Mechanical compression regulates tumor spheroid invasion into a 3D collagen matrix.

Uncontrolled growth of tumor cells in confined spaces leads to the accumulation of compressive stress within the tumor. Although the effects of tension within 3D extracellular matrices (ECMs) on tumor growth and invasion are well established, the role of compression in tumor mechanics and invasion is largely unexplored. In this study, we modified a Transwell assay such that it provides constant compressive loads to spheroids embedded within a collagen matrix. We used microscopic imaging to follow the single cell dynamics of the cells within the spheroids, as well as invasion into the 3D ECMs. Our experimental results showed that malignant breast tumor (MDA-MB-231) and non-tumorigenic epithelial (MCF10A) spheroids responded differently to a constant compression. Cells within the malignant spheroids became more motile within the spheroids and invaded more into the ECM under compression; whereas cells within non-tumorigenic MCF10A spheroids became less motile within the spheroids and did not display apparent detachment from the spheroids under compression. These findings suggest that compression may play differential roles in healthy and pathogenic epithelial tissues and highlight the importance of tumor mechanics and invasion.

Coding long COVID: characterizing a new disease through an ICD-10 lens.

BACKGROUND: Naming a newly discovered disease is a difficult process; in the context of the COVID-19 pandemic and the existence of post-acute sequelae of SARS-CoV-2 infection (PASC), which includes long COVID, it has proven especially challenging. Disease definitions and assignment of a diagnosis code are often asynchronous and iterative. The clinical definition and our understanding of the underlying mechanisms of long COVID are still in flux, and the deployment of an ICD-10-CM code for long COVID in the USA took nearly 2 years after patients had begun to describe their condition. Here, we leverage the largest publicly available HIPAA-limited dataset about patients with COVID-19 in the US to examine the heterogeneity of adoption and use of U09.9, the ICD-10-CM code for "Post COVID-19 condition, unspecified." METHODS: We undertook a number of analyses to characterize the N3C population with a U09.9 diagnosis code (n = 33,782), including assessing person-level demographics and a number of area-level social determinants of health; diagnoses commonly co-occurring with U09.9, clustered using the Louvain algorithm; and quantifying medications and procedures recorded within 60 days of U09.9 diagnosis. We stratified all analyses by age group in order to discern differing patterns of care across the lifespan. RESULTS: We established the diagnoses most commonly co-occurring with U09.9 and algorithmically clustered them into four major categories: cardiopulmonary, neurological, gastrointestinal, and comorbid conditions. Importantly, we discovered that the population of patients diagnosed with U09.9 is demographically skewed toward female, White, non-Hispanic individuals, as well as individuals living in areas with low poverty and low unemployment. Our results also include a characterization of common procedures and medications associated with U09.9-coded patients. CONCLUSIONS: This work offers insight into potential subtypes and current practice patterns around long COVID and speaks to the existence of disparities in the diagnosis of patients with long COVID. This latter finding in particular requires further research and urgent remediation.

Gain-of-function variants in GABRD reveal a novel pathway for neurodevelopmental disorders and epilepsy.

A potential link between GABRD encoding the δ subunit of extrasynaptic GABAA receptors and neurodevelopmental disorders has largely been disregarded due to conflicting conclusions from early studies. However, we identified seven heterozygous missense GABRD variants in 10 patients with neurodevelopmental disorders and generalized epilepsy. One variant occurred in two sibs of healthy parents with presumed somatic mosaicism, another segregated with the disease in three affected family members, and the remaining five occurred de novo in sporadic patients. Electrophysiological measurements were used to determine the functional consequence of the seven missense δ subunit variants in receptor combinations of α1ß3δ and α4ß2δ GABAA receptors. This was accompanied by analysis of electroclinical phenotypes of the affected individuals. We determined that five of the seven variants caused altered function of the resulting α1ß3δ and α4ß2δ GABAA receptors. Surprisingly, four of the five variants led to gain-of-function effects, whereas one led to a loss-of-function effect. The stark differences between the gain-of-function and loss-of function effects were mirrored by the clinical phenotypes. Six patients with gain-of-function variants shared common phenotypes: neurodevelopmental disorders with behavioural issues, various degrees of intellectual disability, generalized epilepsy with atypical absences and generalized myoclonic and/or bilateral tonic-clonic seizures. The EEG showed qualitative analogies among the different gain-of-function variant carriers consisting of focal slowing in the occipital regions often preceding irregular generalized epileptiform discharges, with frontal predominance. In contrast, the one patient carrying a loss-of-function variant had normal intelligence and no seizure history, but has a diagnosis of autism spectrum disorder and suffers from elevated internalizing psychiatric symptoms. We hypothesize that increase in tonic GABA-evoked current levels mediated by δ-containing extrasynaptic GABAA receptors lead to abnormal neurotransmission, which represent a novel mechanism for severe neurodevelopmental disorders. In support of this, the electroclinical findings for the gain-of-function GABRD variants resemble the phenotypic spectrum reported in patients with missense SLC6A1 (GABA uptake transporter) variants. This also indicates that the phenomenon of extrasynaptic receptor overactivity is observed in a broader range of patients with neurodevelopmental disorders, because SLC6A1 loss-of-function variants also lead to overactive extrasynaptic δ-containing GABAA receptors. These findings have implications when selecting potential treatment options, as a substantial portion of available antiseizure medication act by enhancing GABAergic function either directly or indirectly, which could exacerbate symptoms in patients with gain-of-function GABRD variants.

