The HIF signaling pathway in osteoblasts directly modulates erythropoiesis through the production of EPO.

Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.

Chromosomal structural rearrangements implicate long non-coding RNAs in rare germline disorders.

In recent years, there has been increased focus on exploring the role the non-protein-coding genome plays in Mendelian disorders. One class of particular interest is long non-coding RNAs (lncRNAs), which has recently been implicated in the regulation of diverse molecular processes. However, because lncRNAs do not encode protein, there is uncertainty regarding what constitutes a pathogenic lncRNA variant, and thus annotating such elements is challenging. The Developmental Genome Anatomy Project (DGAP) and similar projects recruit individuals with apparently balanced chromosomal abnormalities (BCAs) that disrupt or dysregulate genes in order to annotate the human genome. We hypothesized that rearrangements disrupting lncRNAs could be the underlying genetic etiology for the phenotypes of a subset of these individuals. Thus, we assessed 279 cases with BCAs and selected 191 cases with simple BCAs (breakpoints at only two genomic locations) for further analysis of lncRNA disruptions. From these, we identified 66 cases in which the chromosomal rearrangements directly disrupt lncRNAs. In 30 cases, no genes of any other class aside from lncRNAs are directly disrupted, consistent with the hypothesis that lncRNA disruptions could underly the phenotypes of these individuals. Strikingly, the lncRNAs MEF2C-AS1 and ENSG00000257522 are each disrupted in two unrelated cases. Furthermore, we experimentally tested the lncRNAs TBX2-AS1 and MEF2C-AS1 and found that knockdown of these lncRNAs resulted in decreased expression of the neighboring transcription factors TBX2 and MEF2C, respectively. To showcase the power of this genomic approach for annotating lncRNAs, here we focus on clinical reports and genetic analysis of seven individuals with likely developmental etiologies due to lncRNA disruptions.

Long-term humoral and cellular immunity after primary SARS-CoV-2 infection: a 20-month longitudinal study.

BACKGROUND: SARS-CoV-2 remains a world-wide health issue. SARS-CoV-2-specific immunity is induced upon both infection and vaccination. However, defining the long-term immune trajectory, especially after infection, is limited. In this study, we aimed to further the understanding of long-term SARS-CoV-2-specific immune response after infection. RESULTS: We conducted a longitudinal cohort study among 93 SARS-CoV-2 recovered individuals. Immune responses were continuously monitored for up to 20 months after infection. The humoral responses were quantified by Spike- and Nucleocapsid-specific IgG levels. T cell responses to Spike- and non-Spike epitopes were examined using both intercellular cytokine staining (ICS) assay and Activation-Induced marker (AIM) assay with quantification of antigen-specific IFNγ production. During the 20 months follow-up period, Nucleocapsid-specific antibody levels and non-Spike-specific CD4 + and CD8 + T cell frequencies decreased in the blood. However, a majority of participants maintained a durable immune responses 20 months after infection: 59% of the participants were seropositive for Nucleocapsid-specific IgG, and more than 70% had persisting non-Spike-specific T cells. The Spike-specific response initially decreased but as participants were vaccinated against COVID-19, Spike-specific IgG levels and T cell frequencies were boosted reaching similar or higher levels compared to 1 month post-infection. The trajectory of infection-induced SARS-CoV-2-specific immunity decreases, but for the majority of participants it persists beyond 20 months. The T cell response displays a greater durability. Vaccination boosts Spike-specific immune responses to similar or higher levels as seen after primary infection. CONCLUSIONS: For most participants, the response persists 20 months after infection, and the cellular response appears to be more long-lived compared to the circulating antibody levels. Vaccination boosts the S-specific response but does not affect the non-S-specific response. Together, these findings support the understanding of immune contraction, and with studies showing the immune levels required for protection, adds to the knowledge of durability of protection against future SARS-CoV-2.

Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin.

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.

The Legionella effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the Legionella-containing vacuole.

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein-lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

Glecaprevir/Pibrentasvir: The First 8-Week, Pangenotypic HCV Treatment Regimen for Patients 12 Years of Age and Older.

