Structural Basis of the Activation of Heterotrimeric Gs-Protein by Isoproterenol-Bound ß1-Adrenergic Receptor.

Cardiac disease remains the leading cause of morbidity and mortality worldwide. The ß1-adrenergic receptor (ß1-AR) is a major regulator of cardiac functions and is downregulated in the majority of heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by ß1-ARs, leading to increased heart rate and contractility. Here, we use cryo-electron microscopy and functional studies to investigate the molecular mechanism by which ß1-AR activates Gs. We find that the tilting of α5-helix breaks a hydrogen bond between the sidechain of His373 in the C-terminal α5-helix and the backbone carbonyl of Arg38 in the N-terminal αN-helix of Gαs. Together with the disruption of another interacting network involving Gln59 in the α1-helix, Ala352 in the ß6-α5 loop, and Thr355 in the α5-helix, these conformational changes might lead to the deformation of the GDP-binding pocket. Our data provide molecular insights into the activation of G-proteins by G-protein-coupled receptors.

Risk of liver fibrosis associated with long-term methotrexate therapy may be overestimated.

BACKGROUND & AIMS: The risk of significant liver fibrosis from prolonged methotrexate (MTX) exposure has been estimated at around 5%, prompting intensive monitoring strategies. However, the evidence is derived from retrospective studies that under-reported risk factors for liver disease. We evaluated the risk of long-term MTX therapy on liver fibrosis in a longitudinal cohort study using two non-invasive markers. METHOD: Between 2014-2021, adult patients diagnosed with rheumatoid arthritis (RA) or psoriasis for ≥2 years were recruited prospectively from six UK sites. The MTX group included patients who received MTX for ≥6 months, whereas the unexposed group included those who never received MTX. All patients underwent full liver profiling, with transient elastography (TE) and enhanced liver fibrosis (ELF) marker measurements. RESULTS: A total of 999 patients (mean age 60.8 ± 12 years, 62.3% females) were included. Of 976 with valid TE values, 149 (15.3%) had liver stiffness ≥7.9 kPa. Of 892 with a valid ELF, 262 (29.4%) had ELF ≥9.8. Age and BMI were independently associated with elevated liver stiffness and ELF. Neither MTX cumulative dose nor duration was associated with elevated liver stiffness. Diabetes was the most significant risk factor associated with liver stiffness ≥7.9 kPa (adjusted odds ratio = 3.19; 95% CI 1.95-5.20; p <0.001). Regular use of non-steroidal anti-inflammatory drugs showed the strongest association with ELF ≥9.8 (odds ratio = 1.76; 95% CI 1.20-2.56; p = 0.003), suggesting the degree of joint inflammation in RA may confound ELF as a non-invasive marker of liver fibrosis. CONCLUSION: The risk of liver fibrosis attributed to MTX itself might have been previously overestimated; there is a need to consider modifying current monitoring guidelines for MTX. IMPACT AND IMPLICATIONS: Current guidelines recommend intensive (2-3 monthly) monitoring strategies for patients on long-term methotrexate therapy due to the potential risk of liver fibrosis. Evaluation of the association using two validated non-invasive markers of liver fibrosis, liver stiffness and enhanced liver fibrosis score, in a large cohort of patients with rheumatoid arthritis or psoriasis shows that the reported risk has previously been overestimated. The clinical focus should be to improve patients' metabolic risk factors, diabetes and BMI, that are independently associated with liver stiffness. There is a need to consider modifying current treatment monitoring guidelines for methotrexate.

Expression of sterile-α and armadillo motif containing protein (SARM) in rheumatoid arthritis monocytes correlates with TLR2-induced IL-1ß and disease activity.