A lack of repeatability creates the illusion of a trade-off between basal and plastic cold tolerance.

The thermotolerance-plasticity trade-off hypothesis predicts that ectotherms with greater basal thermal tolerance have a lower acclimation capacity. This hypothesis has been tested at both high and low temperatures but the results often conflict. If basal tolerance constrains plasticity (e.g. through shared mechanisms that create physiological constraints), it should be evident at the level of the individual, provided the trait measured is repeatable. Here, we used chill-coma onset temperature and chill-coma recovery time (CCO and CCRT; non-lethal thermal limits) to quantify cold tolerance of Drosophila melanogaster across two trials (pre- and post-acclimation). Cold acclimation improved cold tolerance, as expected, but individual measurements of CCO and CCRT in non-acclimated flies were not (or only slightly) repeatable. Surprisingly, however, there was still a strong correlation between basal tolerance and plasticity in cold-acclimated flies. We argue that this relationship is a statistical artefact (specifically, a manifestation of regression to the mean; RTM) and does not reflect a true trade-off or physiological constraint. Thermal tolerance trade-off patterns in previous studies that used similar methodology are thus likely to be impacted by RTM. Moving forward, controlling and/or correcting for RTM effects is critical to determining whether such a trade-off or physiological constraint exists.

Phase III study of selpercatinib versus chemotherapy ± pembrolizumab in untreated RET positive non-small-cell lung cancer.

Selpercatinib, a novel, highly selective and potent, inhibitor of RET, demonstrated clinically meaningful antitumor activity with manageable toxicity in heavily pretreated and treatment-naive RET fusion-positive non-small-cell lung cancer patients in a Phase I/II clinical trial. LIBRETTO-431 (NCT04194944) is a randomized, global, multicenter, open-label, Phase III trial, evaluating selpercatinib versus carboplatin or cisplatin and pemetrexed chemotherapy with or without pembrolizumab in treatment-naive patients with locally advanced/metastatic RET fusion-positive nonsquamous non-small-cell lung cancer. The primary end point is progression-free survival by independent review. Key secondary end points include overall survival, response rate, duration of response and progression-free survival. Clinical trial registration: NCT04194944 (ClinicalTrials.gov).

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro.

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43def ), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.

An impressive capacity for cold tolerance plasticity protects against ionoregulatory collapse in the disease vector Aedes aegypti.

The mosquito Aedes aegypti is largely confined to tropical and subtropical regions, but its range has recently been spreading to colder climates. As insect biogeography is tied to environmental temperature, understanding the limits of A. aegypti thermal tolerance and their capacity for phenotypic plasticity is important in predicting the spread of this species. In this study, we report on the chill coma onset (CCO) and recovery time (CCRT), as well as low-temperature survival phenotypes of larvae and adults of A. aegypti that developed or were acclimated to 15°C (cold) or 25°C (warm). Cold acclimation did not affect CCO temperatures of larvae but substantially reduced CCO in adults. Temperature and the duration of exposure both affected CCRT, and cold acclimation strongly mitigated these effects and increased rates of survival following prolonged chilling. Female adults were far less likely to take a blood meal when cold acclimated, and exposing females to blood (without feeding) attenuated some of the beneficial effects of cold acclimation on CCRT. Lastly, larvae suffered from haemolymph hyperkalaemia when chilled, but cold acclimation attenuated the imbalance. Our results demonstrate that A. aegypti larvae and adults have the capacity to acclimate to low temperatures, and do so at least in part by better maintaining ion balance in the cold. This ability for cold acclimation may facilitate the spread of this species to higher latitudes, particularly in an era of climate change.

