and exocytosis. Deterioration of these processes manifests during aging and exposure to stress, which is in part due to the breakdown of the actin cytoskeleton. However, the regulatory mechanisms involved in preservation of cytoskeletal form and function are not well-understood. Here, we performed a multipronged, cross-organismal screen combining a whole-genome CRISPR-Cas9 screen in human fibroblasts with in vivo Caenorhabditis elegans synthetic lethality screening. We identified the bromodomain protein, BET-1, as a key regulator of actin function and longevity. Overexpression of bet-1 preserves actin function at late age and promotes life span and healthspan in C. elegans. These beneficial effects are mediated through actin preservation by the transcriptional regulator function of BET-1. Together, our discovery assigns a key role for BET-1 in cytoskeletal health, highlighting regulatory cellular networks promoting cytoskeletal homeostasis.

Rapid synthesis of drug-encapsulated films by evaporation-induced self-assembly for highly-controlled drug release from biomaterial surfaces.

Infection at the surgical site for dental implants results in failed procedures, patient pain, burdensome economic impact, and the over-prescription of prophylactic antibiotics. Mesoporous silica films as coatings for implants may provide an ideal antimicrobial drug storage and local release vector to the site of infection, however traditional drug loading techniques result in insufficient drug load and short-term release kinetics. In this work, we have applied a method to use a surfactant-antimicrobial drug octenidine dihydrochloride (OCT) as a template for mesostructured silica, to demonstrate silica-OCT composite films. The films are synthesized by evaporation induced self-assembly (EISA) and we explore the effects of synthesis parameters on porous film structure, OCT incorporation, and OCT drug release rates. Drug micelle incorporation into the silica mesostructure was highly dependent on silica precursor pre-reaction to form silica oligomers before film spin-casting. The OCT drug concentration of the synthesis solution dictated the time required for effective incorporation (without phase separation), with total loading in the film of up to 90% by mass. The OCT content in the films was found to directly determine the timescale of drug release, from 2 to 8 h for a single layer film. The total release timescale was increased by the addition of multiple layers of OCT-silica films to nearly 2 weeks. Drug release from films completely inhibited Streptococcus mutans (UA159) growth, while drug-free porous silica films showed no increase in bacterial growth over non-porous control. These OCT-silica films have a significant potential to store and release antimicrobial drugs from dental implant surfaces.

LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization.

The dysfunction of mitochondria is associated with the physiological consequences of aging and many age-related diseases. Therefore, critical quality control mechanisms exist to protect mitochondrial functions, including the unfolded protein response of the mitochondria (UPRMT). However, it is still unclear how UPRMT is regulated in mammals with mechanistic discrepancies between previous studies. Here, we reasoned that a study of conserved mechanisms could provide a uniquely powerful way to reveal previously uncharacterized components of the mammalian UPRMT. We performed cross-species comparison of genetic requirements for survival under­and in response to­mitochondrial stress between karyotypically normal human stem cells and the nematode Caenorhabditis elegans. We identified a role for EPS-8/EPS8 (epidermal growth factor receptor pathway substrate 8), a signaling protein adaptor, in general mitochondrial homeostasis and UPRMT regulation through integrin-mediated remodeling of the actin cytoskeleton. This study also highlights the use of cross-species comparisons in genetic screens to interrogate cellular pathways.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes.

Multimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in hundreds of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Proteins are measured with a ~5-hour immunoassay. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing Latrunculin A detects a unique subpopulation (~2%) exhibiting downregulated F-actin, but upregulated microtubules. Thus, some cells may upregulate other cytoskeletal complexes to counteract the stress of Latrunculin A treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock may dysregulate filamentous and globular actin correlation. In this work, our assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

Brains and brawn: Stress-induced myokine abates nervous system aging.

Skeletal muscle secretes numerous systemic factors, termed myokines, which can regulate homeostasis of distal tissues. In this issue, Rai et al. (2021) identify and characterize a novel myokine, Amyrel, which is secreted under muscle proteasome stress and protects central nervous system health and function by enhancing protein quality control during aging.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2.

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.

Beyond the cell factory: Homeostatic regulation of and by the UPRER.

The endoplasmic reticulum (ER) is commonly referred to as the factory of the cell, as it is responsible for a large amount of protein and lipid synthesis. As a membrane-bound organelle, the ER has a distinct environment that is ideal for its functions in synthesizing these primary cellular components. Many different quality control machineries exist to maintain ER stability under the stresses associated with synthesizing, folding, and modifying complex proteins and lipids. The best understood of these mechanisms is the unfolded protein response of the ER (UPRER), in which transmembrane proteins serve as sensors, which trigger a coordinated transcriptional response of genes dedicated for mitigating the stress. As the name suggests, the UPRER is most well described as a functional response to protein misfolding stress. Here, we focus on recent findings and emerging themes in additional roles of the UPRER outside of protein homeostasis, including lipid homeostasis, autophagy, apoptosis, and immunity.

Hong Kong Society of Clinical Blood Management recommendations for implementation of patient blood management.

Patient blood management (PBM) is a patient-centred, multidisciplinary approach to optimise red cell mass, minimise blood loss, and manage tolerance to anaemia in an effort to improve patient outcomes. Well-implemented PBM improves patient outcomes and reduces demand for blood products. The multidisciplinary approach of PBM can often allow patients to avoid blood transfusions, which are associated with less favourable clinical outcomes. In Hong Kong, there has been increasing demand for blood in the ageing population, and there are simultaneous blood safety and donor issues that are adversely affecting the blood supply. To address these challenges, the Hong Kong Society of Clinical Blood Management recommends implementation of a PBM programme in Hong Kong, including strategies such as optimising red blood cell mass, improving anaemia management, minimising blood loss, and rationalising the use of blood and blood products.

Intraspecific Diversity and Taxonomy of Emmonsia crescens.

Emmonsia crescens is known as an environmental pathogen causing adiaspiromycosis in small rodents. As the generic name Emmonsia is no longer available for this species, its taxonomic position is re-evaluated. The intraspecific variation of Emmonsia crescens was analyzed using molecular, morphological, and physiological data, and the relationship between frequency of adiaspiromycosis and body temperature of host animals was explored. A North American and a pan-global lineage could be discerned, each with subclusters at low genetic distance. European strains produced the classical type of very large adiaspores, while in the North American lineage adiaspores relatively small, resembling the broad-based budding cells of Blastomyces. Members of the closely related genus Emergomyces may exhibit large, broad-based in addition to small, narrow-based budding cells. We conclude that the morphology of the pathogenic phase in these fungi differs gradationally between species and even populations, and is therefore less suitable as a diagnostic criterion for generic delimitation. Two Emmonsia species are reclassified in Emergomyces.