Failure to control oxidative stress is closely related to aging and to a diverse range of human diseases. We have reported that protein kinase C γ (PKCγ) acts as a primary oxidative stress sensor in the lens. PKCγ has a Zn-finger C1B stress switch domain, residues 101-150. Mutation, H101Y, in the C1B domain of PKCγ proteins causes a failure of the PKCγ oxidative stress response (Lin and Takemoto (2005) (100 μM, 3 h) activated a caspase-3 apoptotic pathway in the lens epithelial cells but was more severe in cells expressing PKCγ mutations. The presence of 18α-glycyrrhetinic acid (AGA), an inhibitor of gap junctions, decreased Cx43 and Cx50 protein levels and gap junction plaque number. This reduction in gap junctions by AGA resulted in inhibition of H 2 O 2 -induced apoptosis. Our results demonstrate that there is a dominant negative effect of PKCγ C1B mutations on endogenous PKCγ which results in loss of control of gap junctions. Modeled structures suggest that the severity of C1B mutation effects may be related to extent of loss of C1B structure. Mutations in the C1B domain of PKCγ result in increased apoptosis in lens epithelial cells. This can be prevented by a gap junction inhibitor. Thus, propagation of apoptosis from cellto-cell in lens epithelial cells may be through open gap junctions. The control of gap junctions requires PKCγ.

SUMMARY Cataracts, or lens opacities, are the leading cause of blindness worldwide. Cataracts increase with age and environmental insults, e.g. oxidative stress. Lens homeostasis depends on functional gap junctions. Knockout or missense mutations of lens gap junction proteins, Cx46 or Cx50, result in cataractogenesis in mice. We have previously demonstrated that protein kinase Cγ (PKCγ) regulates gap junctions in the lens epithelium and cortex. In the current study, we further determined whether PKCγ control of gap junctions protects the lens from cataractogenesis induced by oxidative stress in vitro, using PKCγ knockout and control mice as our models. The results demonstrate that PKCγ knockout lenses are normal at 2 days post-natal when compared to control. However, cell damage, but not obvious cataract, was observed in the lenses of 6-week-old PKCγ knockout mice,suggesting that the deletion of PKCγ causes lenses to be more susceptible to damage. Furthermore, in vitro incubation or lens oxidative stress treatment by H2O2 significantly induced lens opacification (cataract) in the PKCγ knockout mice when compared to controls. Biochemical and structural results also demonstrated that H2O2 activation of endogenous PKCγ resulted in phosphorylation of Cx50 and subsequent inhibition of gap junctions in the lenses of control mice, but not in the knockout. Deletion of PKCγaltered the arrangement of gap junctions on the cortical fiber cell surface,and completely abolished the inhibitory effect of H2O2on lens gap junctions. Data suggest that activation of PKCγ is an important mechanism regulating the closure of the communicating pathway mediated by gap junction channels in lens fiber cells. The absence of this regulatory mechanism in the PKCγ knockout mice may cause those lenses to have increased susceptibility to oxidative damage.

The region (101-112) of C1B domain in PKC gamma plays a crucial role in the activation of the enzyme and subsequent gap junction inhibition. Substitution studies on peptides correlating to the C1B region show that a flexible structure and ability to be phosphorylated on serine 109 are critical for this purpose.

BACKGROUND Urinary tract infection (UTI) symptoms are common in primary care, but antibiotics are appropriate only when an infection is present. Urine culture is the reference standard test for infection, but results take >1 day. A machine learning predictor of urine cultures showed high accuracy for an emergency department (ED) population but required urine microscopy features that are not routinely available in primary care (the NeedMicro classifier). METHODSWe redesigned a classifier (NoMicro) that does not depend on urine microscopy and retrospectively validated it internally (ED data set) and externally (on a newly curated primary care [PC] data set) using a multicenter approach including 80,387 (ED) and 472 (PC) adults. We constructed machine learning models using extreme gradient boosting (XGBoost), artificial neural networks, and random forests (RFs). The primary outcome was pathogenic urine culture growing ≥100,000 colony forming units. Predictor variables included age; gender; dipstick urinalysis nitrites, leukocytes, clarity, glucose, protein, and blood; dysuria; abdominal pain; and history of UTI. RESULTSRemoval of microscopy features did not severely compromise performance under internal validation: NoMicro/XGBoost receiver operating characteristic area under the curve (ROC-AUC) 0.86 (95% CI, 0.86-0.87) vs NeedMicro 0.88 (95% CI, 0.87-0.88). Excellent performance in external (PC) validation was also observed: NoMicro/RF ROC-AUC 0.85 (95% CI, 0.81-0.89). Retrospective simulation suggested that NoMicro/RF can be used to safely withhold antibiotics for low-risk patients, thereby avoiding antibiotic overuse. CONCLUSIONSThe NoMicro classifier appears appropriate for PC. Prospective trials to adjudicate the balance of benefits and harms of using the NoMicro classifier are appropriate.

Introduction Implementing a health system-based hypertension programme may lower blood pressure (BP). Methods We performed a randomized, controlled pilot study to assess feasibility, acceptability, and safety of a home-based virtual hypertension programme integrating evidence-based strategies to overcome current barriers to BP control. Trained clinical pharmacists staffed the virtual collaborative care clinic (vCCC) to remotely manage hypertension using a BP dashboard and phone “visits” to monitor BP, adherence, side effects of medications, and prescribe anti-hypertensives. Patients with uncontrolled hypertension were identified via electronic health records. Enrolled patients were randomized to either vCCC or usual care for 3 months. We assessed patients’ home BP monitoring behaviour, and patients’, physicians’, and pharmacists’ perspectives on feasibility and acceptability of individual programme components. Results Thirty-one patients (vCCC = 17, usual care = 14) from six physician clinics completed the pilot study. After 3 months, average BP decreased in the vCCC arm (P = 0.01), but not in the control arm (P = 0.45). The vCCC participants measured BP more (9.9 vs. 1.2 per week, P < 0.001). There were no intervention-related adverse events. Participating physicians (n = 6), pharmacists (n = 5), and patients (n = 31) rated all programme components with average scores of >4.0, a pre-specified benchmark. Nine adaptations in vCCC design and delivery were made based on potential barriers to implementing the programme and suggestions. Conclusion A home-based virtual hypertension programme using team-based care, technology, and a logical integration of evidence-based strategies is safe, acceptable, and feasible to intended users. These pilot data support studies to assess the effectiveness of this programme at a larger scale.