Intracellular lipid surveillance by small G protein geranylgeranylation.

Imbalances in lipid homeostasis can have deleterious effects on health1,2. Yet how cells sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption remains unclear. Here we describe a mechanism for intracellular lipid surveillance in Caenorhabditis elegans that involves transcriptional inactivation of the nuclear hormone receptor NHR-49 through its cytosolic sequestration to endocytic vesicles via geranylgeranyl conjugation to the small G protein RAB-11.1. Defective de novo isoprenoid synthesis caused by lipid depletion limits RAB-11.1 geranylgeranylation, which promotes nuclear translocation of NHR-49 and activation of rab-11.2 transcription to enhance transporter residency at the plasma membrane. Thus, we identify a critical lipid sensed by the cell, its conjugated G protein, and the nuclear receptor whose dynamic interactions enable cells to sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption and lipid metabolism.

The Effects of Human Immunodeficiency Virus Type 1 (HIV-1) Antigen-Expanded Specific T-Cell Therapy and Vorinostat on Persistent HIV-1 Infection in People With HIV on Antiretroviral Therapy.

BACKGROUND: The histone deacetylase inhibitor vorinostat (VOR) can reverse human immunodeficiency virus type 1 (HIV-1) latency in vivo and allow T cells to clear infected cells in vitro. HIV-specific T cells (HXTCs) can be expanded ex vivo and have been safely administered to people with HIV (PWH) on antiretroviral therapy. METHODS: Six PWH received infusions of 2 × 107 HXTCs/m² with VOR 400 mg, and 3 PWH received infusions of 10 × 107 HXTCs/m² with VOR. The frequency of persistent HIV by multiple assays including quantitative viral outgrowth assay (QVOA) of resting CD4+ T cells was measured before and after study therapy. RESULTS: VOR and HXTCs were safe, and biomarkers of serial VOR effect were detected, but enhanced antiviral activity in circulating cells was not evident. After 2 × 107 HXTCs/m² with VOR, 1 of 6 PWH exhibited a decrease in QVOA, and all 3 PWH exhibited such declines after 10 × 107 HXTCs/m² and VOR. However, most declines did not exceed the 6-fold threshold needed to definitively attribute decline to the study intervention. CONCLUSIONS: These modest effects provide support for the strategy of HIV latency reversal and reservoir clearance, but more effective interventions are needed to yield the profound depletion of persistent HIV likely to yield clinical benefit. Clinical Trials Registration. NCT03212989.

Land-use change interacts with island biogeography to alter bird community assembly.

Anthropogenic activities have reshaped biodiversity on islands worldwide. However, it remains unclear how island attributes and land-use change interactively shape multiple facets of island biodiversity through community assembly processes. To answer this, we conducted bird surveys in various land-use types (mainly forest and farmland) using transects on 34 oceanic land-bridge islands in the largest archipelago of China. We found that bird species richness increased with island area and decreased with isolation, regardless of the intensity of land-use change. However, forest-dominated habitats exhibited lower richness than farmland-dominated habitats. Island bird assemblages generally comprised species that share more similar traits or evolutionary histories (i.e. functional and/or phylogenetic clustering) than expected if assemblages were randomly assembled. Contrary to our expectations, we observed that bird assemblages in forest-dominated habitats were more clustered on large and close islands, whereas assemblages in farmland-dominated habitats were more clustered on small islands. These contrasting results indicate that land-use change interacts with island biogeography to alter the community assembly of birds on inhabited islands. Our findings emphasize the importance of incorporating human-modified habitats when examining the community assembly of island biota, and further suggest that agricultural landscapes on large islands may play essential roles in protecting countryside island biodiversity.

Environmental monitoring of current good manufacturing practices cleanroom facilities for manufacturing of cellular therapy products in an academic hospital setting.

