Intelligent image-activated sorting of Chlamydomonas reinhardtii by mitochondrial localization.

Organelle positioning in cells is associated with various metabolic functions and signaling in unicellular organisms. Specifically, the microalga Chlamydomonas reinhardtii repositions its mitochondria, depending on the levels of inorganic carbon. Mitochondria are typically randomly distributed in the Chlamydomonas cytoplasm, but relocate toward the cell periphery at low inorganic carbon levels. This mitochondrial relocation is linked with the carbon-concentrating mechanism, but its significance is not yet thoroughly understood. A genotypic understanding of this relocation would require a high-throughput method to isolate rare mutant cells not exhibiting this relocation. However, this task is technically challenging due to the complex intracellular morphological difference between mutant and wild-type cells, rendering conventional non-image-based high-event-rate methods unsuitable. Here, we report our demonstration of intelligent image-activated cell sorting by mitochondrial localization. Specifically, we applied an intelligent image-activated cell sorting system to sort for C. reinhardtii cells displaying no mitochondrial relocation. We trained a convolutional neural network (CNN) to distinguish the cell types based on the complex morphology of their mitochondria. The CNN was employed to perform image-activated sorting for the mutant cell type at 180 events per second, which is 1-2 orders of magnitude faster than automated microscopy with robotic pipetting, resulting in an enhancement of the concentration from 5% to 56.5% corresponding to an enrichment factor of 11.3. These results show the potential of image-activated cell sorting for connecting genotype-phenotype relations for rare-cell populations, which require a high throughput and could lead to a better understanding of metabolic functions in cells.

The Effect of a Novel Platysma Hammock Flap During Extended Deep Plane Facelift on the Signs of Aging in the Neck.

BACKGROUND: Aging changes in the neck, including platysma banding (PB), skin laxity (SL), and submandibular gland visibility (SGV), have a high degree of recurrence after rhytidectomy. OBJECTIVES: The authors sought to assess the long-term improvement in PB, SL, and SGV with addition of aplatysmal hammock flap to the extended deep-plane facelift and assess patient satisfaction. METHODS: This was a prospective study of 123 consecutive patients undergoing extended deep-plane facelift incorporating platysma hammock flap with or without midline platysmaplasty. Standard 2-dimensional patient photographs were employed to assess PB, SL, and SGV preoperative and >12 months postoperative. A 1-year postoperative patient satisfaction survey was conducted. RESULTS: The platysmal hammock flap without midline platysmaplasty cohort had a significant (P < 0.01) reduction in mean preoperative PB, SL, and SGV scores from 1.03, 1.88, and 1.21 to 0.06, 0.03, and 0.15 at 21 months. The platysmal hammock flap with midline platysmaplasty cohort had a significant (P < 0.01) reduction in preoperative PB, SL, and SGV scores from 1.81, 2.43, and 1.81 to 0.10, 0.15, and 0.48 at 18 months. The platysmal hammock flap with and without midline platysmaplasty cohorts had 96.2% and 88.9% satisfaction, respectively. CONCLUSIONS: Extended deep-plane facelift with a platysmal hammock flap achieves long-term, sustained improvements in PB, SL, and SGV; is well-tolerated; and results in substantial patient satisfaction.

Morphological Indicator for Directed Evolution of Euglena gracilis with a High Heavy Metal Removal Efficiency.

In the past few decades, microalgae-based bioremediation methods for treating heavy metal (HM)-polluted wastewater have attracted much attention by virtue of their environment friendliness, cost efficiency, and sustainability. However, their HM removal efficiency is far from practical use. Directed evolution is expected to be effective for developing microalgae with a much higher HM removal efficiency, but there is no non-invasive or label-free indicator to identify them. Here, we present an intelligent cellular morphological indicator for identifying the HM removal efficiency of Euglena gracilis in a non-invasive and label-free manner. Specifically, we show a strong monotonic correlation (Spearman's ρ = -0.82, P = 2.1 × 10-5) between a morphological meta-feature recognized via our machine learning algorithms and the Cu2+ removal efficiency of 19 E. gracilis clones. Our findings firmly suggest that the morphology of E. gracilis cells can serve as an effective HM removal efficiency indicator and hence have great potential, when combined with a high-throughput image-activated cell sorter, for directed-evolution-based development of E. gracilis with an extremely high HM removal efficiency for practical wastewater treatment worldwide.

