Faculty Bibliography

PURPOSE/OBJECTIVE:Sepsis is a leading cause of morbidity and mortality worldwide and is characterized by vascular leak. Treatment for sepsis, specifically intravenous fluids, may worsen deterioration in the context of vascular leak. We therefore sought to quantify vascular leak in sepsis patients to guide fluid resuscitation. METHODS:We performed a retrospective cohort study of sepsis patients in four ICU databases in North America, Europe, and Asia. We developed an intuitive vascular leak index (VLI) and explored the relationship between VLI and in-hospital death and fluid balance using generalized additive models (GAM). RESULTS:Using a GAM, we found that increased VLI is associated with an increased risk of in-hospital death. Patients with a VLI in the highest quartile (Q4), across the four datasets, had a 1.61-2.31 times increased odds of dying in the hospital compared to patients with a VLI in the lowest quartile (Q1). VLI Q2 and Q3 were also associated with increased odds of dying. The relationship between VLI, treated as a continuous variable, and in-hospital death and fluid balance was statistically significant in the three datasets with large sample sizes. Specifically, we observed that as VLI increased, there was increase in the risk for in-hospital death and 36-84 h fluid balance. CONCLUSIONS:Our VLI identifies groups of patients who may be at higher risk for in-hospital death or for fluid accumulation. This relationship persisted in models developed to control for severity of illness and chronic comorbidities.

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the 'Waffle Method' which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. We propose the Waffle Method as a way to achieve many advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT.

OBJECTIVE:Millions of babies have been conceived by IVF, yet debate about its safety to offspring continues. We hypothesized that superovulation and in vitro fertilization (IVF) promote genomic changes, including altered telomere length (TL) and activation of the retrotransposon LINE-1 (L1), and tested this hypothesis in a mouse model. MATERIAL AND METHODS/METHODS:Experimental study analyzing TL and L1 copy number in C57BL/6 J mouse blastocysts in vivo produced from natural mating cycles (N), in vivo produced following superovulation (S), or in vitro produced following superovulation (IVF). We also examined the effects of prolonged culture on TL and L1 copy number in the IVF group comparing blastocysts cultured 96 h versus blastocysts cultured 120 h. TL and L1 copy number were measured by Real Time PCR. RESULTS:TL in S (n = 77; Mean: 1.50 ± 1.15; p = 0.0007) and IVF (n = 82; Mean: 1.72 ± 1.44; p < 0.0001) exceeded that in N (n = 16; Mean: 0.61 ± 0.27). TL of blastocysts cultured 120 h (n = 15, Mean: 2.14 ± 1.05) was significantly longer than that of embryos cultured for 96 h (n = 67, Mean: 1.63 ± 1.50; p = 0.0414). L1 copy number of blastocysts cultured for 120 h (n = 15, Mean: 1.71 ± 1.49) exceeded that of embryos cultured for 96 h (n = 67, Mean: 0.95 ± 1.03; p = 0.0162). CONCLUSIONS:Intriguingly ovarian stimulation, alone or followed by IVF, produced embryos with significantly longer telomeres compared to in vivo, natural cycle-produced embryos. The significance of this enriched telomere endowment for the health and longevity of offspring born from IVF merit future studies.

The Notch signalling pathway is a master regulator of cell fate transitions in development and disease. In the brain, Notch promotes neural stem cell (NSC) proliferation, regulates neuronal migration and maturation and can act as an oncogene or tumour suppressor. How NOTCH and its transcription factor RBPJ activate distinct gene regulatory networks in closely related cell types in vivo remains to be determined. Here we use Targeted DamID (TaDa), requiring only thousands of cells, to identify NOTCH and RBPJ binding in NSCs and their progeny in the mouse embryonic cerebral cortex in vivo. We find that NOTCH and RBPJ associate with a broad network of NSC genes. Repression of NSC-specific Notch target genes in intermediate progenitors and neurons correlates with decreased chromatin accessibility, suggesting that chromatin compaction may contribute to restricting NOTCH-mediated transactivation.

Adult stem cells coordinate intrinsic and extrinsic, local and systemic, cues to maintain the proper balance between self-renewal and differentiation. However, the precise mechanisms stem cells use to integrate these signals remain elusive. Here, we show that Escargot (Esg), a member of the Snail family of transcription factors, regulates the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis by attenuating the activity of the pro-differentiation insulin receptor (InR) pathway. Esg positively regulates the expression of an antagonist of insulin signaling, ImpL2, while also attenuating the expression of InR. Furthermore, Esg-mediated repression of the InR pathway is required to suppress CySC loss in response to starvation. Given the conservation of Snail-family transcription factors, characterizing the mechanisms by which Esg regulates cell-fate decisions during homeostasis and a decline in nutrient availability is likely to provide insight into the metabolic regulation of stem cell behavior in other tissues and organisms.

Contractile actomyosin and protrusive branched F-actin networks interact in a dynamic balance, repeatedly contracting and expanding apical cell contacts to organize the epithelium of the developing fly retina. Previously we showed that the immunoglobulin superfamily protein Sidekick (Sdk) contributes to contraction by recruiting the actin binding protein Polychaetoid (Pyd) to vertices. Here we show that as tension increases during contraction, Sdk progressively accumulates at vertices, where it toggles to recruit the WAVE regulatory complex (WRC) to promote actin branching and protrusion. Sdk alternately interacts with the WRC and Pyd using the same C-terminal motif. With increasing protrusion, levels of Sdk and the WRC decrease at vertices while levels of Pyd increase paving the way for another round of contraction. Thus, by virtue of dynamic association with vertices and interchangeable associations with contractile and protrusive effectors, Sdk is central to controlling the balance between contraction and expansion that shapes this epithelium.

Heart failure is a multifactorial disease that affects an estimated 38 million people worldwide. Current pharmacotherapy of heart failure with reduced ejection fraction (HFrEF) includes combination therapy with angiotensin-converting enzyme inhibitors (ACEi) and β-adrenergic receptor blockers (β-AR blockers), a therapy also used as treatment for non-cardiac conditions. Our knowledge of the molecular changes accompanying treatment with ACEi and β-AR blockers is limited. Here, we applied proteomics and phosphoproteomics approaches to profile the global changes in protein abundance and phosphorylation state in cardiac left ventricles consequent to combination therapy of β-AR blocker and ACE inhibitor in HFrEF and control hearts. The phosphorylation changes induced by treatment were profoundly different for failing than for non-failing hearts. HFrEF was characterized by profound downregulation of mitochondrial proteins coupled with derangement of β-adrenergic and pyruvate dehydrogenase signaling. Upon treatment, phosphorylation changes consequent to HFrEF were reversed. In control hearts, treatment mainly led to downregulation of canonical PKA signaling. The observation of divergent signaling outcomes depending on disease state underscores the importance of evaluating drug effects within the context of the specific conditions present in the recipient heart.

Segmental bone defects (SBD) are difficult to treat, requiring a comprehensive understanding of the bone and soft tissue injury. Defect size, fracture characteristics, and local and systemic biology all help dictate treatment options. Bone grafting with autograft or allograft, Masquelet technique, and bone transport with external or internal fixation can all be used successfully in the correct patient. When deciding on the best treatment option and addressing any complications throughout the process, it is important to always keep in mind the three principles of bone healing: sterility, stability, and biology. The goal of this review is to present the history of treatment for critical SBD, including the indications and challenges that have been addressed and current and emerging treatment options.