As the field of cell and gene therapy (CGT) continues to grow, so too must the infrastructure and regulatory guidance supporting the manufacture of these potentially life-saving products-especially early-phase products manufactured at an increasing number of academic or hospital-based facilities providing decentralized (or point of care) manufacturing. An important component of current good manufacturing practices, including those regulating cell and gene therapies, is the establishment of an effective environmental monitoring (EM) program. While several guidelines for establishing an EM program are available, these guidelines do not specifically address the unique aspects of manufacturing CGT products and they do not provide real-world evidence demonstrating the effectiveness of the program. Here, we describe the establishment and evolution of an EM program in a cell therapy manufacturing facility at an academic hospital. With 10 years of EM data, we analyze the effectiveness for identifying trends in environmental conditions and highlight important findings, with the aim of providing practical evidence and guidance for the development of future early-phase EM programs.

Genetic context effects can override canonical cis regulatory elements in Escherichia coli.

Recent experiments have shown that in addition to control by cis regulatory elements, the local chromosomal context of a gene also has a profound impact on its transcription. Although this chromosome-position dependent expression variation has been empirically mapped at high-resolution, the underlying causes of the variation have not been elucidated. Here, we demonstrate that 1 kb of flanking, non-coding synthetic sequences with a low frequency of guanosine and cytosine (GC) can dramatically reduce reporter expression compared to neutral and high GC-content flanks in Escherichia coli. Natural and artificial genetic context can have a similarly strong effect on reporter expression, regardless of cell growth phase or medium. Despite the strong reduction in the maximal expression level from the fully-induced reporter, low GC synthetic flanks do not affect the time required to reach the maximal expression level after induction. Overall, we demonstrate key determinants of transcriptional propensity that appear to act as tunable modulators of transcription, independent of regulatory sequences such as the promoter. These findings provide insight into the regulation of naturally occurring genes and an independent control for optimizing expression of synthetic biology constructs.

A global analysis of avian island diversity-area relationships in the Anthropocene.

Research on island species-area relationships (ISAR) has expanded to incorporate functional (IFDAR) and phylogenetic (IPDAR) diversity. However, relative to the ISAR, we know little about IFDARs and IPDARs, and lack synthetic global analyses of variation in form of these three categories of island diversity-area relationship (IDAR). Here, we undertake the first comparative evaluation of IDARs at the global scale using 51 avian archipelagic data sets representing true and habitat islands. Using null models, we explore how richness-corrected functional and phylogenetic diversity scale with island area. We also provide the largest global assessment of the impacts of species introductions and extinctions on the IDAR. Results show that increasing richness with area is the primary driver of the (non-richness corrected) IPDAR and IFDAR for many data sets. However, for several archipelagos, richness-corrected functional and phylogenetic diversity changes linearly with island area, suggesting that the dominant community assembly processes shift along the island area gradient. We also find that archipelagos with the steepest ISARs exhibit the biggest differences in slope between IDARs, indicating increased functional and phylogenetic redundancy on larger islands in these archipelagos. In several cases introduced species seem to have 're-calibrated' the IDARs such that they resemble the historic period prior to recent extinctions.

Optic cup morphogenesis requires neural crest-mediated basement membrane assembly.

Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.

A Morrison stem gekkotan reveals gecko evolution and Jurassic biogeography.

Geckos are a speciose and globally distributed clade of Squamata (lizards, including snakes and amphisbaenians) that are characterized by a host of modifications for nocturnal, scansorial and insectivorous ecologies. They are among the oldest divergences in the lizard crown, so understanding the origin of geckoes (Gekkota) is essential to understanding the origin of Squamata, the most species-rich extant tetrapod clade. However, the poor fossil record of gekkotans has obscured the sequence and timing of the assembly of their distinctive morphology. Here, we describe the first North American stem gekkotan based on a three-dimensionally preserved skull from the Morrison Formation of western North America. Despite its Late Jurassic age, the new species already possesses several key characteristics of the gekkotan skull along with retained ancestral features. We show that this new stem gekkotan, and several previously named species of uncertain phylogenetic relationships, comprise a widespread clade of early crown lizards, substantiating faunal homogeneity in Laurasia during the Late Jurassic that extended across disparate ecological, body-size and physiological classes.