Open Versus Endoscopic Surgery of Zenker's Diverticula: A Systematic Review and Meta-analysis.

Most Zenker's diverticula (ZD) cohort studies are single-institution retrospective observational studies of recurrence rates. There is a gap in the literature regarding patient-reported outcomes after ZD surgery. This study was conducted to compare if open transcervical diverticulectomy (OD) is better than endoscopic laser diverticulectomy (ELD) or endoscopic stapler-assisted diverticulectomy (ESD). The study design is of systematic review and meta-analysis. The following databases were searched: SCOPUS, EMBASE, PubMed, and Word of Science through December 2017. The quality of the studies was evaluated using 22-item STROBE checklist with 3 independent physician reviewers. The Inter-rater reliability was calculated both as a percent and utilizing Cohen's Kappa. For the meta-analysis, Cohen's d for an effect size was calculated for all studies comparing dysphagia results before and after surgery. A total of 865 patients were treated across 11 selected publications, of which 106 patients were treated OD, 310 ELD, and 449 with an ESD approach. Patient-reported dysphagia outcomes were reported as Cohen's d (confidence interval): OD, ELD, and ESD were 1.31 (0.88, 1.74), 1.91 (1.62, 2.20), and 2.45 (2.04, 2.86), respectively. The pooled effect of all studies for dysphagia was 2.22 (1.85, 2.59) and regurgitation 2.20 (1.80, 2.59). We did not prove that OD has superior outcomes compared to ESD and ELD. Any method of surgical intervention yields a large effect (i.e., improvement in dysphagia and regurgitation) comparing patient-reported symptoms before and after surgery. Future research, currently underway, includes a prospective, multi-institutional study comparing standardized outcomes between treatments of ZD including symptom resolution, complications, and recurrences using validated measures to define long-term outcomes.Level of Evidence 3.

Dermabrasion for Scars and Wire Loop Electrocautery for Rhinophyma.

Dermabrasion and wire loop electrocautery are controlled scalpel-less procedures to remove superficial skin layers to treat dermal surface irregularities. Their postprocedure healing involves healing by secondary intention. The purpose of this paper is to discuss the use of diamond fraise dermabrasion to improve scars and the use of wire loop electrocautery to treat rhinophyma surface irregularities. Both techniques are minimally invasive and low cost, and at the same time, can significantly improve facial skin deformities. An advantage in using wire loop electrocautery for rhinophyma excision is that it is a simple, economic, and very effective technique to sculpt the nose, with minimal intraoperative bleeding. With dermabrasion, pretreatment and postregimens can help improve postprocedure results. Future efforts to modulate the healing from both of these techniques include the potential use of topical growth factors, autologous platelet-rich plasma, or using stem cells to accelerate collagen formation and reepithelization during the postprocedure period.

Expression of the ORMDLS, modulators of serine palmitoyltransferase, is regulated by sphingolipids in mammalian cells.

The relationship between serine palmitoyltransferase (SPT) activity and ORMDL regulation of sphingolipid biosynthesis was investigated in mammalian HEK293 cells. Each of the three human ORMDLs reduced the increase in long-chain base synthesis seen after overexpression of wild-type SPT or SPT containing the C133W mutation in hLCB1, which produces the non-catabolizable sphingoid base, 1-deoxySa. ORMDL-dependent repression of sphingoid base synthesis occurred whether SPT was expressed as individual subunits or as a heterotrimeric single-chain SPT fusion protein. Overexpression of the single-chain SPT fusion protein under the control of a tetracycline-inducible promoter in stably transfected cells resulted in increased endogenous ORMDL expression. This increase was not transcriptional; there was no significant increase in any of the ORMDL mRNAs. Increased ORMDL protein expression required SPT activity since overexpression of a catalytically inactive SPT with a mutation in hLCB2a had little effect. Significantly, increased ORMDL expression was also blocked by myriocin inhibition of SPT as well as fumonisin inhibition of the ceramide synthases, suggesting that increased expression is a response to a metabolic signal. Moreover, blocking ORMDL induction with fumonisin treatment resulted in significantly greater increases in in vivo SPT activity than was seen when ORMDLs were allowed to increase, demonstrating the physiological significance of this response.

