Spatiotemporal Investigation of Intercellular Heterogeneity via Multiple Photocaged Probes.

Intercellular heterogeneity occurs widely under both normal physiological environments and abnormal disease-causing conditions. Several attempts to couple spatiotemporal information to cell states in a microenvironment were performed to decipher the cause and effect of heterogeneity. Furthermore, spatiotemporal manipulation can be achieved with the use of photocaged/photoactivatable molecules. Here, we provide a platform to spatiotemporally analyze differential protein expression in neighboring cells by multiple photocaged probes coupled with homemade photomasks. We successfully established intercellular heterogeneity (photoactivable ROS trigger) and mapped the targets (directly ROS-affected cells) and bystanders (surrounding cells), which were further characterized by total proteomic and cysteinomic analysis. Different protein profiles were shown between bystanders and target cells in both total proteome and cysteinome. Our strategy should expand the toolkit of spatiotemporal mapping for elucidating intercellular heterogeneity.

Therapeutic Potential of Tpl2 (Tumor Progression Locus 2) Inhibition on Diabetic Vasculopathy Through the Blockage of the Inflammasome Complex.

OBJECTIVE: Diabetic retinopathy, one of retinal vasculopathy, is characterized by retinal inflammation, vascular leakage, blood-retinal barrier breakdown, and neovascularization. However, the molecular mechanisms that contribute to diabetic retinopathy progression remain unclear. Approach and Results: Tpl2 (tumor progression locus 2) is a protein kinase implicated in inflammation and pathological vascular angiogenesis. Nε-carboxymethyllysine (CML) and inflammatory cytokines levels in human sera and in several diabetic murine models were detected by ELISA, whereas liquid chromatography-tandem mass spectrometry analysis was used for whole eye tissues. The CML and p-Tpl2 expressions on the human retinal pigment epithelium (RPE) cells were determined by immunofluorescence. Intravitreal injection of pharmacological inhibitor or NA (neutralizing antibody) was used in a diabetic rat model. Retinal leukostasis, optical coherence tomography, and H&E staining were used to observe pathological features. Sera of diabetic retinopathy patients had significantly increased CML levels that positively correlated with diabetic retinopathy severity and foveal thickness. CML and p-Tpl2 expressions also significantly increased in the RPE of both T1DM and T2DM diabetes animal models. Mechanistic studies on RPE revealed that CML-induced Tpl2 activation and NADPH oxidase, and inflammasome complex activation were all effectively attenuated by Tpl2 inhibition. Tpl2 inhibition by NA also effectively reduced inflammatory/angiogenic factors, retinal leukostasis in streptozotocin-induced diabetic rats, and RPE secretion of inflammatory cytokines. The attenuated release of angiogenic factors led to inhibited vascular abnormalities in the diabetic animal model. CONCLUSIONS: The inhibition of Tpl2 can block the inflammasome signaling pathway in RPE and has potential clinical and therapeutic implications in diabetes-associated retinal microvascular dysfunction.

Approximate Mortality Risks between Hyperuricemia and Diabetes in the United States.

Aim: This study aimed to compare mortality risks across uric acid (UA) levels between non-diabetes adults and participants with diabetes and to investigate the association between hyperuricemia and mortality risks in low-risk adults. Methods: We analyzed data from adults aged >18 years without coronary heart disease and chronic kidney disease (n = 29,226) from the National Health and Nutrition Examination Survey (1999-2010) and the associated mortality data (up to December 2011). We used the Cox proportional hazards models to examine the risk of all-cause and cause-specific (cardiovascular disease (CVD) and cancer) mortality at different UA levels between adults with and without diabetes. Results: Over a median follow-up of 6.6 years, 2069 participants died (495 from CVD and 520 from cancers). In non-diabetes adults at UA ≥ 5 mg/dL, all-cause and CVD mortality risks increased across higher UA levels (p-for-trend = 0.037 and 0.058, respectively). The lowest all-cause mortality risk in participants with diabetes was at the UA level of 5-7 mg/dL. We set the non-diabetes participants with UA levels of <7 mg/dL as a reference group. Without considering the effect of glycemic control, the all-cause mortality risk in non-diabetes participants with UA levels of ≥7 mg/dL was equivalent to risk among diabetes adults with UA levels of <7 mg/dL (hazard ratio = 1.44 vs. 1.57, p = 0.49). A similar result was shown in CVD mortality risk (hazard ratio = 1.80 vs. 2.06, p = 0.56). Conclusion: Hyperuricemia may be an indicator to manage multifaceted cardiovascular risk factors in low-risk adults without diabetes, but further studies and replication are warranted.

