Developmental regulation of matrix metalloproteinases in response to multi-factorial, severe TBI injuries during immaturity.

INTRODUCTION: A striking pattern in young children after severe TBI is when the entire cortical ribbon displays tissue damage: hemispheric hypodensity (HH). HH is often a result of abusive head trauma (AHT). We previously reported a model of HH in a gyrencephalic species where a combination of injuries consisting of 1) cortical impact, 2) midline shift, 3) subdural hematoma/subarachnoid hemorrhage, 4) traumatic seizures, and 5) brief apnea and hypoventilation, resulted in extensive, hypoxic-ischemic type injury. Importantly, this mechanism closely resembles that seen in children, with relative sparing of the contralateral cortex, thus, ruling out a pure asphyxia mechanism. In this model, piglets of similar developmental stage to human toddlers (postnatal day 30, PND30) have extensive hypoxic-ischemic damage to the cortical ribbon with sparing of the contralateral hemisphere and deep gray matter areas. However, piglets of similar developmental stage to human infants (postnatal day 7, PND7) have less hypoxic-ischemic damage that is notably bilateral and patchy. We therefore sought to discover whether the extensive tissue damage observed in PND30 was due to a greater upregulation of matrix metalloproteinases (MMPs). MATERIALS AND METHODS: In PND 7 or PND 30 piglets receiving AHT injuries (cortical impact, midline shift, subdural hematoma/subarachnoid hemorrhage, traumatic seizures, and brief apnea and hypoventilation) or a sham injury, the pattern of albumin extravasation and MMP-9 upregulation throughout the brain was determined via immunohistochemistry, brain tissue adjacent to the cortical impact where the tissue damage spreads was collected for Western blots, and the gelatinase activity was determined over time in peripheral plasma. EEG was recorded and piglets survived up to 24 hours after injury administration. RESULTS: The pattern of albumin extravasation, indicating vasogenic edema, as well as increase in MMP-9, were both present at the same areas of hypoxic-ischemic tissue damage. Evidence from immunohistochemistry, western blot, and zymogens demonstrate that MMP- 2,- 3 or -9 are constitutively expressed during immaturity and are not different between developmental stages; however, active forms are upregulated in PND30 but not PND7 after in response to AHT model injuries. Furthermore, peripheral active MMP-9 was downregulated after model injuries in PND7. CONCLUSIONS: This differential response to AHT model injuries might confer protection to the PND7 brain. Additionally, we find that immature gyrencephalic species have a greater baseline and array of MMP's than previously demonstrated in rodent species. Treatment with an oral or intravenous broad-spectrum matrix metalloproteinase inhibitor might reduce the extensive spread of injury in PND30, but the exposure to metalloproteinase inhibitors must be acute as to not interfere with the homeostatic role of matrix metalloproteinases in normal postnatal brain development and plasticity as well as post-injury synaptogenesis and tissue repair.

When words are your scalpel, what and how information is exchanged may be differently salient to assessors.

PURPOSE: Variable assessments of learner performances can occur when different assessors determine different elements to be differently important or salient. How assessors determine the importance of performance elements has historically been thought to occur idiosyncratically and thus be amenable to assessor training interventions. More recently, a main source of variation found among assessors was two underlying factors that were differently emphasised: medical expertise and interpersonal skills. This gave legitimacy to the theory that different interpretations of the same performance may represent multiple truths. A faculty development activity introducing assessors to entrustable professional activities in which they estimated a learner's level of readiness for entrustment provided an opportunity to qualitatively explore assessor variation in the context of an interaction and in a setting in which interpersonal skills are highly valued. METHODS: Using a constructivist grounded theory approach, we explored variation in assessment processes among a group of palliative medicine assessors who completed a simulated direct observation and assessment of the same learner interaction. RESULTS: Despite identifying similar learner strengths and areas for improvement, the estimated level of readiness for entrustment varied substantially among assessors. Those who estimated the learner as not yet ready for entrustment seemed to prioritise what information was exchanged and viewed missed information as performance gaps. Those who estimated the learner as ready for entrustment seemed to prioritise how information was exchanged and viewed the same missed information as personal style differences or appropriate clinical judgement. When presented with a summary, assessors expressed surprise and concern about the variation. CONCLUSION: A main source of variation among our assessors was the differential salience of performance elements that align with medical expertise and interpersonal skills. These data support the theory that when assessing an interaction, differential salience for these two factors may be an important and perhaps inevitable source of assessor variation.