OBJECTIVE: Cartilage and bone damage in RA are associated with elevated IL-1ß. The effects of IL-1ß can be reduced by biological therapies that target IL-1ß or TNF-α. However, the mechanisms responsible for increased IL-1ß and the effect of anti-TNF-α have not been fully elucidated. Recently, sterile-α and armadillo motif containing protein (SARM) was identified as a negative regulator of toll-like receptor (TLR) induced IL-1ß secretion through an interaction with the inflammasome. This study set out to investigate SARM during TLR-induced IL-1ß secretion in RA peripheral blood monocytes and in patients commencing anti-TNF-α treatment. METHODS: Monocytes were isolated from RA patients and healthy controls; disease activity was measured by DAS28. IL-1ß secretion was measured by ELISA following TLR1/2, TLR4 and TLR7/8 stimulation. The mRNA expression of SARM1, IL-1ß and the components of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome were measured by quantitative PCR. SARM protein expression was measured by western blotting. RESULTS: TLR1/2 activation induced elevated IL-1ß in RA monocytes compared with healthy controls (P = 0.0009), which negatively correlated with SARM1 expression (P = 0.0086). Lower SARM expression also correlated with higher disease activity (P = 0.0246). Additionally, patients responding to anti-TNF-α treatment demonstrated a rapid upregulation of SARM, which was not observed in non-responders. CONCLUSION: Together, these data highlight a potential contribution from SARM to RA pathophysiology where decreased SARM may lead to elevated IL-1ß associated with RA pathogenesis. Furthermore, the data additionally present a potential mechanism by which TNF-α blockade can modify IL-1ß secretion.

Reducing cryoEM file storage using lossy image formats.

Recent advances in instrumentation and software for cryoEM have increased the applicability and utility of this method. High levels of automation and faster data acquisition rates require hard decisions to be made regarding data retention. Here we investigate the efficacy of data compression applied to aligned summed movie files. Surprisingly, these images can be compressed using a standard lossy method that reduces file storage by 90-95% and yet can still be processed to provide sub-2 Šreconstructed maps. We do not advocate this as an archival method, but it may provide a useful means for retaining images as an historical record, especially at large facilities.

Effects of time-of-day on inhibition of lens-induced myopia by quinpirole, pirenzepine and atropine in chicks.

Injections of the D2 dopamine receptor agonist quinpirole or the acetylcholine muscarinic receptor antagonists pirenzepine and atropine prevent the development of negative-lens-induced myopia in chicks by inhibiting ocular growth. Because ocular growth is diurnally rhythmic, we hypothesized that the efficacy for inhibition may depend on time of day. Chicks wore monocular -10D lenses for 5 days, starting at 12d of age. The light cycle was 12L/12D. The lens-wearing eye received daily intravitreal injections for 4 days, of 20 µl quinpirole (10 nmol), at the following times: 7:30 EST (lights-on; morning; n = 12), 12:00 (mid-day; n = 13), or 19:30 (evening; n = 17). The same protocol was used for pirenzepine (0.2 µmol) and atropine (18 nmol), at the following times: 8:30 EDT (lights-on; n = 10; n = 18), 14:00 (n = 10; n = 12), or 20:30 (n = 18; n = 16). Saline injections were done in separate groups of birds for all groups as controls, and the data combined (n = 28). Ocular dimensions were measured using A-scan ultrasonography on treatment day 1 at 12:00, and again on day 5 at 12:00; growth rate is defined as the change in axial length over 96 h. For quinpirole and pirenzepine, subsets (n's in Methods) of mid-day and evening groups were measured at 6 h intervals on day 5 (from 12:00 to 12:00) to obtain rhythm parameters for axial length and choroidal thickness; for atropine, only the mid-day group was measured. Refractions were measured on day 5 with a Hartinger's refractometer. For quinpirole and pirenzepine, mid-day injections were more effective at inhibiting ocular growth than evening (Exp-fellow: quinpirole: -68 vs 118 µm/96h; post-hoc Bonferroni p = 0.016; pirenzepine: 79 vs 215 µm/96h; p = 0.046). There were no between-group statistically significant differences for atropine. For quinpirole, the mid-day amplitude of the axial rhythm was smaller than for evening (95 vs 142 µm; p < 0.05), but there were no time-dependent effects on the rhythms for pirenzepine. For atropine, the amplitude of the axial-length rhythm was significantly larger than that for pirenzepine at mid-day. We conclude that there is a phase-dependent efficacy for quinpirole and pirenzepine, with mid-day injections being most effective. There were no consistent time-dependent alterations in rhythm parameters for any of the drugs.