RAGE Signaling in Skeletal Biology.

PURPOSE OF REVIEW: The receptor for advanced glycation end products (RAGE) and several of its ligands have been implicated in the onset and progression of pathologies associated with aging, chronic inflammation, and cellular stress. In particular, the role of RAGE and its ligands in bone tissue during both physiological and pathological conditions has been investigated. However, the extent to which RAGE signaling regulates bone homeostasis and disease onset remains unclear. Further, RAGE effects in the different bone cells and whether these effects are cell-type specific is unknown. The objective of the current review is to describe the literature over RAGE signaling in skeletal biology as well as discuss the clinical potential of RAGE as a diagnostic and/or therapeutic target in bone disease. RECENT FINDINGS: The role of RAGE and its ligands during skeletal homeostasis, tissue repair, and disease onset/progression is beginning to be uncovered. For example, detrimental effects of the RAGE ligands, advanced glycation end products (AGEs), have been identified for osteoblast viability/activity, while others have observed that low level AGE exposure stimulates osteoblast autophagy, which subsequently promotes viability and function. Similar findings have been reported with HMGB1, another RAGE ligand, in which high levels of the ligand are associated with osteoblast/osteocyte apoptosis, whereas low level/short-term administration stimulates osteoblast differentiation/bone formation and promotes fracture healing. Additionally, elevated levels of several RAGE ligands (AGEs, HMGB1, S100 proteins) induce osteoblast/osteocyte apoptosis and stimulate cytokine production, which is associated with increased osteoclast differentiation/activity. Conversely, direct RAGE-ligand exposure in osteoclasts may have inhibitory effects. These observations support a conclusion that elevated bone resorption observed in conditions of high circulating ligands and RAGE expression are due to actions on osteoblasts/osteocytes rather than direct actions on osteoclasts, although additional work is required to substantiate the observations. Recent studies have demonstrated that RAGE and its ligands play an important physiological role in the regulation of skeletal development, homeostasis, and repair/regeneration. Conversely, elevated levels of RAGE and its ligands are clearly related with various diseases associated with increased bone loss and fragility. However, despite the recent advancements in the field, many questions regarding RAGE and its ligands in skeletal biology remain unanswered.

Connexins and Pannexins in Bone and Skeletal Muscle.

PURPOSE OF REVIEW: To discuss current knowledge on the role of connexins and pannexins in the musculoskeletal system. RECENT FINDINGS: Connexins and pannexins are crucial for the development and maintenance of both bone and skeletal muscle. In bone, the presence of connexin and more recently of pannexin channels in osteoblasts, osteoclasts, and osteocytes has been described and shown to be essential for normal skeletal development and bone adaptation. In skeletal muscles, connexins and pannexins play important roles during development and regeneration through coordinated regulation of metabolic functions via cell-to-cell communication. Further, under pathological conditions, altered expression of these proteins can promote muscle atrophy and degeneration by stimulating inflammasome activity. In this review, we highlight the important roles of connexins and pannexins in the development, maintenance, and regeneration of musculoskeletal tissues, with emphasis on the mechanisms by which these molecules mediate chemical (e.g., ATP and prostaglandin E2) and physical (e.g., mechanical stimulation) stimuli that target the musculoskeletal system and their involvement in the pathophysiological changes in both genetic and acquired diseases.

Inhibition of osteocyte apoptosis prevents the increase in osteocytic receptor activator of nuclear factor κB ligand (RANKL) but does not stop bone resorption or the loss of bone induced by unloading.