Emerging frontiers in immuno- and gene therapy for cancer.

BACKGROUND AIMS: The field of cell and gene therapy in oncology has moved rapidly since 2017 when the first cell and gene therapies, Kymriah followed by Yescarta, were approved by the Food and Drug Administration in the United States, followed by multiple other countries. Since those approvals, several new products have gone on to receive approval for additional indications. Meanwhile, efforts have been made to target different cancers, improve the logistics of delivery and reduce the cost associated with novel cell and gene therapies. Here, we highlight various cell and gene therapy-related technologies and advances that provide insight into how these new technologies will speed the translation of these therapies into the clinic. CONCLUSIONS: In this review, we provide a broad overview of the current state of cell and gene therapy-based approaches for cancer treatment - discussing various effector cell types and their sources, recent advances in both CAR and non-CAR genetic modifications, and highlighting a few promising approaches for increasing in vivo efficacy and persistence of therapeutic drug products.

Rates and Correlates of Uptake of Continuous Glucose Monitors Among Adults with Type 2 Diabetes in Primary Care and Endocrinology Settings.

BACKGROUND: Clinical trials indicate continuous glucose monitor (CGM) use may benefit adults with type 2 diabetes, but CGM rates and correlates in real-world care settings are unknown. OBJECTIVE: We sought to ascertain prevalence and correlates of CGM use and to examine rates of new CGM prescriptions across clinic types and medication regimens. DESIGN: Retrospective cohort using electronic health records in a large academic medical center in the Southeastern US. PARTICIPANTS: Adults with type 2 diabetes and a primary care or endocrinology visit during 2021. MAIN MEASURES: Age, gender, race, ethnicity, insurance, clinic type, insulin regimen, hemoglobin A1c values, CGM prescriptions, and prescribing clinic type. KEY RESULTS: Among 30,585 adults with type 2 diabetes, 13% had used a CGM. CGM users were younger and more had private health insurance (p < .05) as compared to non-users; 72% of CGM users had an intensive insulin regimen, but 12% were not taking insulin. CGM users had higher hemoglobin A1c values (both most recent and most proximal to the first CGM prescription) than non-users. CGM users were more likely to receive endocrinology care than non-users, but 23% had only primary care visits in 2021. For each month in 2021, a mean of 90.5 (SD 12.5) people started using CGM. From 2020 to 2021, monthly rates of CGM prescriptions to new users grew 36% overall, but 125% in primary care. Most starting CGM in endocrinology had an intensive insulin regimen (82% vs. 49% starting in primary care), whereas 28% starting CGM in primary care were not using insulin (vs. 5% in endocrinology). CONCLUSION: CGM uptake for type 2 diabetes is increasing rapidly, with most growth in primary care. These trends present opportunities for healthcare system adaptations to support CGM use and related workflows in primary care to support growth in uptake.

An extradural cyst in a French Bulldog.

CASE HISTORY: A 7-year-old, male neutered French Bulldog was referred to a specialist veterinary hospital for evaluation of progressive paraparesis of 6-months' duration. The owners reported both faecal and urinary incontinence at home. CLINICAL FINDINGS: The dog presented with ambulatory paraparesis and pelvic limb ataxia that was more pronounced in the right pelvic limb. The pelvic limb withdrawal response and sciatic myotatic response were reduced bilaterally. Postural reaction responses were delayed in both pelvic limbs, and this was more obvious in the right pelvic limb. The anal tone and perineal sensation were normal at the time of examination.An L4-S3 myelopathy was suspected. CT of the spine revealed a compressive, bilobed, extramedullary, cyst-like structure within the vertebral canal, between L7 and S3. Surgical removal of the cyst via a L7-S1 dorsal laminectomy was performed. Histopathological examination and additional immunohistochemistry of the excised structure indicated a probable ependymal cyst with a ciliated lining. The dog recovered well post-operatively, and at follow-up 3 weeks later had some improvement of his neurological signs. The paraparesis and pelvic limb ataxia had improved; however, the remaining neurological examination was similar to the pre-surgical examination. DIAGNOSIS: Extradural cyst. CLINICAL RELEVANCE: Spinal cysts can contribute to clinical signs that resemble other common chronic spinal cord diseases, such as intervertebral disc disease. Therefore, this disease should be considered as a differential when dealing with cases of progressive paraparesis and pelvic limb ataxia. This case report may potentially provide opportunities in the future for further understanding of the pathogenesis, behaviour, outcomes and subclassification of spinal cysts in dogs.