High resolution single particle cryo-electron microscopy using beam-image shift.

Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (±0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.

Brief hyperopic defocus or form deprivation have varying effects on eye growth and ocular rhythms depending on the time-of-day of exposure.

It is generally accepted that myopic defocus is a more potent signal to the emmetropization system than hyperopic defocus: one hour per day of myopic defocus cancels out 11 h of hyperopic defocus. However, we have recently shown that the potency of brief episodes of myopic defocus at inhibiting eye growth depends on the time of day of exposure. We here ask if this will also be true of the responses to brief periods of hyperopic defocus: may integration of the signal depend on time of day? If so, are the rhythms in axial length and choroidal thickness altered? Hyperopic defocus: Birds had one eye exposed to hyperopic defocus by the wearing of -10D lenses for 2 or 6 h at one of 3 times of day for 5 days: Morning (7 am - 9 am: n = 13; 7 am - 1 pm: n = 6), Mid-day (12 pm - 2 pm: n = 20; 10 am - 4 pm: n = 8), or Evening (7 pm - 9 pm: n = 12; 2 pm - 8 pm: n = 11). A separate group wore monocular lenses continually as a control (n = 12). Form deprivation: Birds wore a diffuser over one eye for 2 h at one of 3 times of day for 5 days: Morning (n = 12); Mid-day (n = 19) or Evening (n = 6). For all groups, ocular dimensions were measured using high-frequency A-scan ultrasonography at noon on the first day, under inhalation anesthesia. On day 5, eye dimensions were re-measured at 12 pm, and refractive errors were measured using a Hartinger's refractometer. A subset of birds in the 2-h lens group (morning, n = 8; mid-day, n = 8; evening, n = 6), and the deprivation group (n = 6 per time point), were also measured at 6 pm, 12 am, 6 am and 12 pm on the last day of exposure, to obtain the parameters of the diurnal rhythms in axial length and choroidal thickness. The effects of 2 h of defocus depended on time of day of exposure: it stimulated eye growth when exposure was in the morning and inhibited it when it was at mid-day (change in vitreous chamber, X-C; ANOVA p < 0.0005; 120 µm vs -77 µm/5d, respectively; t-tests: p = 0.001; p = 0.01; post-hoc tests: p = 0.002). For mid-day, experimental eyes were more hyperopic (1.4 D; p < 0.0001). Similar to 2 h defocus, 6 h exposures at mid-day inhibited growth and produced hyperopia (X-C: -167 µm; t-test p = 0.005; RE: 1.8 D; p = 0.03). The effects of 2 h of FD were similar to those of hyperopic defocus in inhibiting growth for mid-day exposures, but FD inhibited growth for the morning exposures as well (Axial length: X-C: Morning: -122 µm; mid-day: -92 µm; ttests p = 0.006 and p = 0.016 respectively). Experimental eyes were more hyperopic (1.8 D; 1.0 D; p < 0.05). The rhythms in axial length were altered for the morning exposures in both conditions. Form deprivation in the morning, which caused inhibition, caused the phases of the two rhythms to shift toward one another (peaks at 6:00 am and 10:45 am for choroid and axial length respectively). Our findings imply that the retinal "integrator", and/or scleral growth regulator exhibit diurnal rhythms. Furthermore, they suggest that reading activities early in the day may be contraindicated in school children at risk of becoming myopic.

Eicosapentaenoic Acid Modulates Trichomonas vaginalis Activity.

