Heme-Induced Macrophage Phenotype Switching and Impaired Endogenous Opioid Homeostasis Correlate with Chronic Widespread Pain in HIV.

Chronic widespread pain (CWP) is associated with a high rate of disability and decreased quality of life in people with HIV-1 (PWH). We previously showed that PWH with CWP have increased hemolysis and elevated plasma levels of cell-free heme, which correlate with low endogenous opioid levels in leukocytes. Further, we demonstrated that cell-free heme impairs ß-endorphin synthesis/release from leukocytes. However, the cellular mechanisms by which heme dampens ß-endorphin production are inconclusive. The current hypothesis is that heme-dependent TLR4 activation and macrophage polarization to the M1 phenotype mediate this phenomenon. Our novel findings showed that PWH with CWP have elevated M1-specific macrophage chemokines (ENA-78, GRO-α, and IP-10) in plasma. In vitro, hemin-induced polarization of M0 and M2 macrophages to the M1 phenotype with low ß-endorphins was mitigated by treating cells with the TLR4 inhibitor, TAK-242. Similarly, in vivo phenylhydrazine hydrochloride (PHZ), an inducer of hemolysis, injected into C57Bl/6 mice increased the M1/M2 cell ratio and reduced ß-endorphin levels. However, treating these animals with the heme-scavenging protein hemopexin (Hx) or TAK-242 reduced the M1/M2 ratio and increased ß-endorphins. Furthermore, Hx attenuated heme-induced mechanical, heat, and cold hypersensitivity, while TAK-242 abrogated hypersensitivity to mechanical and heat stimuli. Overall, these results suggest that heme-mediated TLR4 activation and M1 polarization of macrophages correlate with impaired endogenous opioid homeostasis and hypersensitivity in people with HIV.

Chest CT Findings and Their Temporal Evolution in COVID-19 Pneumonia.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic originated in China in November 2019 and is caused by the SARS-CoV-2 virus. The virus binds to nasal and pharyngeal epithelial cells and migrates to the lower respiratory tract. The confirmatory test for COVID-19 infection is the reverse transcription-polymerase chain reaction (RT-PCR). Chest CT plays an important role in the diagnosis, triage, and treatment of affected individuals. We describe the findings on chest CT and their temporal evolution in COVID-19 pneumonia. METHODS: We conducted a retrospective, cross-sectional study on COVID-19-positive patients who underwent chest CT. CT images of the patients were reviewed for ground-glass opacities, consolidation, crazy-paving appearance, vascular dilatation, traction bronchiectasis, architectural distortion, and subpleural and parenchymal bands. Distribution of opacities on axial sections, ancillary findings, and co-existent lung diseases were recorded. To assess the temporal evolution of CT findings, the time in days between the onset of the first symptom and the date of the CT scan of each patient was recorded. Statistical analysis was performed. RESULTS: Ground-glass opacities, consolidation, and a combination of both were the most important features in COVID-19 pneumonia. Patients in the early stage showed simple ground-glass opacities; in the progressive stage showed consolidation and ground-glass opacities with crazy-paving appearance, subpleural and parenchymal bands, and architectural distortion; in the peak stage showed progression of these findings; and in the late stage showed interval resolution of these findings. Axial distribution of these opacities was asymmetric, with peripheral subpleural predominance involving posterior, lateral, and both these locations, associated with apicobasal gradient. CONCLUSION: Chest CT permits rapid diagnosis of COVID-19 pneumonia, enabling appropriate treatment to be instituted at the earliest. Thus, it is life-saving in resource-constrained environments.

Can You Hear Me Now? The Impact of Hearing Loss on Patient Health Literacy.