Autophagy regulates sphingolipid levels in the liver.

Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.

Topological and functional characterization of the ssSPTs, small activating subunits of serine palmitoyltransferase.

The topological and functional organization of the two isoforms of the small subunits of human serine palmitoyltransferase (hssSPTs) that activate the catalytic hLCB1/hLCB2 heterodimer was investigated. A variety of experimental approaches placed the N termini of the ssSPTs in the cytosol, their C termini in the lumen, and showed that they contain a single transmembrane domain. Deletion analysis revealed that the ability to activate the heterodimer is contained in a conserved 33-amino acid core domain that has the same membrane topology as the full-length protein. In combination with analysis of isoform chimera and site-directed mutagenesis, a single amino acid residue in this core (Met(25) in ssSPTa and Val(25) in ssSPTb) was identified which confers specificity for palmitoyl- or stearoyl-CoA, respectively, in both yeast and mammalian cells. This same residue also determines which isoform is a better activator of a mutant heterodimer, hLCB1(S331F)/hLCB2a, which has increased basal SPT activity and decreased amino acid substrate selectivity. This suggests that the role of the ssSPTs is to increase SPT activity without compromising substrate specificity. In addition, the observation that the C-terminal domains of ssSPTa and ssSPTb, which are highly conserved within each subfamily but are the most divergent regions between isoform subfamilies, are not required for activation of the heterodimer or for acyl-CoA selectivity suggests that the ssSPTs have additional roles that remain to be discovered.

Sphingolipids in the root play an important role in regulating the leaf ionome in Arabidopsis thaliana.

Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodihydrosphinganine (3-KDS), which is reduced by a 3-KDS reductase to dihydrosphinganine. Ser palmitoyltransferase is essential for plant viability. Arabidopsis thaliana contains two genes (At3g06060/TSC10A and At5g19200/TSC10B) encoding proteins with significant similarity to the yeast 3-KDS reductase, Tsc10p. Heterologous expression in yeast of either Arabidopsis gene restored 3-KDS reductase activity to the yeast tsc10Δ mutant, confirming both as bona fide 3-KDS reductase genes. Consistent with sphingolipids having essential functions in plants, double mutant progeny lacking both genes were not recovered from crosses of single tsc10A and tsc10B mutants. Although the 3-KDS reductase genes are functionally redundant and ubiquitously expressed in Arabidopsis, 3-KDS reductase activity was reduced to 10% of wild-type levels in the loss-of-function tsc10a mutant, leading to an altered sphingolipid profile. This perturbation of sphingolipid biosynthesis in the Arabidopsis tsc10a mutant leads an altered leaf ionome, including increases in Na, K, and Rb and decreases in Mg, Ca, Fe, and Mo. Reciprocal grafting revealed that these changes in the leaf ionome are driven by the root and are associated with increases in root suberin and alterations in Fe homeostasis.

Killian Jamieson Diverticulum, the Great Mimicker: A Case Series and Contemporary Review.