Objective: To review the pharmacology, pharmacokinetics, efficacy, safety, dosing, and cost information of glecaprevir/pibrentasvir in the treatment of hepatitis C virus (HCV). Data Sources: A literature search was conducted between September 2018 and July 2019 using PubMed and Google Scholar with the search terms glecaprevir, pibrentasvir, Mavyret, Maviret, and hepatitis C. Clinicaltrials.gov was searched using the same terms. References of published articles were assessed for additional information. Study Selection and Data Extraction: English-language preclinical and clinical studies on the chemistry, pharmacology, pharmacokinetics, safety, and efficacy of glecaprevir/pibrentasvir were evaluated. Data Synthesis: Food and Drug Administration-approved glecaprevir/pibrentasvir is considered both safe and efficacious for the treatment of HCV genotypes 1 to 6 and in several patient populations, such as those with treatment-naïve or treatment-experienced HCV; with or without compensated cirrhosis, HIV-1 coinfection, or renal impairment; post-liver or post-kidney transplant; and ≥12 years of age. Sustained virological response rates ranged from 83% to 100% in clinical trials, and safety outcomes appear similar to other guideline-recommended HCV treatment options. Relevance to Patient Care and Clinical Practice: This review discusses the pharmacological, efficacy, and safety data found in glecaprevir/pibrentasvir clinical trials and relates this to guideline recommendations and the practical use of this medication for treatment of HCV. Conclusions: With HCV infection rates remaining elevated, it is important to have safe and efficacious treatment options. Glecaprevir/pibrentasvir is a safe and efficacious guideline-recommended, 8-week treatment for HCV in several patient populations, with these populations likely growing in the near future given ongoing and future studies.

Forging our understanding of lncRNAs in the brain.

During both development and adulthood, the human brain expresses many thousands of long noncoding RNAs (lncRNAs), and aberrant lncRNA expression has been associated with a wide range of neurological diseases. Although the biological significance of most lncRNAs remains to be discovered, it is now clear that certain lncRNAs carry out important functions in neurodevelopment, neural cell function, and perhaps even diseases of the human brain. Given the relatively inclusive definition of lncRNAs-transcripts longer than 200 nucleotides with essentially no protein coding potential-this class of noncoding transcript is both large and very diverse. Furthermore, emerging data indicate that lncRNA genes can act via multiple, non-mutually exclusive molecular mechanisms, and specific functions are difficult to predict from lncRNA expression or sequence alone. Thus, the different experimental approaches used to explore the role of a lncRNA might each shed light upon distinct facets of its overall molecular mechanism, and combining multiple approaches may be necessary to fully illuminate the function of any particular lncRNA. To understand how lncRNAs affect brain development and neurological disease, in vivo studies of lncRNA function are required. Thus, in this review, we focus our discussion upon a small set of neural lncRNAs that have been experimentally manipulated in mice. Together, these examples illustrate how studies of individual lncRNAs using multiple experimental approaches can help reveal the richness and complexity of lncRNA function in both neurodevelopment and diseases of the brain.

Sex-specific role for the long noncoding RNA Pnky in mouse behavior.

The aberrant expression of specific long noncoding RNAs (lncRNAs) has been associated with cognitive and psychiatric disorders. Although a growing number of lncRNAs are now known to regulate neural cell development and function, relatively few lncRNAs have been shown to underlie animal behavior. Pnky is an evolutionarily conserved, neural lncRNA that regulates brain development. Using mouse genetic strategies, we show that Pnky has sex-specific roles in mouse behavior and that this lncRNA can underlie specific behavior by functioning in trans. Male Pnky-knockout mice have decreased context generalization in a paradigm of associative fear learning and memory. In female Pnky-knockout mice, there is an increase in the acoustic startle response, a behavior that is altered in affective disorders. Remarkably, expression of Pnky from a bacterial artificial chromosome transgene decreases the acoustic startle response in female Pnky-knockout mice, demonstrating that Pnky can modulate specific animal behavior by functioning in trans. More broadly, these studies illustrate how specific lncRNAs can underlie cognitive and mood disorders.