Hidden species diversity in an iconic living fossil vertebrate.

Ancient, species-poor lineages persistently occur across the Tree of life. These lineages are likely to contain unrecognized species diversity masked by the low rates of morphological evolution that characterize living fossils. Halecomorphi is a lineage of ray-finned fishes that diverged from its closest relatives before 200 Ma and is represented by only one living species in eastern North America, the bowfin, Amia calva Linnaeus. Here, we use double digest restriction-site-associated DNA sequencing and morphology to illuminate recent speciation in bowfins. Our results support the delimitation of a second living species of Amia, with the timing of diversification dating to the Plio-Pleistocene. This delimitation expands the species diversity of an ancient lineage that is integral to studies of vertebrate genomics and development, yet is facing growing conservation threats driven by the caviar fishery.

Long-term declines in bird populations in tropical agricultural countryside.

Tropical agriculture is a major driver of biodiversity loss, yet it can provide conservation opportunities, especially where protected areas are inadequate. To investigate the long-term biodiversity capacity of agricultural countryside, we quantified bird population trends in Costa Rica by mist netting 57,255 birds of 265 species between 1999 and 2010 in sun coffee plantations, riparian corridors, secondary forests, forest fragments, and primary forest reserves. More bird populations (69) were declining than were stable (39) or increasing (4). Declines were common in resident, insectivorous, and more specialized species. There was no relationship between the species richness of a habitat and its conservation value. High-value forest bird communities were characterized by their distinct species composition and habitat and dietary functional signatures. While 49% of bird species preferred forest to coffee, 39% preferred coffee to forest and 12% used both habitats, indicating that coffee plantations have some conservation value. Coffee plantations, although lacking most of the forest specialists, hosted 185 bird species, had the highest capture rates, and supported increasing numbers of some forest species. Coffee plantations with higher tree cover (7% vs. 13%) had more species with increasing capture rates, twice as many forest specialists, and half as many nonforest species. Costa Rican countryside habitats, especially those with greater tree cover, host many bird species and are critical for connecting bird populations in forest remnants. Diversified agricultural landscapes can enhance the biodiversity capacity of tropical countryside, but, for the long-term persistence of all forest bird species, large (>1,000 ha) protected areas are essential.

Treatment of acute traumatic nasopharyngeal stenosis with balloon dilation in two dogs.

CASE HISTORY: Two dogs were referred to Veterinary Specialists Aotearoa for evaluation and treatment after sustaining significant head trauma. Case 1 was a 7-month-old, female Staffordshire Bull Terrier who was hit by a car at low speed. Case 2 was a 2-year old, male neutered German Shepherd who sustained a gunshot wound to the head whilst on duty for the New Zealand Police Dog Unit. CLINICAL FINDINGS: The dog in Case 1 suffered numerous facial fractures which caused collapse of the ventral nasal meatus and dorsal nasopharyngeal wall. The dog in Case 2 had extensive osseous and soft tissue damage to the nose, nasopharynx and cervical region with severe narrowing of the ventral meatuses, nasopharyngeal meatus and rostral nasopharynx due to multiple fracture fragments and shrapnel pieces. A diagnosis of traumatic nasopharyngeal stenosis was made in each case by computed tomography. Mechanical balloon dilation was used to treat the stenosis in both dogs. The balloon dilations were performed using a 12-mm balloon dilation catheter inserted in an antegrade fashion. In the first dog, the procedure was performed blind and was repeated three times with 5- and 9-day intervals between dilations. In the second dog, the procedure was performed under endoscopic guidance and again, was repeated three times with a 7-day interval between dilations. Clinical success was reported in both patients following treatment and the second dog also underwent a follow-up computed tomography scan which confirmed resolution of the stenosis. DIAGNOSIS: Traumatic nasopharyngeal stenosis that was successfully treated with balloon dilation. CLINICAL RELEVANCE: Findings suggest that balloon dilation may be an effective technique for the treatment of traumatic nasopharyngeal stenosis in dogs. Multiple dilation procedures are likely required, but the procedure can ultimately result in long term clinical resolution.Abbreviations: CT: Computed tomography; VSA: Veterinary Specialists Aotearoa.