Association of Visual Impairment With Neuropsychiatric Symptoms of Dementia.

BACKGROUND: Visual impairment is a risk factor for cognitive impairment and hallucinations in older adults, but associations with other neuropsychiatric symptoms (NPS) of dementia have not been examined. METHODS: We analyzed cross-sectional data from the Aging, Demographics, and Memory Study (ADAMS), a nationally representative sample of the US population aged 70+ years. Vision was measured by self-report and using a near card. Dementia was ascertained through cognitive testing with expert consensus, and NPS were screened using the Neuropsychiatric Inventory. We used logistic regression to measure the association between visual impairment and prevalent NPS adjusting for sociodemographic factors and comorbidities. Analyses incorporated sample weights to account for the complex survey design of ADAMS. RESULTS: Of 624 participants with dementia, 332 (53%) had self-reported visual impairment and 193 (31%) had best-corrected acuity of 20/40 or worse. In unadjusted models, self-reported visual impairment was significantly associated with hallucinations (OR 2.88; 95% CI 1.12-7.44), depression (OR 2.79; 95% CI 1.7-4.57), and agitation (OR 1.61; 95% CI 1.05-2.48). Reduced visual acuity was associated with hallucinations (OR 10.13; 95% CI 2.93-34.98), psychosis (OR 6.69, 95% CI 2.53-17.7), and mania (OR 5.92, 95% CI 1.77-19.82). However, these associations did not remain significant after covariate adjustment. CONCLUSIONS: Visual impairment was associated with hallucinations, depression, agitation, psychosis, and mania in patients with dementia, but at least some of this relationship is explained by age, comorbidities, and other factors.

Excitonic Beam Steering in an Active van der Waals Metasurface.

Two-dimensional transition metal dichalcogenides (2D TMDCs) are promising candidates for ultrathin active nanophotonic elements due to the strong tunable excitonic resonances that dominate their optical response. Here, we demonstrate dynamic beam steering by an active van der Waals metasurface that leverages large complex refractive index tunability near excitonic resonances in monolayer molybdenum diselenide (MoSe2). Through varying the radiative and nonradiative rates of the excitons, we can dynamically control both the reflection amplitude and phase profiles, resulting in an excitonic phased array metasurface. Our experiments show reflected light steering to angles between -30° and 30° at different resonant wavelengths corresponding to the A exciton and B exciton. This active van der Waals metasurface relies solely on the excitonic resonances of the monolayer MoSe2 material rather than geometric resonances of patterned nanostructures, suggesting the potential to harness the tunability of excitonic resonances for wavefront shaping in emerging photonic applications.

Refractive Index Modulation in Monolayer Molybdenum Diselenide.

Two-dimensional transition metal dichalcogenides are promising candidates for ultrathin light modulators due to their highly tunable excitonic resonances at visible and near-infrared wavelengths. At cryogenic temperatures, large excitonic reflectivity in monolayer molybdenum diselenide (MoSe2) has been shown, but the permittivity and index modulation have not been studied. Here, we demonstrate large gate-tunability of complex refractive index in monolayer MoSe2 by Fermi level modulation and study the doping dependence of the A and B excitonic resonances for temperatures between 4 and 150 K. By tuning the charge density, we observe both temperature- and carrier-dependent epsilon-near-zero response in the permittivity and transition from metallic to dielectric near the A exciton energy. We attribute the dynamic control of the refractive index to the interplay between radiative and non-radiative decay channels that are tuned upon gating. Our results suggest the potential of monolayer MoSe2 as an active material for emerging photonics applications.

CDSeq: A novel complete deconvolution method for dissecting heterogeneous samples using gene expression data.