OBJECTIVE: To elucidate the impact of hearing loss on patient health literacy. STUDY DESIGN: Prospective, cross-sectional study. SETTING: Academic otology practice at a university hospital. PATIENTS: Consecutive, adult, English-speaking patients. MAIN OUTCOME MEASURES: Inadequate health literacy, defined as a composite score of less than or equal to nine on the brief health literacy screen (BHLS), was compared with patient hearing data utilizing the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) hearing classification. Secondary outcome measures included comparisons of inadequate BHLS scores according to patient demographic and clinical information. RESULTS: There were 300 consecutive adult (>18 years old) patients evaluated with the BHLS at a university otology practice between February and March 2019. The median patient age was 60-years (range, 18-91 yr), a slight majority (160, 53.3%) were women, and most patients were White (241, 86.7%) and non-Hispanic (260, 91.6%). Overall, 9.7% of patients were found to have inadequate health literacy. Men had higher rates of inadequate health literacy as compared with women (13.6% versus 6.3%, odds ratio [OR] = 2.35, 95% confidence interval [CI] 1.06-5.25). Audiometric data was available for 284 (95%) patients, of which 235 (82.7%) had class A or B hearing and 49 (17.3%) had class C or D hearing. Patients with Class C or D hearing had a lower median composite BHLS score compared with patients with Class A or B hearing (11.6 versus 13.6, p < 0.0001) and an increased rate of inadequate health literacy (28.6% versus 4.7%, OR = 8.15, 95% CI 3.42-19.37). Increased age, female sex, and better hearing were independent predictors of higher BHLS scores on multivariable analysis. CONCLUSIONS: Hearing loss is an independent risk factor for inadequate health literacy. Providers should be aware of this risk and consider implementing strategies to improve counseling for this at-risk group of patients.

Cardiorespiratory Fitness Is Associated With Better Cardiometabolic Health and Lower PTSD Severity in Post-9/11 Veterans.

INTRODUCTION: Post-traumatic stress disorder (PTSD) is associated with an increased risk of cardiovascular and metabolic diseases and physical inactivity. Cardiorespiratory fitness (CRF), which is modifiable by physical activity, is a strong independent predictor of cardiometabolic health. However, the relationship between CRF and cardiometabolic health in veterans with PTSD is unknown. Thus, this study aimed to explore the cross-sectional relationships among CRF, indices of cardiometabolic health (ie, HbA1c, blood lipids, blood pressure, waist-hip ratio, and body mass index), and PTSD severity in veterans with PTSD. MATERIALS AND METHODS: This study was approved by the local Institutional Review Board. All participants were informed of the study risks and provided consent prior to participation. Participants (n = 13) completed a cardiopulmonary exercise test, a fasting blood draw, and the Clinician Administered PTSD Scale. Correlations between CRF and cardiometabolic health were examined with Spearman's rank correlations, and differences in PTSD symptom severity were explored as a function of CRF (ie, low-to-moderate vs. high CRF), using multiple linear regression. RESULTS: Peak oxygen uptake ($\dot{\mathrm{V}}$O2peak) was correlated with high-density lipoproteins rho = 0.60, P = 0.04 and diastolic blood pressure rho = -0.56, P = 0.05. Ventilatory threshold was correlated with HbA1c rho = -0.61, P = 0.03 and diastolic blood pressure rho = -0.56, P = 0.05. Higher CRF was associated with lower total PTSD severity standardized ß = -0.84, P = 0.01, adjusted R2 = 0.47, total Cluster C symptoms (avoidance/numbing) ß = -0.71, P = 0.02, adjusted R2 = 0.49, and total Cluster D symptoms (hyperarousal) ß = -0.89, P = 0.01, adjusted R2 = 0.41, while adjusting for age and smoking status. CONCLUSIONS: These preliminary findings suggest that CRF and by proxy physical activity may be important factors in understanding the increased risk of cardiovascular and metabolic disease associated with PTSD.

Better defining best-aided condition: The role of hearing aids on cochlear implantation qualification rates.