Insulinoma in a young female patient with systemic lupus erythematosus: a case report.

OBJECTIVE: Fasting hypoglycemia may occur in subjects with systemic lupus erythematosus (SLE) when accompanied with insulin-binding antibodies or insulin-receptor antibodies. However, insulinoma has not been reported in SLE subjects with hypoglycemia. METHODS: We present a case report and review the relevant literature. RESULTS: A 26-year-old female with underlying SLE experienced several episodes of neuropsychiatric symptoms in a fasting state. The steroid dosage was titrated up, but in vain. Timely imaging studies showed a pancreatic tumor, and insulinoma was proven by pathology. Hypoglycemia did not recur after surgery. CONCLUSION: Physicians should distinguish insulinoma from autoimmunity-mediated hypoglycemia in SLE patients with fasting hypoglycemia.

Live-cell dynamic sensing of Cd(2+) with a FRET-based indicator.

Cd(2+) causes damages to several human tissues. Although the toxicological and carcinogenetic mechanisms of Cd(2+) have been previously established, some basic questions on this toxicant remain unclear. In this study, we constructed Met-cad 1.57, a new fluorescent resonance energy transfer (FRET)-based Cd(2+) indicator, which contains a portion of a Cd(2+)-binding protein (CadR) obtained from Pseudomonas putida as the Cd(2+) sensing key. We produced a human embryonic kidney cell line HEK-MCD157 which stably expresses the Met-cad 1.57 for further investigations. Both fluorescence spectroscopy and FRET microscopic ratio imaging were used to monitor the Cd(2+) concentration within the living HEK-MCD157 cells. The dissociation constant of Met-cad 1.57 was approximately 271 nM. The function of Ca(2+) channels as a potential Cd(2+) entry gateway was further confirmed in the HEK-MCD157 cells. The organelle-targeted property of the protein-based Cd(2+) indicator directly reveals the nucleus accumulation phenomena. In summary, a human kidney cell line that stably expresses the FRET-based Cd(2+) indicator Met-cad 1.57 was constructed for reliable and convenient investigations to determine the Cd(2+) concentration within living cells, including the identification of the entry pathway of Cd(2+) and sub-cellular sequestration.

A Surgery-first approach in surgical-orthodontic treatment of mandibular prognathism--a case report.

The conventional approach in orthodontic surgery treatment of dentofacial anomalies requires a varied period of pre-surgical orthodontic treatment. This presurgical period is considered to be important for adequate surgical treatment and stable results. This period is usually long bothersome for patients because dental decompensation is required and there is consequent deterioration of aesthetics and function, especially in cases of skeletal Class III occlusion. At Chang Gung Craniofacial Center, a surgery-first approach (SFA), i.e. minimal pre-surgical orthodontics, is one of the treatment choices for Class III patients. In this report, we present a 19-year-old man with mandibular prognathism, an anterior open bite and severe dental crowding treated with SFA. The patient received orthognathic surgery a week after bracing of the teeth. The operation and recovery were uneventful as well as the following orthodontic treatment. The total treatment time was only four months, much shorter than with the conventional approach. The patient benefitted from immediate improvement of the facial profile after surgery, and a much shorter total treatment, and the results were not compromised. We believe in selected cases, SFA is a good and effective treatment alternative.