OBJECTIVE: To assess barium esophagram (BAS) as a diagnostic marker for patients with Killian Jamieson diverticula (KJD). METHODS: Prospective, multicenter cohort study of individuals enrolled in the Prospective OUtcomes of Cricopharyngeus Hypertonicity (POUCH) Collaborative. Patient demographics, comorbidities, radiographic imaging reports, laryngoscopy findings, patient-reported outcome measures (PROM), and operative reporting were abstracted from a REDCap database and summarized using means, medians, percentages, frequencies. Paired t-tests and Wilcoxon Signed Rank test were used to test pre- to post-operative differences in RSI, EAT-10, and VHI-10 scores. Diagnostic test evaluation including sensitivity, specificity, positive, and negative predictive value with 95% confidence intervals were calculated comparing BAS findings to operative report. RESULTS: A total of 287 persons were enrolled; 13 (4%) patients were identified with confirmed KJD on operative reports. 100% underwent open transcervical excision. BAS has a 46.2% (95% confidence interval [CI]: 23.2, 70.9) sensitivity and 97.8% (95% CI: 95.3, 99.0) specificity in detecting a KJD and 50% (95% CI: 25.4, 74.6) positive predictive value but 97.4% (95%CI: 94.8, 98.7) negative predictive value. Preoperatively, patients reported mean (SD) RSI and EAT-10 of 19.4 (9) and 8.3 (7.5) accordingly. Postoperatively, patients reported mean (SD) RSI and EAT-10 as 5.4 (6.2) and 2.3 (3.3). Both changes in RSI and EAT-10 were statistically significant (p = 0.008, p = 0.03). CONCLUSION: KJD are rare and represent <5% of hypopharyngeal diverticula undergoing surgical intervention. Open transcervical surgery significantly improves symptoms of dysphagia. BAS has high specificity but low sensitivity in detecting KJD. LEVEL OF EVIDENCE: 4 Laryngoscope, 133:2110-2115, 2023.

Cricopharyngeus Muscle Dysfunction and Hypopharyngeal Diverticula (e.g., Zenker): A Multicenter Study.

OBJECTIVE: To describe demographics and imaging and compare findings and symptoms at presentation in a large cohort of persons with cricopharyngeus muscle dysfunction (CPMD) with and without hypopharyngeal diverticula. METHODOLOGY: Prospective, multicenter cohort study of all individuals enrolled in the Prospective OUtcomes of Cricopharyngeal Hypertonicity (POUCH) Collaborative. Patient survey, comorbidities, radiography, laryngoscopy findings, and patient-reported outcome measures (e.g., Eating Assessment Tool [EAT-10]) data were abstracted from a REDCap database and summarized using means, medians, percentages, and frequencies. Diagnostic categories were compared using analysis of variance. RESULTS: A total of 250 persons were included. The mean age (standard deviation [SD]) of the cohort was 69.0 (11.2). Forty-two percent identified as female. Zenker diverticula (ZD) was diagnosed in 85.2%, 9.2% with CPMD without diverticula, 4.4% with a Killian Jamieson diverticula (KJD), and 1.2% traction-type diverticula. There were no differences between diagnostic categories in regard to age, gender, and duration of symptoms (p = 0.25, 0.19, 0.45). The mean (SD) EAT-10 score for each group was 17.1 (10.1) for ZD, 20.2 (9.3) for CPMD, and 10.3 (9.4) for KJD. Patients with isolated CPMD had significantly greater EAT-10 scores compared to the other diagnostic groups (p = 0.03). CONCLUSION: ZD is the most common, followed by CPMD without diverticula, KJD, and traction-type. Patients with isolated obstructing CPMD may be more symptomatic than persons with ZD or KJD. LEVEL OF EVIDENCE: 4 Laryngoscope, 133:1349-1355, 2023.

Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities.

Serine palmitoyltransferase (SPT) catalyzes the first committed step in sphingolipid biosynthesis. In yeast, SPT is composed of a heterodimer of 2 highly-related subunits, Lcb1p and Lcb2p, and a third subunit, Tsc3p, which increases enzyme activity markedly and is required for growth at elevated temperatures. Higher eukaryotic orthologs of Lcb1p and Lcb2p have been identified, but SPT activity is not highly correlated with coexpression of these subunits and no ortholog of Tsc3p has been identified. Here, we report the discovery of 2 proteins, ssSPTa and ssSPTb, which despite sharing no homology with Tsc3p, each substantially enhance the activity of mammalian SPT expressed in either yeast or mammalian cells and therefore define an evolutionarily conserved family of low molecular weight proteins that confer full enzyme activity. The 2 ssSPT isoforms share a conserved hydrophobic central domain predicted to reside in the membrane, and each interacts with both hLCB1 and hLCB2 as assessed by positive split ubiquitin 2-hybrid analysis. The presence of these small subunits, along with 2 hLCB2 isofoms, suggests that there are 4 distinct human SPT isozymes. When each SPT isozyme was expressed in either yeast or CHO LyB cells lacking endogenous SPT activity, characterization of their in vitro enzymatic activities, and long-chain base (LCB) profiling revealed differences in acyl-CoA preference that offer a potential explanation for the observed diversity of LCB seen in mammalian cells.