Quantifying cell-type proportions and their corresponding gene expression profiles in tissue samples would enhance understanding of the contributions of individual cell types to the physiological states of the tissue. Current approaches that address tissue heterogeneity have drawbacks. Experimental techniques, such as fluorescence-activated cell sorting, and single cell RNA sequencing are expensive. Computational approaches that use expression data from heterogeneous samples are promising, but most of the current methods estimate either cell-type proportions or cell-type-specific expression profiles by requiring the other as input. Although such partial deconvolution methods have been successfully applied to tumor samples, the additional input required may be unavailable. We introduce a novel complete deconvolution method, CDSeq, that uses only RNA-Seq data from bulk tissue samples to simultaneously estimate both cell-type proportions and cell-type-specific expression profiles. Using several synthetic and real experimental datasets with known cell-type composition and cell-type-specific expression profiles, we compared CDSeq's complete deconvolution performance with seven other established deconvolution methods. Complete deconvolution using CDSeq represents a substantial technical advance over partial deconvolution approaches and will be useful for studying cell mixtures in tissue samples. CDSeq is available at GitHub repository (MATLAB and Octave code): https://github.com/kkang7/CDSeq.

Nuclear export factor 3 regulates localization of small nucleolar RNAs.

Small nucleolar RNAs (snoRNAs) guide chemical modifications of ribosomal and small nuclear RNAs, functions that are carried out in the nucleus. Although most snoRNAs reside in the nucleolus, a growing body of evidence indicates that snoRNAs are also present in the cytoplasm and that snoRNAs move between the nucleus and cytoplasm by a mechanism that is regulated by lipotoxic and oxidative stress. Here, in a genome-wide shRNA-based screen, we identified nuclear export factor 3 (NXF3) as a transporter that alters the nucleocytoplasmic distribution of box C/D snoRNAs from the ribosomal protein L13a (Rpl13a) locus. Using RNA-sequencing analysis, we show that NXF3 associates not only with Rpl13a snoRNAs, but also with a broad range of box C/D and box H/ACA snoRNAs. Under homeostatic conditions, gain- or loss-of-function of NXF3, but not related family member NXF1, decreases or increases cytosolic Rpl13a snoRNAs, respectively. Furthermore, treatment with the adenylyl cyclase activator forskolin diminishes cytosolic localization of the Rpl13a snoRNAs through a mechanism that is dependent on NXF3 but not NXF1. Our results provide evidence of a new role for NXF3 in regulating the distribution of snoRNAs between the nuclear and cytoplasmic compartments.

Understanding the Impact of Fluid Restriction on Growth Outcomes in Infants Following Cardiac Surgery.

OBJECTIVE: Fluid restriction is reported to be a barrier in providing adequate nutrition following cardiac surgery. The specific aim of this study was to evaluate the adequacy of nutritional intake during the postoperative period using anthropometrics by comparing preoperative weight status, as measured by weight-for-age z scores, to weight status at discharge home. DESIGN: Prospective cohort study. SETTING: Cardiac ICU at Miami Children's Hospital. PATIENTS: Infants from birth to 12 months old who were scheduled for cardiac surgery at Miami Children's Hospital between December 2013 and September 2014 were followed during the postoperative stay. INTERVENTIONS: Observational study. MEASUREMENTS AND MAIN RESULTS: Preoperative and discharge weight-for-age z scores were analyzed. The Risk Adjustment for Congenital Heart Surgery 1 categories were obtained to account for the individual complexity of each case. In patients who had preoperative and discharge weights available (n = 40), the mean preoperative weight-for-age z score was -1.3 ± 1.43 and the mean weight-for-age z score at hospital discharge was -1.89 ± 1.35 with a mean difference of 0.58 ± 0.5 (p < 0.001). A higher Risk Adjustment for Congenital Heart Surgery 1 category was correlated with a greater decrease in weight-for-age z scores (r = -0.597; p = 0.002). CONCLUSIONS: Nutritional status during the postoperative period was found inadequate through the use of objective anthropometric measures and by comparing them with normal growth curves. Increase in surgical risk categories predicted a greater decrease in weight-for-age z scores. The development of future protocols for nutritional intervention should consider surgical risk categories.

Cytosolic accumulation of small nucleolar RNAs (snoRNAs) is dynamically regulated by NADPH oxidase.