OBJECTIVE: Describe the effect of hearing aid type used during cochlear implantation evaluation on qualification rates. METHODS: Consecutive adult patients at an academic cochlear implant program undergoing cochlear implantation evaluation were identified to determine cochlear implantation qualification rate according to history of hearing aid use and type of hearing aid used during evaluation. RESULTS: 609 patients met criteria. 90.1% of patients reported prior use of a hearing aid, and 77.4% reported current use of a hearing aid. Patients were most likely to undergo cochlear implantation evaluation utilizing their own personal hearing aids exclusively (61.6%) followed by loaner hearing aids fitted at the time of the evaluation (28.2%). White patients were more likely to be tested using personal hearing aids (OR = 2.60, 95% CI 1.43 to 4.71). Married patients were more likely to be current hearing aid users (OR 1.62, 95% CI 1.04 to 2.51) and were more likely to be tested using personal hearing aids (OR = 1.68, 95% CI 1.10 to 2.56). Patients with a history of any hearing aid use (OR = 2.50, 95% CI 1.42 to 4.40) and current hearing aid use (OR = 1.62, 95% CI 1.06 to 2.49) were more likely to qualify for cochlear implantation. Patients tested using personal hearing aids were 1.5 times more likely to qualify for cochlear implantation (95% CI 0.99 to 2.27). CONCLUSION: History of hearing amplification and current amplification predict cochlear implant qualification. Hearing aids fitted at the time of cochlear implantation evaluation may result in lower qualification rates.

Notch Signaling in Vascular Smooth Muscle Cells.

The Notch signaling pathway is a highly conserved pathway involved in cell fate determination in embryonic development and also functions in the regulation of physiological processes in several systems. It plays an especially important role in vascular development and physiology by influencing angiogenesis, vessel patterning, arterial/venous specification, and vascular smooth muscle biology. Aberrant or dysregulated Notch signaling is the cause of or a contributing factor to many vascular disorders, including inherited vascular diseases, such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, associated with degeneration of the smooth muscle layer in cerebral arteries. Like most signaling pathways, the Notch signaling axis is influenced by complex interactions with mediators of other signaling pathways. This complexity is also compounded by different members of the Notch family having both overlapping and unique functions. Thus, it is vital to fully understand the roles and interactions of each Notch family member in order to effectively and specifically target their exact contributions to vascular disease. In this chapter, we will review the Notch signaling pathway in vascular smooth muscle cells as it relates to vascular development and human disease.

Identification of a CArG box-dependent enhancer within the cysteine-rich protein 1 gene that directs expression in arterial but not venous or visceral smooth muscle cells.

Smooth muscle cells (SMCs) are heterogeneous with respect to their contractile, synthetic, and proliferative properties, though the regulatory factors responsible for their phenotypic diversity remain largely unknown. To further our understanding of smooth muscle gene regulation, we characterized the cis-regulatory elements of the murine cysteine-rich protein 1 gene (CRP1/Csrp1). CRP1 is expressed in all muscle cell types during embryogenesis and predominates in vascular and visceral SMCs in the adult. We identified a 5-kb enhancer within the CRP1 gene that is sufficient to drive expression in arterial but not venous or visceral SMCs in transgenic mice. This enhancer also exhibits region-specific activity in the outflow tract of the heart and the somites. Within the 5-kb CRP1 enhancer, we found a single CArG box that binds serum response factor (SRF), and by mutational analysis, demonstrate that the activity of the enhancer is dependent on this CArG element. Our findings provide further evidence for the existence of distinct regulatory programs within SMCs and suggest a role for SRF in the activation of the CRP1 gene.

The LIM homeodomain protein dLim1 defines a subclass of neurons within the embryonic ventral nerve cord of Drosophila.

Members of the LIM homeodomain family of transcription factors have been implicated in specifying cell identity in a range of species. In Drosophila three LIM homeobox genes, apterous, lim3 and isl, have been shown to control axon pathfinding of subsets of neurons within the embryo. Here we describe the isolation and characterization of another LIM homeobox gene in Drosophila termed dlim1, a homolog of the vertebrate Lim-1 gene. The sequence and expression of dLim1 is highly related to its vertebrate homologs. Within the Drosophila embryo, dLim1 is expressed in the head primordia, the brain lobes, and in distinct sets of motorneurons and interneurons within the ventral nerve cord. Comparatively in vertebrates, Lim-1 (Lhx1) along with Lim-3 (Lhx3), Gsh-4 (Lhx4), Isl-1 and Isl-2 are expressed in developing motorneurons along the spinal column, where their overlapping expression suggests a role for these genes in the establishment of specific motorneuron subtypes. dLim1 is absent from all cells expressing Isl, Lim3, and Apterous, indicating that these proteins function independently within the Drosophila embryo. To investigate the function of dlim1, we generated loss-of-function mutations within the locus. Our findings show that dlim1 is an essential gene that when mutated results in lethality near the larval-pupal boundary. In contrast to vertebrate Lim-1, dlim1 has no apparent role in anterior patterning of the Drosophila embryo. Our analysis shows that dlim1 has been evolutionarily conserved, however the Drosophila lim1 gene exhibits unique properties that distinguishes it from its vertebrate homologs.

Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis.

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.

Independent regulatory elements in the upstream region of the Drosophila beta 3 tubulin gene (beta Tub60D) guide expression in the dorsal vessel and the somatic muscles.

The beta 3 tubulin gene (beta Tub60D) is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm while enhancers localized in the first intron guide expression in the visceral mesoderm. Deletion analysis carried out in transgenic flies revealed independent regulatory elements for the dorsal vessel and the somatic mesoderm. For expression in the somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for the Drosophila myocyte-specific enhancer binding factor 2 (D-MEF2), a MADS-box transcription factor known to be essential for mesoderm development. Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of beta 3 tubulin in D-mef2 mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearby cis regulatory elements. These data show that the upstream control region of the beta 3 tubulin gene is an early target of the D-MEF2 transcriptional activator.

A novel A/T-rich element mediates ANF gene expression during cardiac myocyte hypertrophy.

The induction of the atrial natriuretic factor (ANF) gene during alpha 1-adrenergic stimulation of neonatal rat ventricular myocytes has served as a model for gene expression during cardiac muscle cell hypertrophy. This study describes and identifies a single regulatory element that mediates expression of the ANF gene. Deletional mutations were generated in a 639-bp fragment of the ANF promoter that confers alpha 1-adrenergic inducibility to a luciferase reporter gene in transient transfection assays in ventricular myocytes. The results of gel mobility shift and diethylpyrocarbonate (DEPC) interference studies with nuclear cardiac cell extracts identified the nucleotide contract points for a novel A/T-rich element (ANF-AT) at positions -582/-575 that partially mediates alpha 1-adrenergic inducibility. Mutations in the ANF-AT element reduced alpha-adrenergic inducibility of an ANF-TK-luciferase fusion gene in cardiac cells by 35% but had no effect on expression in other muscle and non-muscle cells tested. Gel mobility supershift assays with antibodies directed against the MEF-2 protein, the homeobox protein MHox, or the zinc finger protein HF-1b, document that these factors are not major components of the endogenous ANF-AT binding activity in cardiac muscle cells. The current study provides evidence for a role for a novel A/T-rich element in the regulation of ANF gene expression in cardiac ventricular myocytes.

Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila.

Members of the myocyte enhancer binding factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, and serum response factor) box transcription factors are expressed in the skeletal, cardiac, and smooth muscle lineages of vertebrate and Drosophila embryos. These factors bind an adenine-thymidine-rich DNA sequence associated with muscle-specific genes. The function of MEF2 was determined by generating a loss-of-function of the single mef2 gene in Drosophila (D-mef2). In loss-of-function embryos, somatic, cardiac, and visceral muscle cells did not differentiate, but myoblasts were normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2.

D-MEF2: a MADS box transcription factor expressed in differentiating mesoderm and muscle cell lineages during Drosophila embryogenesis.

The myocyte enhancer factor (MEF) 2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates. We have cloned a protein from Drosophila, termed D-MEF2, that shares extensive amino acid homology with the MADS (MCM1, Agamous, Deficiens, and serum-response factor) domains of the vertebrate MEF2 proteins. D-mef2 gene expression is first detected during Drosophila embryogenesis within mesodermal precursor cells prior to specification of the somatic and visceral muscle lineages. Expression of D-mef2 is dependent on the mesodermal determinants twist and snail but independent of the homeobox-containing gene tinman, which is required for visceral muscle and heart development. D-mef2 expression precedes that of the MyoD homologue, nautilus, and, in contrast to nautilus, D-mef2 appears to be expressed in all somatic and visceral muscle cell precursors. Its temporal and spatial expression patterns suggest that D-mef2 may play an important role in commitment of mesoderm to myogenic lineages.

Homeodomain protein MHox and MADS protein myocyte enhancer-binding factor-2 converge on a common element in the muscle creatine kinase enhancer.