Deep imaging flow cytometry.

Imaging flow cytometry (IFC) has become a powerful tool for diverse biomedical applications by virtue of its ability to image single cells in a high-throughput manner. However, there remains a challenge posed by the fundamental trade-off between throughput, sensitivity, and spatial resolution. Here we present deep-learning-enhanced imaging flow cytometry (dIFC) that circumvents this trade-off by implementing an image restoration algorithm on a virtual-freezing fluorescence imaging (VIFFI) flow cytometry platform, enabling higher throughput without sacrificing sensitivity and spatial resolution. A key component of dIFC is a high-resolution (HR) image generator that synthesizes "virtual" HR images from the corresponding low-resolution (LR) images acquired with a low-magnification lens (10×/0.4-NA). For IFC, a low-magnification lens is favorable because of reduced image blur of cells flowing at a higher speed, which allows higher throughput. We trained and developed the HR image generator with an architecture containing two generative adversarial networks (GANs). Furthermore, we developed dIFC as a method by combining the trained generator and IFC. We characterized dIFC using Chlamydomonas reinhardtii cell images, fluorescence in situ hybridization (FISH) images of Jurkat cells, and Saccharomyces cerevisiae (budding yeast) cell images, showing high similarities of dIFC images to images obtained with a high-magnification lens (40×/0.95-NA), at a high flow speed of 2 m s-1. We lastly employed dIFC to show enhancements in the accuracy of FISH-spot counting and neck-width measurement of budding yeast cells. These results pave the way for statistical analysis of cells with high-dimensional spatial information.

A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity.

The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 subunit of serine palmitoyltransferase (SPT). Serum from patients and transgenic mice expressing a disease-causing mutation (C133W) contain elevated levels of 1-deoxysphinganine (1-deoxySa), which presumably arise from inappropriate condensation of alanine with palmitoyl-CoA. Mutant heterodimeric SPT is catalytically inactive. However, mutant heterotrimeric SPT has approximately 10-20% of wild-type activity and supports growth of yeast cells lacking endogenous SPT. In addition, long chain base profiling revealed the synthesis of significantly more 1-deoxySa in yeast and mammalian cells expressing the heterotrimeric mutant enzyme than in cells expressing wild-type enzyme. Wild-type and mutant enzymes had similar affinities for serine. Surprisingly, the enzymes also had similar affinities for alanine, indicating that the major affect of the C133W mutation is to enhance activation of alanine for condensation with the acyl-CoA substrate. In vivo synthesis of 1-deoxySa by the mutant enzyme was proportional to the ratio of alanine to serine in the growth media, suggesting that this ratio can be used to modulate the relative synthesis of sphinganine and 1-deoxySa. By expressing SPT as a single-chain fusion protein to ensure stoichiometric expression of all three subunits, we showed that GADD153, a marker for endoplasmic reticulum stress, was significantly elevated in cells expressing mutant heterotrimers. GADD153 was also elevated in cells treated with 1-deoxySa. Taken together, these data indicate that the HSAN1 mutations perturb the active site of SPT resulting in a gain of function that is responsible for the HSAN1 phenotype.

Massive image-based single-cell profiling reveals high levels of circulating platelet aggregates in patients with COVID-19.