Trichomonas vaginalis is a sexually transmitted parasite and, while it is often asymptomatic in males, the parasite is associated with disease in both sexes. Metronidazole is an effective treatment for trichomoniasis, but resistant strains have evolved and, thus, it has become necessary to investigate other possible therapies. In this study, we examined the effects of native and oxidized forms of the sodium salts of eicosapentaenoic, docosahexaenoic, and arachidonic acids on T. vaginalis activity. Eicosapentaenoic acid was the most toxic with 190 and 380 µM causing approximately 90% cell death in Casu2 and ATCC 50142 strains, respectively. In contrast, oxidized eicosapentaenoic acid was the least toxic, requiring > 3 mM to inhibit activity, while low levels (10 µM) were associated with increased parasite density. Mass spectrometric analysis of oxidized eicosapentaenoic acid revealed C20 products containing one to six additional oxygen atoms and various degrees of bond saturation. These results indicate that eicosapentaenoic acid has different effects on T. vaginalis survival, depending on whether it is present in the native or oxidized form. A better understanding of lipid metabolism in T. vaginalis may facilitate the design of synthetic fatty acids that are effective for the treatment of metronidazole-resistant T. vaginalis.

Factors Related to Human Papillomavirus Positivity among Oral Cavity and Pharynx Cancers from Surveillance, Epidemiology and End Results (SEER) Program Data.

BACKGROUND: As human papillomavirus positive (HPV+) oral cavity and pharynx cancer (OCPC) incidence increases significantly, our objective was to determine whether selected sociodemographic and clinical factors were associated with HPV+ OCPCs overall and by oropharyngeal and non-oropharyngeal sites. METHODS: Surveillance, Epidemiology and End Results (SEER) Program data were used in this study. Specifically, univariate and logistic regression models were used to examine the relationships between HPV+ and HPV- OCPC cases and age, sex, race, ethnicity, marital status, factors of neighborhood socioeconomic status (i.e., nSES/Yost index) and rurality/urbanity, first malignancy status, histology, reporting source, stage at diagnosis, and OCPC anatomic site. The same approach was used to identify risk factors for HPV positivity for oropharyngeal and non-oropharyngeal OCPCs separately. RESULTS: In all OCPCs, cases that were male, <80 years old, lived in the four highest nSES categories, diagnosed with a non-"gum and other mouth" OCPC (ref = hypopharynx), not locally staged at diagnosis, and a first malignancy had higher odds of being HPV+. Cases that were American Indian/Alaska Native and Asian or Pacific Islander (ref = White), Spanish-Hispanic-Latino ethnicity, non-married/partnered, and not reported by a hospital/clinic had lower odds of being HPV+. Associations were maintained in oropharyngeal OCPCs and only age and race remained significant for non-oropharyngeal OCPCs. CONCLUSIONS: Sociodemographic and clinical differences in HPV+ and HPV- OCPC, overall and for (non)oropharyngeal, cases exist. IMPACT: Identification of OCPC and (non)oropharyngeal risk factors for HPV positivity may assist in discovering high-risk groups that should receive enhanced public health efforts to reduce the U.S. OCPC burden.

Up-to-date management of gout.

PURPOSE OF REVIEW: Gout is a true crystal deposition disease, extremely painful and bone and tissue damaging if untreated. It is the only curable form of arthritis. Although we have many treatments to cure gout, it is a disease that is consistently undertreated/mismanaged and perceived by clinicians and the lay public as a 'laughable condition' with the patients' lifestyle often held erroneously to account. This article would give you a good understanding of modern and established pharmacological and nonpharmacological treatments used in the management of acute and chronic gout and how to 'treat to target' to cure the disease. RECENT FINDINGS: Many of the drugs we use to manage patients with gouty arthritis have been in existence since the 1970s and 1980s. In the past few years, because of the improved physiological understanding of gout, new innovative treatments such as anti-IL inhibitors, a nonxanthine oxidase inhibitor and the uricase enzymes have been developed adding to our armamentarium of drugs. SUMMARY: With the introduction of new research, we have been able to explore how to also use established treatments more effectively, raising the profile of gout and its best management and introducing the principle of treating the patient to urate target.