MHox is a mesoderm-specific homeodomain protein that binds an A/T-rich element that is essential for activity of the muscle creatine kinase (MCK) enhancer. The MHox binding site also binds the ubiquitous homeodomain protein Oct-1 as well as myocyte enhancer-binding factor-2 (MEF2), which belongs to the MADS superfamily of transactivators. To determine which of these proteins activates MCK transcription through the A/T element, we mutated this sequence such that it would selectively bind MHox, MEF2, or Oct-1 and tested the activities of the mutant enhancers in skeletal muscle cells. These mutant enhancers revealed that only MEF2 is able to activate the MCK enhancer through the A/T element. The convergence of homeodomain and MADS proteins on the A/T element in the MCK enhancer provides a mechanism through which a single DNA sequence can mediate positive and negative regulation of gene transcription and is reminiscent of the roles of these two classes of transcription factors in the control of other cell-specific genes.

The expression pattern of the chick homeobox gene gMHox suggests a role in patterning of the limbs and face and in compartmentalization of somites.

MHox is a homeodomain protein that binds an essential element in the core of the muscle creatine kinase enhancer. In the mouse embryo, MHox expression is restricted to mesenchymal cells; in adult mice the gene is highly expressed in skeletal and cardiac muscle. To further define the functions of MHox during embryogenesis, we have cloned its chicken homolog, termed gMHox, and analyzed its properties and detailed expression patterns. Our studies show that the amino acid sequence and DNA-binding properties of the avian and murine gene products are very similar. Furthermore, the sites of expression are alike with high levels of expression in the splanchnic mesoderm, in the somatic mesoderm, in the limb bud mesoderm, in the dermatome and in the dermis, and in the ectomesenchyme of the face. gMHox became downregulated as chondrogenesis proceeded, whereas its expression was maintained in perichondrium and undifferentiated mesenchymal cells beneath the surface ectoderm. Such a pattern of expression suggests that gMHox may participate in maintenance of mesenchymal cell lineages derived from both mesoderm and the neural crest and in patterning of the limbs and the face. Removal of the surface ectoderm overlying the somites has no visible effect on the architecture of somites but results in the failure of gMHox to be expressed in the underlying dermatome, suggesting that regulation of gMHox expression in these cells is dependent on cues emanating from the surface ectoderm.

MHox: a mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer.

Myogenic helix-loop-helix (HLH) proteins, such as myogenin and MyoD, can activate muscle-specific transcription when introduced into a variety of nonmuscle cell types. Whereas cells of mesodermal origin are especially permissive to the actions of these myogenic regulators, many other cell types are refractory to myogenic conversion by them. Here we describe a novel homeodomain protein, MHox, that binds an A+T-rich element in the muscle creatine kinase (MCK) enhancer that is essential for muscle-specific transcription and trans-activation by myogenic HLH proteins. MHox is completely restricted to mesodermally derived cell types during embryogenesis and to established cell lines of mesodermal origin. In contrast to most other homeobox genes, MHox expression is excluded from the nervous system, with the highest levels observed in limb bud and visceral arches. In adult mice, MHox is expressed at high levels in skeletal muscle, heart and uterus. The DNA-binding properties and pattern of MHox expression are unique among homeobox genes and suggest a role for MHox as a transcriptional regulator that participates in the establishment of diverse mesodermal cell types.

Carbonic anhydrase II gene expression in cell lines from human pancreatic adenocarcinoma.

Current evidence suggests that carbonic anhydrase II (CA II) is produced by pancreatic duct cells but not by pancreatic acinar or islet cells. The aim of this study was to determine whether CA II homologous RNA and CA II immunoreactive protein are produced by cell lines established from human pancreatic adenocarcinomas. A 1.7-Kb CA II homologous RNA was detected in poly(A+) RNA isolated from normal human pancreas, normal human liver, and to varying degrees in the cell lines examined. The CA II immunoreactivity corresponding to approximately 30 kD (consistent with the established molecular mass of CA II) was also detected by immunoblotting in normal human pancreas, normal human liver, and some of the cell lines. We also found that the levels of CA II homologous RNA increase in the pancreatic adenocarcinoma cell lines following treatment with the differentiating agent, retinoic acid.