Small nucleolar RNAs (snoRNAs) guide nucleotide modifications of cellular RNAs in the nucleus. We previously showed that box C/D snoRNAs from the Rpl13a locus are unexpected mediators of physiologic oxidative stress, independent of their predicted ribosomal RNA modifications. Here we demonstrate that oxidative stress induced by doxorubicin causes rapid cytoplasmic accumulation of the Rpl13a snoRNAs through a mechanism that requires superoxide and a nuclear splice variant of NADPH oxidase. RNA-sequencing analysis reveals that box C/D snoRNAs as a class are present in the cytoplasm, where their levels are dynamically regulated by NADPH oxidase. These findings suggest that snoRNAs may orchestrate the response to environmental stress through molecular interactions outside of the nucleus.

Endothelin-1 overexpression exacerbates atherosclerosis and induces aortic aneurysms in apolipoprotein E knockout mice.

OBJECTIVE: Endothelin (ET)-1 plays a role in vascular reactive oxygen species production and inflammation. ET-1 has been implicated in human atherosclerosis and abdominal aortic aneurysm (AAA) development. ET-1 overexpression exacerbates high-fat diet-induced atherosclerosis in apolipoprotein E(-/-) (Apoe(-/-)) mice. ET-1-induced reactive oxygen species and inflammation may contribute to atherosclerosis progression and AAA development. APPROACH AND RESULTS: Eight-week-old male wild-type mice, transgenic mice overexpressing ET-1 selectively in endothelium (eET-1), Apoe(-/-) mice, and eET-1/Apoe(-/-) mice were fed high-fat diet for 8 weeks. eET-1/Apoe(-/-) had a 45% reduction in plasma high-density lipoprotein (P<0.05) and presented ≥ 2-fold more aortic atherosclerotic lesions compared with Apoe(-/-) (P<0.01). AAAs were detected only in eET-1/Apoe(-/-) (8/21; P<0.05). Reactive oxygen species production was increased ≥ 2-fold in perivascular fat, media, or atherosclerotic lesions in the ascending aorta and AAAs of eET-1/Apoe(-/-) compared with Apoe(-/-) (P<0.05). Monocyte/macrophage infiltration was enhanced ≥ 2.5-fold in perivascular fat of ascending aorta and AAAs in eET-1/Apoe(-/-) compared with Apoe(-/-) (P<0.05). CD4(+) T cells were detected almost exclusively in perivascular fat (3/6) and atherosclerotic lesions (5/6) in ascending aorta of eET-1/Apoe(-/-) (P<0.05). The percentage of spleen proinflammatory Ly-6C(hi) monocytes was enhanced 26% by ET-1 overexpression in Apoe(-/-) (P<0.05), and matrix metalloproteinase-2 was increased 2-fold in plaques of eET-1/Apoe(-/-) (P<0.05) compared with Apoe(-/-). CONCLUSIONS: ET-1 plays a role in progression of atherosclerosis and AAA formation by decreasing high-density lipoprotein, and increasing oxidative stress, inflammatory cell infiltration, and matrix metalloproteinase-2 in perivascular fat, vascular wall, and atherosclerotic lesions.

Entrustable Professional Activities in Palliative Medicine: A Faculty and Learner Development Activity.

Background: Faculty development (FD) is critical to the implementation of competency-based medical education (CBME) and yet evidence to guide the design of FD activities is limited. Our aim with this study was to describe and evaluate an FD activity as part of CBME implementation. Methods: Palliative medicine faculty were introduced to entrustable professional activities (EPAs) and gained experience estimating a learner's level of readiness for entrustment by directly observing a simulated encounter. The variation that was found among assessments was discussed in facilitated debrief sessions. Attitudes and confidence levels were measured 1 week and 6 months following debriefs. Results: Participants were able to use the EPA framework when estimating the learner's readiness level for entrustment. Significant improvements in attitudes and level of confidence for several knowledge, skill, and behavior domains were maintained over time. Conclusions: Simulated direct observation and facilitated debriefs contributed to preparing both faculty and learners for CBME and EPA implementation.

Preservation of endothelium-dependent relaxation in atherosclerotic mice with endothelium-restricted endothelin-1 overexpression.