Apoptosis of osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because osteocytes are major RANKL producers, we hypothesized that apoptotic osteocytes signal to neighboring osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in osteocyte apoptosis and osteocytic RANKL expression. The BPs also inhibited osteoblast apoptosis but did not prevent the increase in osteoblastic RANKL. Unloaded mice exhibited high serum levels of the bone resorption marker C-telopeptide fragments of type I collagen (CTX), elevated osteoclastogenesis, and increased osteoclasts in bone. Aln, but not IG9402, prevented all of these effects. In addition, Aln prevented the reduction in spinal and femoral bone mineral density, spinal bone volume/tissue volume, trabecular thickness, mechanical strength, and material strength induced by unloading. Although IG9402 did not prevent the loss of bone mass, it partially prevented the loss of strength, suggesting a contribution of osteocyte viability to strength independent of bone mass. These results demonstrate that osteocyte apoptosis leads to increased osteocytic RANKL. However, blockade of these events is not sufficient to restrain osteoclast formation, inhibit resorption, or stop bone loss induced by skeletal unloading.

Monotherapy with minocycline or trimethoprim/sulfamethoxazole for treatment of Stenotrophomonas maltophilia infections.

OBJECTIVES: Stenotrophomonas maltophilia is a Gram-negative bacillus intermittently isolated from hospitalized patients. Trimethoprim/sulfamethoxazole is considered the treatment of choice for S. maltophilia infections, though limited by toxicities. Minocycline is utilized at our institution for S. maltophilia infections due to its improved tolerability and in vitro susceptibility rates. Our objective was to evaluate the effectiveness of minocycline monotherapy compared with trimethoprim/sulfamethoxazole monotherapy for treatment of S. maltophilia infections. METHODS: Patients were identified via microbiology laboratory data and those with at least one positive culture for S. maltophilia were cross-referenced with pharmacy data to detect patients who received trimethoprim/sulfamethoxazole or minocycline. Patients initially receiving combination therapy were excluded. Our primary outcome was treatment failure, defined as receipt of alternative antibiotics with in vitro activity against S. maltophilia, isolation of S. maltophilia on repeat culture or death within 30 days of treatment. RESULTS: Forty-five patients were evaluated. Overall mortality rate was 9% and equal between groups; 41% of patients (9/22) who received trimethoprim/sulfamethoxazole and 30% (7/23) of patients who received minocycline experienced treatment failure (P = 0.67). Patients who received minocycline were more likely to have had a recent acute kidney injury (AKI) (43.5% versus 9%; P = 0.017) or chronic lung disease (52% versus 9%; P = 0.003). Logistic regression showed consistent results of non-inferiority of the primary outcome when controlling for rates of underlying lung pathology and recent AKI (P = 0.728). CONCLUSIONS: Treatment failure did not differ between patients receiving trimethoprim/sulfamethoxazole or minocycline monotherapy for treatment of S. maltophilia infections.

High bone mass in mice lacking Cx37 because of defective osteoclast differentiation.

Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37(-/-)) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37(-/-) mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37(-/-) mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37(+/+) littermates. sRANKL/M-CSF treatment of nonadherent Cx37(-/-) bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37(+/+) cell cultures. Further, Cx37(-/-) osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37(-/-) osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37(-/-) mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.

Decoding the role of tRNA modifications in cancer progression.

Epitranscriptomic modification of tRNA has recently gained traction in the field of cancer biology. The presence of such modifications on tRNA appears to allow for translational control of processes central to progression and malignant transformation. Methyltransferase Like 1 protein (METTL1), along with other epitranscriptomic writers (e.g. NSUN3, NAT10, ELP3, etc.), has recently been investigated in multiple cancer types. Here, we review the impact of such tRNA modifications in tumorigenesis and the progression of cancer toward drug resistance and metastasis. Regulation of central cellular processes relied upon by malignant cancer cells through modulation of the tRNA epitranscriptome represents an area with great potential to bring novel first-in-class therapies to the clinic.

Variation in nutritional quality in UK retail eggs.

Fatty acid (FA), carotenoid and vitamin contents of UK eggs were assessed for four production systems (caged (CA), free-range (FR), organic (OR) and extensive organic (EO)) as well as season. The impact of enforced housing, due to avian influenza, was also investigated. Production system did not alter vitamin D3, B2 or B9 content, but significantly influenced nutritionally desirable FA, carotenoid and vitamins A and E - concentrations decreased as production intensity increased, although for most, CA and FR did not differ significantly. Vitamin E and FA profiles for OR and EO were also similar, although carotenoids were higher in EO eggs. In contrast, FA, carotenoids, vitamins E and B9 were consistent throughout the year, unlike vitamins A, D3 and B2, which fluctuated with season; D and B2 were higher in July than January and lower vitamin A was the only detected implication from enforced housing of FR and OR birds.