Retrospective comparison of outcomes following tibial plateau levelling osteotomy and lateral fabello-tibial suture stabilisation of cranial cruciate ligament disease in small dogs with high tibial plateau angles.

AIMS: To compare short and long-term outcomes after tibial plateau levelling osteotomy (TPLO) and lateral fabello-tibial suture (LFTS) techniques for the management of cranial cruciate ligament disease in small dogs with high tibial plateau angles (TPA). METHODS: In this retrospective study, the medical records of two veterinary specialist practices in the United Kingdom were searched for dogs (<20 kg) that underwent TPLO or LFTS between 2000 and 2010, and had a preoperative radiographic TPA >30° with either short-term (6 weeks) and/or long-term (>3 months) follow-up data. Data collected at a 6-week post-surgical re-examination was derived from orthopaedic examination and radiographic assessment and included the incidence of major and minor complications and scoring of the short-term outcome. Long-term outcome was scored based on results of a subjective owner questionnaire and veterinary orthopaedic examination. RESULTS: A total of 61 (84 stifles) dogs were included in the study: 24 (30 stilfes) and 37 (54 stifles) dogs underwent LFTS and TPLO, respectively. Long-term clinical outcome was different (p = 0.017) between groups; 15/15 stifles in the TPLO group had a good or excellent long-term clinical outcome, compared to 4/8 (50%) in the LFTS group. There was no evidence of a difference in short-term post-operative outcome or owner subjective long-term outcome between treatment groups.Stifles in the LFTS group were more likely (p = 0.027) to have palpable stifle pain at long-term follow-up. Owners reported that 5/16 (31.3%) dogs in the LFTS group required oral non-steroidal anti-inflammatory drug (NSAID) treatment at least monthly (4/5 required daily treatment), whereas no dogs in the TPLO group required treatment with NSAID more frequently than three times per year (p = 0.011).No correlation was found between short-term outcome and owner subjective long-term outcome but there was a positive correlation between short-term outcome and long-term clinical outcome.There was no evidence of a difference in overall major complication rates between treatment groups. The occurrence of complications was associated with heavier body weight at the time of surgery. No other variables were shown to be risk factors for complications. CONCLUSION AND CLINICAL RELEVANCE: Small breed dogs with high TPA that underwent TPLO had better long-term clinical outcomes and were less likely to require NSAID administration than those that underwent LFTS. The risk of complication increased with the weight of the dog at surgery. There was a positive correlation between short-term outcome and long-term clinical outcome.

LongAxis: A MATLAB-based program for 3D quantitative analysis of epithelial cell shape and orientation.

Epithelial morphogenesis, a fundamental aspect of development, generates 3-dimensional tissue structures crucial for organ function. Underlying morphogenetic mechanisms are, in many cases, poorly understood, but mutations that perturb organ development can affect epithelial cell shape and orientation - difficult features to quantify in three dimensions. The basic structure of the eye is established via epithelial morphogenesis: in the embryonic optic cup, the retinal progenitor epithelium enwraps the lens. We previously found that loss of the extracellular matrix protein laminin-alpha1 (lama1) led to mislocalization of apical polarity markers and apparent misorientation of retinal progenitors. We sought to visualize and quantify this phenotype, and determine whether loss of the apical polarity determinant pard3 might rescue the phenotype. To this end, we developed LongAxis, a MATLAB-based program optimized for the retinal progenitor neuroepithelium. LongAxis facilitates 3-dimensional cell segmentation, visualization, and quantification of cell orientation and morphology. Using LongAxis, we find that retinal progenitors in the lama1-/- optic cup are misoriented and slightly less elongated. In the lama1;MZpard3 double mutant, cells are still misoriented, but larger. Therefore, loss of pard3 does not rescue loss of lama1, and in fact uncovers a novel cell size phenotype. LongAxis enables population-level visualization and quantification of retinal progenitor cell orientation and morphology. These results underscore the importance of visualizing and quantifying cell orientation and shape in three dimensions within the retina.