In human atherosclerosis, which is associated with elevated plasma and coronary endothelin (ET)-1 levels, ETA receptor antagonists improve coronary endothelial function. Mice overexpressing ET-1 specifically in the endothelium (eET-1) crossed with atherosclerosis-prone apolipoprotein E knockout mice (Apoe(-/-)) exhibit exaggerated high-fat diet (HFD)-induced atherosclerosis. Since endothelial dysfunction often precedes atherosclerosis development, we hypothesized that mice overexpressing endothelial ET-1 on a genetic background deficient in apolipoprotein E (eET-1/Apoe(-/-)) would have severe endothelial dysfunction. To test this hypothesis, we investigated endothelium-dependent relaxation (EDR) to acetylcholine in eET-1/Apoe(-/-) mice. EDR in mesenteric resistance arteries from 8- and 16-week-old mice fed a normal diet or HFD was improved in eET-1/Apoe(-/-) compared with Apoe(-/-) mice. Nitric oxide synthase (NOS) inhibition abolished EDR in Apoe(-/-). EDR in eET-1/Apoe(-/-) mice was resistant to NOS inhibition irrespective of age or diet. Inhibition of cyclooxygenase, the cytochrome P450 pathway, and endothelium-dependent hyperpolarization (EDH) resulted in little or no inhibition of EDR in eET-1/Apoe(-/-) compared with wild-type (WT) mice. In eET-1/Apoe(-/-) mice, blocking of EDH or soluble guanylate cyclase (sGC), in addition to NOS inhibition, decreased EDR by 36 and 30%, respectively. The activation of 4-aminopyridine-sensitive voltage-dependent potassium channels (Kv) during EDR was increased in eET-1/Apoe(-/-) compared with WT mice. We conclude that increasing eET-1 in mice that develop atherosclerosis results in decreased mutual dependence of endothelial signaling pathways responsible for EDR, and that NOS-independent activation of sGC and increased activation of Kv are responsible for enhanced EDR in this model of atherosclerosis associated with elevated endothelial and circulating ET-1.

Modified chest wall lateral intercostal artery perforator (MCW-LICAP) flap: a versatile flap in the era of oncoplastic breast surgery.

BACKGROUND: Breast reconstruction following oncological resection is becoming more common in recent years. In some ladies, implant reconstruction is not ideal due to significant implant visibility or palpability. Autologous reconstruction addresses the limitations of implant reconstruction but results in potential donor site morbidities. To date, there is no clear advantage ascribed to any technique. With appropriate selection, patients with adequate lateral mammary fold have the option of reconstruction with MCW-LICAP flap. We present our techniques and outcomes from a series of 29 patients who underwent MCW-LICAP flap. METHODS: A retrospective review of consecutive patients who underwent curative resection for breast cancer with immediate MCW-LICAP flap reconstruction, between July 2018 to April 2022 was conducted. The techniques used with its variations along with video demonstrations are presented. RESULTS: A total of 29 patients underwent 34 procedures. Nineteen breast conserving surgeries and 15 mastectomies were completed, and immediate reconstruction performed in all cases. Twenty-three patients had MCW-LICAP, 1 with a Stacked intercostal artery perforator (STICAP) flap, and 5 had MCW-LICAP combined with a Goldilocks mastectomy. There were no cases of complications requiring re-operation. All patients had acceptable time to adjuvant therapy with a median of 36 days. Learning curve analysis showed a significant reduction in operative time after the 6th case. CONCLUSION: In our preliminary experience, MCW-LICAP flap is a safe, reliable, and versatile oncoplastic reconstruction option.

Replacing an 'In-Person' Global Health Annual Conference With a Virtual Format: A Case Study from the Consortium of Universities for Global Health.

Background: Beginning in 2020, the COVID pandemic disrupted many planned annual meetings that relied on travel to a destination for sharing scholarship, networking, and planning future collaborations. As with many organizations, the Consortium of Universities for Global Health (CUGH) began exploring the utilization of a virtual platform on which to conduct the annual conference. Objective: We sought to understand the value of conducting an annual conference virtually and to evaluate the added benefit of utilizing a learning management system. Methods: Routinely collected registration data was used for the CUGH 2021 annual conference, which was completely virtual, and compared to in-person registration data from prior years. In addition, tracking and engagement data from a learning management system was reviewed to understand participation. Findings: The virtual conference attracted the greatest number of registrations, from the largest number of countries since the organization began in 2008. Analyzing the engagement of participants with specific sessions through the on-line learning management system allowed a deeper understanding of the popularity and value of topics. Conclusion: A virtual format is an efficient and effective venue for scholarly conferences. The additional information gained from an on-line learning management system can provide valuable information for future conference planning.