Insights from an N3C RECOVER EHR-based cohort study characterizing SARS-CoV-2 reinfections and Long COVID.

BACKGROUND: Although the COVID-19 pandemic has persisted for over 3 years, reinfections with SARS-CoV-2 are not well understood. We aim to characterize reinfection, understand development of Long COVID after reinfection, and compare severity of reinfection with initial infection. METHODS: We use an electronic health record study cohort of over 3 million patients from the National COVID Cohort Collaborative as part of the NIH Researching COVID to Enhance Recovery Initiative. We calculate summary statistics, effect sizes, and Kaplan-Meier curves to better understand COVID-19 reinfections. RESULTS: Here we validate previous findings of reinfection incidence (6.9%), the occurrence of most reinfections during the Omicron epoch, and evidence of multiple reinfections. We present findings that the proportion of Long COVID diagnoses is higher following initial infection than reinfection for infections in the same epoch. We report lower albumin levels leading up to reinfection and a statistically significant association of severity between initial infection and reinfection (chi-squared value: 25,697, p-value: <0.0001) with a medium effect size (Cramer's V: 0.20, DoF = 3). Individuals who experienced severe initial and first reinfection were older in age and at a higher mortality risk than those who had mild initial infection and reinfection. CONCLUSIONS: In a large patient cohort, we find that the severity of reinfection appears to be associated with the severity of initial infection and that Long COVID diagnoses appear to occur more often following initial infection than reinfection in the same epoch. Future research may build on these findings to better understand COVID-19 reinfections.

More than three years after the start of the COVID-19 pandemic, individuals are frequently reporting multiple COVID-19 infections. However, these reinfections remain poorly understood. Here, we investigate COVID-19 reinfections in a large electronic health record cohort of over 3 million patients. We use data summary techniques and statistical tests to characterize reinfections and their relationships with disease severity, biomarkers, and Long COVID. We find that individuals with severe initial infection are more likely to experience severe reinfection, that some protein levels are lower, leading to reinfection, and that a lower proportion of individuals are diagnosed with Long COVID following reinfection than initial infection. Our work highlights the prevalence and impact of reinfections and suggests the need for further research.

Spatial Mapping of Hematopoietic Clones in Human Bone Marrow.

Clonal hematopoiesis (CH) is the expansion of somatically mutated cells in the hematopoietic compartment of individuals without hematopoietic dysfunction. Large CH clones (i.e., >2% variant allele fraction) predispose to hematologic malignancy, but CH is detected at lower levels in nearly all middle-aged individuals. Prior work has extensively characterized CH in peripheral blood, but the spatial distribution of hematopoietic clones in human bone marrow is largely undescribed. To understand CH at this level, we developed a method for spatially aware somatic mutation profiling and characterized the bone marrow of a patient with polycythemia vera. We identified the complex clonal distribution of somatic mutations in the hematopoietic compartment, the restriction of somatic mutations to specific subpopulations of hematopoietic cells, and spatial constraints of these clones in the bone marrow. This proof of principle paves the way to answering fundamental questions regarding CH spatial organization and factors driving CH expansion and malignant transformation in the bone marrow. SIGNIFICANCE: CH occurs commonly in humans and can predispose to hematologic malignancy. Although well characterized in blood, it is poorly understood how clones are spatially distributed in the bone marrow. To answer this, we developed methods for spatially aware somatic mutation profiling to describe clonal heterogeneity in human bone marrow. See related commentary by Austin and Aifantis, p. 139.

Finding Long-COVID: Temporal Topic Modeling of Electronic Health Records from the N3C and RECOVER Programs.

Post-Acute Sequelae of SARS-CoV-2 infection (PASC), also known as Long-COVID, encompasses a variety of complex and varied outcomes following COVID-19 infection that are still poorly understood. We clustered over 600 million condition diagnoses from 14 million patients available through the National COVID Cohort Collaborative (N3C), generating hundreds of highly detailed clinical phenotypes. Assessing patient clinical trajectories using these clusters allowed us to identify individual conditions and phenotypes strongly increased after acute infection. We found many conditions increased in COVID-19 patients compared to controls, and using a novel method to associate patients with clusters over time, we additionally found phenotypes specific to patient sex, age, wave of infection, and PASC diagnosis status. While many of these results reflect known PASC symptoms, the resolution provided by this unprecedented data scale suggests avenues for improved diagnostics and mechanistic understanding of this multifaceted disease.