A characteristic clinical feature of COVID-19 is the frequent incidence of microvascular thrombosis. In fact, COVID-19 autopsy reports have shown widespread thrombotic microangiopathy characterized by extensive diffuse microthrombi within peripheral capillaries and arterioles in lungs, hearts, and other organs, resulting in multiorgan failure. However, the underlying process of COVID-19-associated microvascular thrombosis remains elusive due to the lack of tools to statistically examine platelet aggregation (i.e., the initiation of microthrombus formation) in detail. Here we report the landscape of circulating platelet aggregates in COVID-19 obtained by massive single-cell image-based profiling and temporal monitoring of the blood of COVID-19 patients (n = 110). Surprisingly, our analysis of the big image data shows the anomalous presence of excessive platelet aggregates in nearly 90% of all COVID-19 patients. Furthermore, results indicate strong links between the concentration of platelet aggregates and the severity, mortality, respiratory condition, and vascular endothelial dysfunction level of COVID-19 patients.

Overexpression of the wild-type SPT1 subunit lowers desoxysphingolipid levels and rescues the phenotype of HSAN1.

Mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT) cause an adult-onset, hereditary sensory, and autonomic neuropathy type I (HSAN1). We previously reported that mice bearing a transgene-expressing mutant SPTLC1 (tgSPTLC1(C133W)) show a reduction in SPT activity and hyperpathia at 10 months of age. Now analyzed at a later age, we find these mice develop sensory loss with a distal small fiber neuropathy and peripheral myelinopathy. This phenotype is largely reversed when these mice are crossed with transgenic mice overexpressing wild-type SPTLC1 showing that the mutant SPTLC1 protein is not inherently toxic. Simple loss of SPT activity also cannot account for the HSAN1 phenotype, since heterozygous SPTLC1 knock-out mice have reduced SPT activity but are otherwise normal. Rather, the presence of two newly identified, potentially deleterious deoxysphingoid bases in the tgSPTLC1(C133W), but not in the wild-type, double-transgenic tgSPTLC1(WT + C133W) or SPTLC1(+/-) mice, suggests that the HSAN1 mutations alter amino acid selectivity of the SPT enzyme such that palmitate is condensed with alanine and glycine, in addition to serine. This observation is consistent with the hypothesis that HSAN1 is the result of a gain-of-function mutation in SPTLC1 that leads to accumulation of a toxic metabolite.

AI on a chip.

Artificial intelligence (AI) has dramatically changed the landscape of science, industry, defence, and medicine in the last several years. Supported by considerably enhanced computational power and cloud storage, the field of AI has shifted from mostly theoretical studies in the discipline of computer science to diverse real-life applications such as drug design, material discovery, speech recognition, self-driving cars, advertising, finance, medical imaging, and astronomical observation, where AI-produced outcomes have been proven to be comparable or even superior to the performance of human experts. In these applications, what is essentially important for the development of AI is the data needed for machine learning. Despite its prominent importance, the very first process of the AI development, namely data collection and data preparation, is typically the most laborious task and is often a limiting factor of constructing functional AI algorithms. Lab-on-a-chip technology, in particular microfluidics, is a powerful platform for both the construction and implementation of AI in a large-scale, cost-effective, high-throughput, automated, and multiplexed manner, thereby overcoming the above bottleneck. On this platform, high-throughput imaging is a critical tool as it can generate high-content information (e.g., size, shape, structure, composition, interaction) of objects on a large scale. High-throughput imaging can also be paired with sorting and DNA/RNA sequencing to conduct a massive survey of phenotype-genotype relations whose data is too complex to analyze with traditional computational tools, but is analyzable with the power of AI. In addition to its function as a data provider, lab-on-a-chip technology can also be employed to implement the developed AI for accurate identification, characterization, classification, and prediction of objects in mixed, heterogeneous, or unknown samples. In this review article, motivated by the excellent synergy between AI and lab-on-a-chip technology, we outline fundamental elements, recent advances, future challenges, and emerging opportunities of AI with lab-on-a-chip technology or "AI on a chip" for short.

Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals.