Distinct sociodemographic differences in incidence and survival rates for human papillomavirus (HPV)-like, non-HPV-like, and "other"-like oral cavity and pharynx cancers: An analysis of Surveillance, Epidemiology and End Results (SEER) Program data.

Purpose: Oral cavity and pharynx cancer (OCPC) cases are traditionally dichotomized into human papillomavirus (HPV) and non-HPV types. Using a proxy for HPV status, the objective was to evaluate differences in incidence and survival rates of OCPC anatomic sub-sites identified as: 1) HPV-like; 2) non-HPV-like (i.e., tobacco/alcohol-related); and 3) "other"-like (i.e., not predominantly HPV-like nor tobacco/alcohol-like) OCPCs. Methods: Data from the Surveillance, Epidemiology and End Results Program were used to examine incidence and survival rates for OCPC categories over time and according to age, sex, race, ethnicity, stage at diagnosis, neighborhood socioeconomic status (i.e., nSES or Yost Index), and rurality/urbanity (i.e., Rural Urban Commuting Area (RUCA) codes). Although HPV status was unavailable in this dataset, OCPC anatomies and histologies were classified into three sub-categories, based on potential risk factors. Frequencies, average annual, age-adjusted incidence rates, five-year relative survival rates, and 95% confidence intervals were examined across and within OCPC categories. Results: HPV-like OCPC incidence rates sharply increased from 1975 through 2015 while non-HPV-like and "other"-like OCPC rates decreased, all converging to similar rates from 2016 through 2018. Increasing over time for both categories, survival was highest for HPV-like and lowest for non-HPV-like OCPCs; survival for "other"-like OCPCs remained stable. Generally, across OCPC categories, incidence and survival rates were significantly higher among males vs. females, Whites vs. African Americans, and non-Hispanics vs. Hispanics. "Other"-like OCPC incidence decreased with increasing nSES tertiles, while no nSES differences were observed for HPV-like and non-HPV-like OCPCs. Incidence rates were significantly lower among urban (vs. rural) residents. For all OCPC categories, survival rates were significantly higher with increasing nSES and variable across RUCA categories. Conclusion/Impact: HPV-like and non-HPV-like OCPC cases had distinct sociodemographic differences; "other"-like OCPC cases were a sociodemographic blend of HPV-like and non-HPV-like OCPC cases, resembling more of the sociodemographic makeup of non-HPV-like OCPC cases. To prevent new OCPCs, additional studies are needed to epidemiologically and clinically differentiate between OCPC categories so that high-risk groups can be better targeted in future public health interventions.

Low Rates of Dual-Site and Concordant Oral-Cervical Human Papillomavirus Infections and Cancers: A Systematic Review.

Objective: The oral-cervical human papillomavirus (HPV) infection/cancer relationship is not well established. Oral-cervical HPV studies were reviewed to assess dual-site occurrence, HPV type concordance, and study quality/deficiencies. Methods: PubMed, EMBASE, Ovid Medline, and Web of Science were searched between 1/1/1990 and 8/10/2021 for studies investigating HPV infections/cancers and type concordance between the oral cavity/oropharynx and cervix. Dual-site and concordant HPV infection rates were summarized as percentages; cancer diagnoses studies were summarized using standardized incidence ratios (SIR). The Quality Assessment Tool for Quantitative Studies (QATQS) evaluated study methodology. Results: One hundred fourteen papers were identified. Most were cross-sectional (n=79, 69%), involved synchronous dual-site HPV testing (n=80, 70%), did not report HPV type concordance (n=62, 54%), and achieved moderate methodological QATQS ratings (n=81, 71%). The overall dual-site infection rate averaged 16%; the HPV type concordance rate averaged 41%, among those dually-infected women. Most HPV-related cancer diagnoses studies reported increased secondary cancer risk, with SIRs generally ranging from 1.4 to 29.4 for secondary cervical cancer after primary oral cancer and from 1.4 to 6.3 for secondary oral cancer after primary cervical cancer. Conclusion/Impact: Oral-cervical HPV infections/cancers remain understudied. Future research should use stronger methodologies and HPV concordance analyses to better understand oral-cervical HPV epidemiology.