Ocular versus generalized myasthenia gravis: a continuum associated with acetylcholine receptor antibody titers.

The aim of this study was to evaluate clinical and serological differences between the ocular myasthenia gravis (oMG) and generalized MG (gMG). This study is a retrospective chart review, in which data was collected from patients fulfilling 2 of 3 diagnostic MG criteria (positive antibodies, evidence of neuromuscular transmission defect on neurophysiological examination, positive effect of pyridostigmine treatment). 350 patients were included and data concerning demographics and MG medical history were collected. Patients with oMG accounted for 15.7 % of the included patients. The two subgroups differed significantly in oMG having a later age at onset, lower AChR antibody-titers, longer doctor-to-diagnosis delay and less intensive MG treatment. Additionally, patients with oMG were faster at reaching a well-controlled disease state. Thymus pathology, number of antibody-positive (95.9 % of gMG and 94.5 % of oMG), sex, number of other autoimmune diseases and delay before drug stability did not differ between oMG and gMG. In conclusion, oMG is presumably a milder form of gMG characterized by lower AChR antibody-titers, a milder phenotype, and a quicker response to a less aggressive treatment. But otherwise, oMG and gMG show very similar characteristics, including the same frequency of positive AChR antibodies, which seems new compared to previous reports.

Rare germline disorders implicate long non-coding RNAs disrupted by chromosomal structural rearrangements.

In recent years, there has been increased focus on exploring the role the non-protein-coding genome plays in Mendelian disorders. One class of particular interest is long non-coding RNAs (lncRNAs), which has recently been implicated in the regulation of diverse molecular processes. However, because lncRNAs do not encode protein, there is uncertainty regarding what constitutes a pathogenic lncRNA variant, and thus annotating such elements is challenging. The Developmental Genome Anatomy Project (DGAP) and similar projects recruit individuals with apparently balanced chromosomal abnormalities (BCAs) that disrupt or dysregulate genes in order to annotate the human genome. We hypothesized that rearrangements disrupting lncRNAs could be the underlying genetic etiology for the phenotypes of a subset of these individuals. Thus, we assessed 279 cases with BCAs and selected 191 cases with simple BCAs (breakpoints at only two genomic locations) for further analysis of lncRNA disruptions. From these, we identified 66 cases in which the chromosomal rearrangements directly disrupt lncRNAs. Strikingly, the lncRNAs MEF2C-AS1 and ENSG00000257522 are each disrupted in two unrelated cases. Furthermore, in 30 cases, no genes of any other class aside from lncRNAs are directly disrupted, consistent with the hypothesis that lncRNA disruptions could underly the phenotypes of these individuals. To showcase the power of this genomic approach for annotating lncRNAs, here we focus on clinical reports and genetic analysis of two individuals with BCAs and additionally highlight six individuals with likely developmental etiologies due to lncRNA disruptions.

Rare variation in non-coding regions with evolutionary signatures contributes to autism spectrum disorder risk.

Little is known about the role of non-coding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of non-coding regions: human accelerated regions (HARs), which show signatures of positive selection in humans; experimentally validated neural VISTA enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole-genome analysis of >16,600 samples and >4,900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly contribute, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in VEs near OTX1 and SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved non-coding regions in ASD risk and suggest potential mechanisms of how regulatory changes can modulate social behavior.

Spatial Single-cell Analysis Decodes Cortical Layer and Area Specification.