In yeast, Tsc10p catalyzes reduction of 3-ketosphinganine to dihydrosphingosine. In mammals, it has been proposed that this reaction is catalyzed by FVT1, which despite limited homology and a different predicted topology, can replace Tsc10p in yeast. Silencing of FVT1 revealed a direct correlation between FVT1 levels and reductase activity, showing that FVT1 is the principal 3-ketosphinganine reductase in mammalian cells. Localization and topology studies identified an N-terminal membrane-spanning domain in FVT1 (absent in Tsc10p) oriented to place it in the endoplasmic reticulum (ER) lumen. In contrast, protease digestion studies showed that the N terminus of Tsc10p is cytoplasmic. Fusion of the N-terminal domain of FVT1 to green fluorescent protein directed the fusion protein to the ER, demonstrating that it is sufficient for targeting. Although both proteins have two predicted transmembrane domains C-terminal to a cytoplasmic catalytic domain, neither had an identifiable lumenal loop. Nevertheless, both Tsc10p and the residual fragment of FVT1 produced by removal of the N-terminal domain with factor Xa protease behave as integral membrane proteins. In addition to their topological differences, mutation of conserved catalytic residues had different effects on the activities of the two enzymes. Thus, while FVT1 can replace Tsc10p in yeast, there are substantial differences between the two enzymes that may be important for regulation of sphingolipid biosynthesis in higher eukaryotes.

The ORMs interact with transmembrane domain 1 of Lcb1 and regulate serine palmitoyltransferase oligomerization, activity and localization.

Serine palmitoyltransferase (SPT), an endoplasmic reticulum-localized membrane enzymecomposed of acatalytic LCB1/LCB2 heterodimer and a small activating subunit (Tsc3 in yeast; ssSPTs in mammals), is negatively regulated by the evolutionarily conserved family of proteins known as the ORMs. In yeast, SPT, the ORMs, and the PI4P phosphatase Sac1, copurify in the "SPOTs" complex. However, neither the mechanism of ORM inhibition of SPT nor details of the interactions of the ORMs and Sac1 with SPT are known. Here we report that the first transmembrane domain (TMD1) of Lcb1 is required for ORM binding to SPT. Loss of binding is not due to altered membrane topology of Lcb1 since replacing TMD1 with a heterologous TMD restores membrane topology but not ORM binding. TMD1 deletion also eliminates ORM-dependent formation of SPT oligomers as assessed by co-immunoprecipitation assays and in vivo imaging. Expression of ORMs lacking derepressive phosphorylation sites results in constitutive SPT oligomerization, while phosphomimetic ORMs fail to induce oligomerization under any conditions. Significantly, when LCB1-RFP and LCB1ΔTMD1-GFP were coexpressed, more LCB1ΔTMD1-GFP was in the peripheral ER, suggesting ORM regulation is partially accomplished by SPT redistribution. Tsc3 deletion does not abolish ORM inhibition of SPT, indicating the ORMs do not simply prevent activation by Tsc3. Binding of Sac1 to SPT requires Tsc3, but not the ORMs, and Sac1 does not influence ORM-mediated oligomerization of SPT. Finally, yeast mutants lacking ORM regulation of SPT require the LCB-P lyase Dpl1 to maintain long-chain bases at sublethal levels.

Acute effects of selective fetoscopic laser photocoagulation on recipient cardiac function in twin-twin transfusion syndrome.

OBJECTIVE: This study evaluated the acute effects of selective fetoscopic laser photocoagulation (SFLP) on recipient cardiovascular function in a twin-twin transfusion syndrome (TTTS) pregnancy. STUDY DESIGN: This was a retrospective chart review of echocardiographic data in TTTS including right (RV) and left (LV) ventricular Doppler myocardial performance index (MPI); LV and RV end diastolic wall thickness; and umbilical artery (UA), vein (UV), and ductus venosus (DV) Dopplers. The primary outcome measures were improved MPI defined as greater than 10% interval decrease in left and/or right MPI. Data were analyzed by Student t test and Fisher's exact test. RESULTS: Sixty-five patients met inclusion criteria. SFLP results in a significant improvement in UV and DV Doppler and an increase in both RV and LV wall thickness. A 10% or greater improvement in recipient LV MPI after SFLP is associated with improved recipient survival as compared with unimproved LV MPI (100% vs 86.1%, P = .05). CONCLUSION: Improved recipient myocardial performance index after SFLP is associated with improvement in recipient survival.