Analysis of Sex-Specific Prostanoid Production Using a Mouse Model of Selective Cyclooxygenase-2 Inhibition.

Background: Prostanoids are a family of lipid mediators formed from arachidonic acid by cyclooxygenase enzymes and serve as biomarkers of vascular function. Prostanoid production may be different in males and females indicating that different therapeutic approaches may be required during disease. Objectives: We examined sex-dependent differences in COX-related metabolites in genetically modified mice that produce a cyclooxygenase-2 (COX2) enzyme containing a tyrosine 385 to phenylalanine (Y385F) mutation. This mutation renders the COX2 enzyme unable to form a key intermediate radical required for complete arachidonic acid metabolism and provides a model of selective COX2 inhibition. Design and Methods: Mice heterozygous for the Y385F mutation in COX2 were mated to produce cohorts of wild-type, heterozygous, and COX2 mutant mice. We investigated whether the genotype distribution followed Mendelian genetics and studied whether sex-specific differences could be found in certain prostanoid levels measured in peritoneal macrophages and in urinary samples. Results: The inheritance of the COX2 mutation displayed a significant deviation with respect to Mendel's laws of genetics, with a lower-than-expected progeny of weaned COX2 mutant pups. In macrophages, prostaglandin E2 (PGE2) production following lipopolysaccharide (LPS) and interferon gamma (IFNγ) stimulation was COX2-dependent in both males and females, and data indicated that crosstalk between the nitric oxide (NO) and COX2 pathways may be sex specific. We observed significant differences in urinary PGE2 production by male and female COX2 mutant mice, with the loss of COX2 activity in male mice decreasing their ability to produce urinary PGE2. Finally, female mice across all 3 genotypes produced similar levels of urinary thromboxane (measured as 11-dehydro TxB2) at significantly higher levels than males, indicating a sex-related difference that is likely COX1-derived. Conclusions: Our findings clearly demonstrate that sex-related differences in COX-derived metabolites can be observed, and that other pathways (such as the NO pathway) are affected.

Body Mass Index and Early Childhood Caries in High Caries Risk Children: A Nested Case-Control Methodological Investigation.

Purpose: To evaluate body mass index (BMI) - early childhood caries (ECC) relationships with various BMI expressions.
Methods: Healthy eight- to 18-month children with unerupted molar(s) were conveniently sampled from Uniontown, Ala., USA, a high caries risk community (i. e., rural, poor, racial minority). Staff measured height/weight, dentists conducted oral exams, and parents completed questionnaires annually (from 2008 to 2014) for BMI, ECC (decayed, missing due to caries, filled primary tooth surfaces [dmfs] score), and sociodemographic values, respectively. Nationally recognized standard (underweight-normal-overweight-obese), crude (overweight/obese-not), and continuous BMI variables were evaluated. Logistic regressions (with restricted cubic splines) assessed BMI-ECC relationships, producing odds ratios (ORs) and 95 percent confidence intervals (95% CIs).
Results: Male and female ECC ORs supported positive and negative parabolic functions, respectively, for increasing standard BMI categories; underweight males were associated with ECC (OR=4.59; 95% CI=1.06 to 19.85). Crudely expressed, overweight/obese males and females had lower and slightly increased odds of ECC, respectively. A continuous BMI produced a similar OR across sexes, while spline models suggested nonlinearity for each.
Conclusion: BMI-ECC associations might be nonlinear; being underweight could be a male ECC risk factor. Studies should include extreme BMI values without collapsing BMI categories.

Sequential endogenous endophthalmitis, fungal keratitis, bacteremia and vertebral osteomyelitis in a person who injects drugs.