Deep Sequencing Reveals Compartmentalized HIV-1 in the Semen of Men with and without Sexually Transmitted Infection-Associated Urethritis.

Concurrent sexually transmitted infections (STI) can increase the probability of HIV-1 transmission primarily by increasing the viral load present in semen. In this study, we explored the relationship of HIV-1 in blood and seminal plasma in the presence and absence of urethritis and after treatment of the concurrent STI. Primer ID deep sequencing of the V1/V3 region of the HIV-1 env gene was done for paired blood and semen samples from antiretroviral therapy (ART)-naive men living in Malawi with (n = 19) and without (n = 5) STI-associated urethritis; for a subset of samples, full-length env genes were generated for sequence analysis and to test entry phenotype. Cytokine concentrations in the blood and semen were also measured, and a reduction in the levels of proinflammatory cytokines was observed following STI treatment. We observed no difference in the prevalence of diverse compartmentalized semen-derived lineages in men with or without STI-associated urethritis, and these viral populations were largely stable during STI treatment. Clonal amplification of one or a few viral sequences accounted for nearly 50% of the viral population, indicating a recent bottleneck followed by limited viral replication. We conclude that the male genital tract is a site where virus can be brought in from the blood, where localized sustained replication can occur, and where specific genotypes can be amplified, perhaps initially by cellular proliferation but further by limited viral replication.IMPORTANCE HIV-1 infection is a sexually transmitted infection that coexists with other STI. Here, we examined the impact of a concurrent STI resulting in urethritis on the HIV-1 population within the male genital tract. We found that viral populations remain largely stable even with treatment of the STI. These results show that viral populations within the male genital tract are defined by factors beyond transient inflammation associated with a concurrent STI.

Integrated Assessment of Viral Transcription, Antigen Presentation, and CD8+ T Cell Function Reveals Multiple Limitations of Class I-Selective Histone Deacetylase Inhibitors during HIV-1 Latency Reversal.

Clinical trials investigating histone deacetylase inhibitors (HDACi) to reverse HIV-1 latency aim to expose reservoirs in antiretroviral (ARV)-treated individuals to clearance by immune effectors, yet have not driven measurable reductions in the frequencies of infected cells. We therefore investigated the effects of the class I-selective HDACi nanatinostat and romidepsin on various blocks to latency reversal and elimination, including viral splicing, antigen presentation, and CD8+ T cell function. In ex vivo CD4+ T cells from ARV-suppressed individuals, both HDACi significantly induced viral transcription, but not splicing nor supernatant HIV-1 RNA. In an HIV-1 latency model using autologous CD8+ T cell clones as biosensors of antigen presentation, neither HDACi-treated CD4+ T cell condition induced clone degranulation. Both HDACi also impaired the function of primary CD8+ T cells in viral inhibition assays, with nanatinostat causing less impairment. These findings suggest that spliced or cell-free HIV-1 RNAs are more indicative of antigen expression than unspliced HIV-RNAs and may help to explain the limited abilities of HDACi to generate CD8+ T cell targets in vivoIMPORTANCE Antiretroviral (ARV) drug regimens suppress HIV-1 replication but are unable to cure infection. This leaves people living with HIV-1 burdened by a lifelong commitment to expensive daily medication. Furthermore, it has become clear that ARV therapy does not fully restore health, leaving individuals at elevated risk for cardiovascular disease, certain types of cancers, and neurocognitive disorders, as well as leaving them exposed to stigma. Efforts are therefore under way to develop therapies capable of curing infection. A key focus of these efforts has been on a class of drugs called histone deacetylase inhibitors (HDACi), which have the potential of exposing hidden reservoirs of HIV-1 to elimination by the immune system. Unfortunately, clinical trial results with HDACi have thus far been disappointing. In the current study, we integrate a number of experimental approaches to build a model that provides insights into the limited activity of HDACi in clinical trials and offers direction for future approaches.