Integration of primary care and behavioral health services in midwestern community health centers: A mixed methods study.

INTRODUCTION: Integrating behavioral health (BH) and primary care is an important strategy to improve health behaviors, mental health, and substance misuse, particularly at community health centers (CHCs) where disease burden is high and access to mental health services is low. Components of different integrated BH models are often combined in practice. It is unknown which components distinguish developing versus established integrated BH programs. METHOD: A survey was mailed to 128 CHCs in 10 Midwestern states in 2016. Generalized estimating equation models were used to assess associations between program characteristics and stage of integration implementation (precontemplation, contemplation, preparation, action, or maintenance). Content analysis of open-ended responses identified integration barriers. RESULTS: Response rate was 60% (N = 77). Most CHCs had colocated BH and primary care services, warm hand-offs from primary care to BH clinicians, shared scheduling and electronic health record (EHR) systems, and depression and substance use disorder screening. Thirty-two CHCs (42%) indicated they had completed integration and were focused on quality improvement (maintenance). Being in the maintenance stage was associated with having a psychologist on staff (odds ratio [OR] = 7.16, 95% confidence interval [CI] [2.76, 18.55]), a system for tracking referrals (OR = 3.42, 95% CI [1.03, 11.36]), a registry (OR = 2.71, 95% CI [1.86, 3.94]), PCMH designation (OR = 2.82, 95% CI [1.48, 5.37]), and a lower proportion of Black/African American patients (OR = .82, 95% CI [.75, .89]). The most common barriers to integration were difficulty recruiting and retaining BH clinicians and inadequate reimbursement. DISCUSSION: CHCs have implemented many foundational components of integrated BH. Future work should address barriers to integration and racial disparities in access to integrated BH. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Vascular gene expression in mice overexpressing human endothelin-1 targeted to the endothelium.

Endothelin (ET)-1 plays an important pathophysiological role in several vascular diseases including hypertension and atherosclerosis. Transgenic mice overexpressing human preproET-1 selectively in the endothelium (eET-1) exhibit vascular injury in the absence of blood pressure elevation. ET-1 overexpression may induce vascular injury by inducing changes in gene expression. To understand mechanisms whereby ET-1 induces vascular damage, vascular gene expression profiling was performed using DNA microarrays. RNA from mesenteric arteries of male and female young (6-7 wk) and mature (6-8 mo) eET-1 and wild-type (WT) mice was isolated, and changes in gene expression were determined by genome-wide expression profiling using Illumina microarray and FlexArray software. Data were analyzed using a relaxed and a stringent statistical approach. The gene lists were compared and analyzed as well with Ingenuity Pathway Analysis. The most common change was an increase in the expression of lipid metabolism genes. Four of these genes were validated by qPCR, cyp51, dgat2, and scd1 genes in young and elovl6 in both young and mature male mice, supporting a role of ET-1 in atherosclerosis. To test the hypothesis that ET-1 participates in mechanisms leading to atherosclerosis, we crossed eET-1 with atherosclerosis-prone apoE(-/-) mice to determine whether ET-1 overexpression exacerbates high-fat diet (HFD)-induced atherosclerosis using oil red O staining of descending thoracic aorta. HFD increased lipid plaques by 3-, 27-, and 86-fold in eET-1, apoE(-/-), and crossed mice, respectively, vs. WT. This suggests that increased endothelial ET-1 expression results in early changes in gene expression in the vascular wall that enhance lipid biosynthesis and accelerate progression of atherosclerosis.

Public Health Crises In Comparison: China's Epidemic Response Policies From SARS To COVID-19.