PURPOSE: To describe multiple ocular (and non-ocular) manifestations of disease that can present in a person who injects drugs (PWID). We report a case of a patient consecutively presenting across multiple visits to an ambulatory eye care clinic as the initial point of contact for endogenous endophthalmitis, fungal keratitis, bacteremia, and psoas abscess with vertebral osteomyelitis within a matter of weeks. OBSERVATIONS: A 51-year-old male with past medical history of alcohol use disorder and injection drug use was initially seen in an eye clinic three days after suffering vision loss in the left eye associated with floaters, photophobia, and eye pain. After initial workup and treatment for panuveitis, endogenous endophthalmitis was suspected. A pars plana vitrectomy was performed, and intravitreal medications were given. A pathogen was never isolated from vitreous samples. Two weeks later, the patient presented with complaints of pain, blurry vision, and foreign body sensation in his opposite (right) eye. Examination revealed a corneal ulcer later identified as a Paecliomyces fungal infection. Two weeks after this, he developed fever, chills, and right-sided flank pain radiating to his testicles. Following evaluation by the emergency department and subsequent hospitalization after bacteremia was noted, he was found to have a right-sided psoas abscess with lumbar vertebral osteomyelitis. Fluid was drained, cultured, and grew methicillin-sensitive Staphylococcus aureus (MSSA). At his last visit, his best-corrected visual acuity was 20/20 OS and 20/30 OD despite central corneal scarring. It was only after hospitalization that he affirmed recent injection drug use, despite being queried about it through the course of his infections. CONCLUSIONS AND IMPORTANCE: Injection drug use is an increasingly common concern for all healthcare providers as the opioid crisis in the United States remains widespread. This case highlights multiple potential infectious processes which may impact persons who inject drugs when seen by eye care providers. It also describes difficulties in caring for people who inject drugs who may not provide critical and timely information relating to their injection drug use and/or may delay care even when faced with potentially vision- and/or life-threatening conditions.

YAP and TAZ Promote Periosteal Osteoblast Precursor Expansion and Differentiation for Fracture Repair.

In response to bone fracture, periosteal progenitor cells proliferate, expand, and differentiate to form cartilage and bone in the fracture callus. These cellular functions require the coordinated activation of multiple transcriptional programs, and the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) regulate osteochondroprogenitor activation during endochondral bone development. However, recent observations raise important distinctions between the signaling mechanisms used to control bone morphogenesis and repair. Here, we tested the hypothesis that YAP and TAZ regulate osteochondroprogenitor activation during endochondral bone fracture healing in mice. Constitutive YAP and/or TAZ deletion from Osterix-expressing cells impaired both cartilage callus formation and subsequent mineralization. However, this could be explained either by direct defects in osteochondroprogenitor differentiation after fracture or by developmental deficiencies in the progenitor cell pool before fracture. Consistent with the second possibility, we found that developmental YAP/TAZ deletion produced long bones with impaired periosteal thickness and cellularity. Therefore, to remove the contributions of developmental history, we next generated adult onset-inducible knockout mice (using Osx-CretetOff ) in which YAP and TAZ were deleted before fracture but after normal development. Adult onset-induced YAP/TAZ deletion had no effect on cartilaginous callus formation but impaired bone formation at 14 days post-fracture (dpf). Earlier, at 4 dpf, adult onset-induced YAP/TAZ deletion impaired the proliferation and expansion of osteoblast precursor cells located in the shoulder of the callus. Further, activated periosteal cells isolated from this region at 4 dpf exhibited impaired osteogenic differentiation in vitro upon YAP/TAZ deletion. Finally, confirming the effects on osteoblast function in vivo, adult onset-induced YAP/TAZ deletion impaired bone formation in the callus shoulder at 7 dpf before the initiation of endochondral ossification. Together, these data show that YAP and TAZ promote the expansion and differentiation of periosteal osteoblast precursors to accelerate bone fracture healing. © 2020 American Society for Bone and Mineral Research (ASBMR).

Structural basis and mechanism of activation of two different families of G proteins by the same GPCR.

The ß1-adrenergic receptor (ß1-AR) can activate two families of G proteins. When coupled to Gs, ß1-AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey ß1-AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gαi and Gαs interact similarly with ß1-AR, the overall interacting modes between ß1-AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by ß1-AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4).