The human cerebral cortex, pivotal for advanced cognitive functions, is composed of six distinct layers and dozens of functionally specialized areas1,2. The layers and areas are distinguished both molecularly, by diverse neuronal and glial cell subtypes, and structurally, through intricate spatial organization3,4. While single-cell transcriptomics studies have advanced molecular characterization of human cortical development, a critical gap exists due to the loss of spatial context during cell dissociation5,6,7,8. Here, we utilized multiplexed error-robust fluorescence in situ hybridization (MERFISH)9, augmented with deep-learning-based cell segmentation, to examine the molecular, cellular, and cytoarchitectural development of human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing 16 million single cells, spans eight cortical areas across four time points in the second and third trimesters. We uncovered an early establishment of the six-layer structure, identifiable in the laminar distribution of excitatory neuronal subtypes by mid-gestation, long before the emergence of cytoarchitectural layers. Notably, while anterior-posterior gradients of neuronal subtypes were generally observed in most cortical areas, a striking exception was the sharp molecular border between primary (V1) and secondary visual cortices (V2) at gestational week 20. Here we discovered an abrupt binary shift in neuronal subtype specification at the earliest stages, challenging the notion that continuous morphogen gradients dictate mid-gestation cortical arealization6,10. Moreover, integrating single-nuclei RNA-sequencing and in situ whole transcriptomics revealed an early upregulation of synaptogenesis in V1-specific Layer 4 neurons, suggesting a role of synaptogenesis in this discrete border formation. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This work not only provides a valuable resource for the field, but also establishes a spatially resolved single-cell analysis paradigm that paves the way for a comprehensive developmental atlas of the human brain.

Substance misuse training in pharmacy education: Results from a pilot study on the integration of an interprofessional experience.

Background: Studies show that pharmacists are unsure in their ability to screen patients for substance abuse. This study evaluates the efficacy of incorporating interprofessional education (IPE) into a substance misuse training program on pharmacy students' learning outcomes in providing screening and counseling for substance misuse. Methods: Pharmacy students from 2019 to 2020 completed 3 substance misuse training modules. Students from the class of 2020 completed an additional IPE event. Both cohorts completed pre- and post-surveys that evaluated knowledge of content and comfort level with patient screening and counseling regarding substance misuse. Paired student t-tests and difference-in-difference analyses were used to evaluate the impact of the IPE event. Findings: Both cohorts (n = 127) showed statistically significant improvement in learning outcomes in providing substance misuse screening and counseling. IPE yielded extremely positive feedback from all students, but its addition to the overall training did not improve learning outcomes. This may be attributed to the differences in baseline knowledge of each class cohort. Conclusion: Substance misuse training successfully improved pharmacy student knowledge and comfort level with providing patient screening and counseling services. Though the IPE event did not improve learning outcomes, qualitative student feedback was overwhelmingly positive and supports the continued incorporation of IPE.

Sex-specific role for the long noncoding RNA Pnky in mouse behavior.

The human brain expresses thousands of different long noncoding RNAs (lncRNAs), and aberrant expression of specific lncRNAs has been associated with cognitive and psychiatric disorders. While a growing number of lncRNAs are now known to regulate neural cell development and function, relatively few have been shown to underlie animal behavior, particularly with genetic strategies that establish lncRNA function in trans. Pnky is an evolutionarily conserved, neural lncRNA that regulates brain development. Using mouse genetic strategies, we show that Pnky has sex-specific roles in mouse behavior and that this lncRNA underlies specific behavior by functioning in trans. Male Pnky-knockout (KO) mice have deficits in cued fear recall, a type of Pavlovian associative memory. In female Pnky-KO mice, the acoustic startle response (ASR) is increased and accompanied by a decrease in prepulse inhibition (PPI), both of which are behaviors altered in affective disorders. Remarkably, expression of Pnky from a bacterial artificial chromosome (BAC) transgene reverses the ASR phenotype of female Pnky-KO mice, demonstrating that Pnky underlies specific animal behavior by functioning in trans. More broadly, these data provide genetic evidence that a lncRNA gene and its function in trans can play a key role in the behavior of adult mammals, contributing fundamental knowledge to our growing understanding of the association between specific lncRNAs and disorders of cognition and mood.

Rare variation in noncoding regions with evolutionary signatures contributes to autism spectrum disorder risk.

Little is known about the role of noncoding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of noncoding regions: Human Accelerated Regions (HARs), which show signatures of positive selection in humans; experimentally validated neural Vista Enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole genome analysis of >16,600 samples and >4900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in a VE near SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved noncoding regions in ASD risk and suggest potential mechanisms of how changes in regulatory regions can modulate social behavior.

Enteroaggregative Escherichia coli O78:H10, the cause of an outbreak of urinary tract infection.