The rate of infectious disease outbreaks has been accelerating over the past two decades, from the SARS epidemic in 2003 to COVID-19 in 2020. Termed by some as the twenty-first century's first pandemic, SARS originated in China and alerted the country to the importance of public health and epidemic response. After SARS, China improved its health infrastructure and reformed its political and legal health governance system. The emergence of COVID-19 from Wuhan in late 2019 put those reforms to the test. This paper analyses China's public health and epidemic response policies from a historical perspective, tracing the evolution of Chinese public health policies after the SARS outbreak in 2003. This paper assesses China's response to COVID-19 and how post-SARS policy reforms, particularly in epidemic response, played out on the ground in Wuhan. What policies worked well? What were the challenges faced? Based on the policy analysis, this paper presents recommendations for how China can improve its epidemic response through strengthened infectious disease surveillance, more transparent political coordination, and expanded public health infrastructure.

Alterations in mitochondrial dynamics with age-related Sirtuin1/Sirtuin3 deficiency impair cardiomyocyte contractility.

Sirtuin1 (SIRT1) and Sirtuin3 (SIRT3) protects cardiac function against ischemia/reperfusion (I/R) injury. Mitochondria are critical in response to myocardial I/R injury as disturbance of mitochondrial dynamics contributes to cardiac dysfunction. It is hypothesized that SIRT1 and SIRT3 are critical components to maintaining mitochondria homeostasis especially mitochondrial dynamics to exert cardioprotective actions under I/R stress. The results demonstrated that deficiency of SIRT1 and SIRT3 in aged (24-26 months) mice hearts led to the exacerbated cardiac dysfunction in terms of cardiac systolic dysfunction, cardiomyocytes contractile defection, and abnormal cardiomyocyte calcium flux during I/R stress. Moreover, the deletion of SIRT1 or SIRT3 in young (4-6 months) mice hearts impair cardiomyocyte contractility and shows aging-like cardiac dysfunction upon I/R stress, indicating the crucial role of SIRT1 and SIRT3 in protecting myocardial contractility from I/R injury. The biochemical and seahorse analysis showed that the deficiency of SIRT1/SIRT3 leads to the inactivation of AMPK and alterations in mitochondrial oxidative phosphorylation (OXPHOS) that causes impaired mitochondrial respiration in response to I/R stress. Furthermore, the remodeling of the mitochondria network goes together with hypoxic stress, and mitochondria undergo the processes of fusion with the increasing elongated branches during hypoxia. The transmission electron microscope data showed that cardiac SIRT1/SIRT3 deficiency in aging alters mitochondrial morphology characterized by the impairment of mitochondria fusion under I/R stress. Thus, the age-related deficiency of SIRT1/SIRT3 in the heart affects mitochondrial dynamics and respiration function that resulting in the impaired contractile function of cardiomyocytes in response to I/R.

Development of a Quantitative Immunoassay for Tear Lacritin Proteoforms.

Purpose: Lacritin is a tear glycoprotein with pro-tearing and pro-ocular surface homeostasis activities that is selectively deficient in most dry eye tears. Proteoforms include an active monomer, inactive polymers, and a splice variant termed lacritin-c. Quantitation of the different proteoforms of tear lacritin may provide a diagnostic tool for ocular diseases. Here, we report the development of an immunoassay for the quantification of multiple lacritin proteoforms in human tear samples. Methods: Basal tears collected on Schirmer test strips with anesthesia were eluted by diffusion and centrifugation under optimized conditions. Tear protein concentrations were determined, and 2.56 µg of each sample was separated by SDS-PAGE followed by western blot analysis. Blots were challenged with anti-Pep Lac N-term antibodies. Detection was with fluorescent secondary antibodies visualized by the LI-COR Odyssey CLx imaging system and quantified with standard curves of recombinant lacritin. Results: The percent total lacritin (ng lacritin/100 ng total protein) ranged from 1.8% to 14.8%. Monomer, lacritin-c, and polymer proteoform percent total protein ranged from 1.1% to 6.3%, 0.3% to 5.4%, and 0.7% to 5.7%, respectively. Monomer lacritin was detected at concentrations of 6 to 176 µM, with lacritin-c and polymer proteoforms at 2 to 46 µM and 1 to 23 µM, respectively. Conclusions: This assay greatly exceeds the power and sensitivity of our prior lacritin enzyme-linked immunosorbent assay that was not capable of distinguishing monomer from polymers and lacritin-c proteoforms. Translational Relevance: A new method has been developed to quantitate multiple proteoforms of tear lacritin in preparation for analyses of samples from clinical trials.