In Vivo Subcellular Mass Spectrometry Enables Proteo-Metabolomic Single-Cell Systems Biology in a Chordate Embryo Developing to a Normally Behaving Tadpole (X. laevis)*.

We report the development of in vivo subcellular high-resolution mass spectrometry (HRMS) for proteo-metabolomic molecular systems biology in complex tissues. With light microscopy, we identified the left-dorsal and left-ventral animal cells in cleavage-stage non-sentient Xenopus laevis embryos. Using precision-translated fabricated microcapillaries, the subcellular content of each cell was double-probed, each time swiftly (<5 s/event) aspirating <5 % of cell volume (≈10 nL). The proteins and metabolites were analyzed by home-built ultrasensitive capillary electrophoresis electrospray ionization employing orbitrap or time-of-flight HRMS. Label-free detection of ≈150 metabolites (57 identified) and 738 proteins found proteo-metabolomic networks with differential quantitative activities between the cell types. With spatially and temporally scalable sampling, the technology preserved the integrity of the analyzed cells, the neighboring cells, and the embryo. 95 % of the analyzed embryos developed into sentient tadpoles that were indistinguishable from their wild-type siblings based on anatomy and visual function in a background color preference assay.

Cerebrospinal fluid and plasma biomarkers in individuals at risk for genetic prion disease.

BACKGROUND: Prion disease is neurodegenerative disease that is typically fatal within months of first symptoms. Clinical trials in this rapidly declining symptomatic patient population have proven challenging. Individuals at high lifetime risk for genetic prion disease can be identified decades before symptom onset and provide an opportunity for early therapeutic intervention. However, randomizing pre-symptomatic carriers to a clinical endpoint is not numerically feasible. We therefore launched a cohort study in pre-symptomatic genetic prion disease mutation carriers and controls with the goal of evaluating biomarker endpoints that may enable informative trials in this population. METHODS: We collected cerebrospinal fluid (CSF) and blood from pre-symptomatic individuals with prion protein gene (PRNP) mutations (N = 27) and matched controls (N = 16), in a cohort study at Massachusetts General Hospital. We quantified total prion protein (PrP) and real-time quaking-induced conversion (RT-QuIC) prion seeding activity in CSF and neuronal damage markers total tau (T-tau) and neurofilament light chain (NfL) in CSF and plasma. We compared these markers cross-sectionally, evaluated short-term test-retest reliability over 2-4 months, and conducted a pilot longitudinal study over 10-20 months. RESULTS: CSF PrP levels were stable on test-retest with a mean coefficient of variation of 7% for both over 2-4 months in N = 29 participants and over 10-20 months in N = 10 participants. RT-QuIC was negative in 22/23 mutation carriers. The sole individual with positive RT-QuIC seeding activity at two study visits had steady CSF PrP levels and slightly increased tau and NfL concentrations compared with the others, though still within the normal range, and remained asymptomatic 1 year later. T-tau and NfL showed no significant differences between mutation carriers and controls in either CSF or plasma. CONCLUSIONS: CSF PrP will be interpretable as a pharmacodynamic readout for PrP-lowering therapeutics in pre-symptomatic individuals and may serve as an informative surrogate biomarker in this population. In contrast, markers of prion seeding activity and neuronal damage do not reliably cross-sectionally distinguish mutation carriers from controls. Thus, as PrP-lowering therapeutics for prion disease advance, "secondary prevention" based on prodromal pathology may prove challenging; instead, "primary prevention" trials appear to offer a tractable paradigm for trials in pre-symptomatic individuals.