In 1991, multiresistant Escherichia coli O78:H10 strains caused an outbreak of urinary tract infections in Copenhagen, Denmark. The phylogenetic origin, clonal background, and virulence characteristics of the outbreak isolates, and their relationship to nonoutbreak O78:H10 strains according to these traits and resistance profiles, are unknown. Accordingly, we extensively characterized 51 archived E. coli O78:H10 isolates (48 human isolates from seven countries, including 19 Copenhagen outbreak isolates, and 1 each of calf, avian, and unknown-source isolates), collected from 1956 through 2000. E. coli O78:H10 was clonally heterogeneous, comprising one dominant clonal group (61% of isolates, including all 19 outbreak isolates) from ST10 (phylogenetic group A) plus several minor clonal groups (phylogenetic groups A and D). All ST10 isolates, versus 25% of non-ST10 isolates, were identified by molecular methods as enteroaggregative E. coli (EAEC) (P < 0.001). Genes present in >90% of outbreak isolates included fimH (type 1 fimbriae; ubiquitous in E. coli); fyuA, traT, and iutA (associated with extraintestinal pathogenic E. coli [ExPEC]); and sat, pic, aatA, aggR, aggA, ORF61, aaiC, aap, and ORF3 (associated with EAEC). An outbreak isolate was lethal in a murine subcutaneous sepsis model and exhibited characteristic EAEC "stacked brick" adherence to cultured epithelial cells. Thus, the 1991 Copenhagen outbreak was caused by a tight, non-animal-associated subset within a broadly disseminated O78:H10 clonal group (ST10; phylogenetic group A), members of which exhibit both ExPEC and EAEC characteristics, whereas O78:H10 isolates overall are phylogenetically diverse. Whether ST10 O78:H10 EAEC strains are both uropathogenic and diarrheagenic warrants further investigation.

Climate change and population growth in Timor Leste: implications for food security.

The climate in Timor Leste (East Timor) is predicted to become about 1.5 °C warmer and about 10 % wetter on average by 2050. By the same year, the population is expected to triple from 1 to 2.5-3 million. This article maps the predicted changes in temperature and rainfall and reviews the implications of climate change and population growth on agricultural systems. Improved cultivars of maize, rice, cassava, sweet potato and peanuts with high yield performance have been introduced, but these will need to be augmented in the future with better adapted cultivars and new crops, such as food and fodder legumes and new management practices. The requirements for fertilizers to boost yields and terracing and/or contour hedgerows to prevent soil erosion of steeply sloping terrain are discussed. Contour hedges can also be used for fodder for improved animal production to provide protein to reduce malnutrition.

Defining obstacles to emergency transfer of trauma patients: An evaluation of retriage processes from nontrauma and lower-level Illinois trauma centers.

BACKGROUND: Retriage is the emergency transfer of severely injured patients from nontrauma and lower-level trauma centers to higher-level trauma centers. We identified the barriers to retriage at sending centers in a single health system. METHODS: We conducted a failure modes effects and criticality analysis at 4 nontrauma centers and 5 lower-level trauma centers in a single health system. Clinicians from each center described the steps in the trauma assessment and retriage process to create a process map. We used standardized scoring to characterize each failure based on frequency, impact on retriage, and prevention safeguards. We ranked each failure using the scores to calculate a risk priority number. RESULTS: We identified 26 steps and 93 failures. The highest-risk failure was refusal by higher-level trauma centers (receiving hospitals) to accept a patient. The most critical failures in the retriage process based on total risk, frequency, and safeguard scores were (1) refusal from a receiving higher-level trauma center to accept a patient (risk priority number = 191), (2) delay in a sending center's consultant examination of a patient in the emergency department (risk priority number = 177), and (3) delay in receiving hospital's consultant calling back (risk priority number = 177). CONCLUSION: We identified (1) addressing obstacles to determining clinical indications for retriage and (2) identifying receiving level I trauma centers who would accept the patient as opportunities to increase timely retriage. Establishing clear clinical indications for retriage that sending and receiving hospitals agree on represents an opportunity for intervention that could improve the retriage of injured patients.