Risk factors and outcomes for surgical repair of obstructed total anomalous pulmonary venous connection.

Background: Surgical repair of obstructed total anomalous pulmonary venous connection (TAPVC) is a high-risk surgical cohort. This study aimed to assess surgical risk factors and outcomes in infants with TAPVC treated at a single centre. Methods: This was a prospective single-centre, observational study that included consecutive patients presenting with obstructed TAPVC. Patients with obstructed supra-cardiac, cardiac, and infra-cardiac TAPVC were included in the study. In-hospital mortality was the primary outcome. Multivariable logistic regression was carried out to identify risk factors for in-hospital mortality. Results: Of the 41 patients, 31 (75.6%) were males. Ages were distributed as follows: 20 (48.8%) between 1 and 3 months, 11 (26.88%) between >3 months, and 10 (24.4%) neonates. The in-hospital mortality was 19.5% (n=8). Of the remaining 33 patients, there were no deaths during the follow-up. Short-term follow-up was achieved for 100% of cases (up to 6 months post-operatively). The mean duration of follow-up was 43.6± 3.6 months. One (3%) early and 2 (6%) late pulmonary vein obstructions were observed during follow-up. Age less than 30 days and pre-operative ventilation were associated with significantly increased risk of in-hospital mortality. Conclusions: Despite logistical challenges, reasonable surgical outcomes are attainable in obstructed TAPVC cases. Age less than 30 days and need for pre-operative ventilation were independent predictors of in-hospital mortality, while pulmonary venous obstruction in the post-operative period contributed to re-interventions.

Public-Private Partnership for Treatment of Congenital Heart Diseases: Experiences From an Indian State.

Background: Treatment of congenital heart disease (CHD), being the most common congenital anomaly, puts immense financial burden in low- and middle-income countries (LMICs) and contributes significantly to infant mortality. We report experiences of treatment of CHD in the Indian state of West Bengal by a public-private partnership (PPP) model. Methods: Under the Rashtriya Bal Swasthya Karyakram, the government of the state of West Bengal in India launched a program called the "Sishu Sathi Scheme" to provide free treatment to children who need heart surgeries, irrespective of economic status. Treatment was provided in selected private hospitals and some public hospitals in a reimbursement model where government compensated the hospitals. Data were collected on such procedures from 2013 to 2022 and analyzed. Results: A total of 27,844 patients with CHD received treatment under the Sishu Sathi Scheme from August 2013 to December 2022. The average number of patients per year was 3,093. Detailed data of procedures from January 2016 to December 2022 showed a total of 22,572 procedures (6,249 device interventions, 4,840 cardiac catheterizations, and 11,483 surgical interventions). The in-hospital mortality of surgical procedures and catheterization lab procedures were 5.2% and 0.9%, respectively. Conclusions: A large number of patients with CHD were successfully treated under a PPP in the state of West Bengal in India. In spite of its inherent challenges, this model is of special relevance in LMICs where access and affordability for treatment of CHD always remain a challenge.

Psychological Impact of COVID-19 and Its Influence on Parental Willingness to Vaccinate Children: A Cross-Sectional Study at a Tertiary Care Hospital in Kolkata.

Background COVID-19 has affected the physical and mental health of people globally, and vaccination is seen as a crucial tool in controlling the pandemic. However, the readiness to vaccinate children remains a concern, particularly in India. Aim The study aimed to investigate the association between the psychological impact of COVID-19 and willingness to vaccinate their children among attendees of the COVID-19 vaccination clinic at Nil Ratan Sircar Medical College, Kolkata. Method The study used an observational, cross-sectional design and collected data from 356 participants between August and September 2022. The COVID-19 Perceived Stress Scale-10 was used to assess participants' psychological impact, and willingness to vaccinate was determined using a survey. Results Approximately 64% (n=227) and 71% (n=253) of the participants exhibited a high level of perceived stress and willingness to vaccinate their children. The vaccine acceptance was significantly associated with perceived stress level and other factors such as family type, presence of chronic illness, and history of acquaintances suffering from COVID-19. Conclusion The study highlights the importance of addressing parental stress and anxiety to enhance vaccination rates among children. To achieve this, population-level awareness of vaccine safety measures and benefits should be raised to alleviate stress and increase vaccine uptake.

Palmitoylation of the Glucagon-like Peptide-1 Receptor Modulates Cholesterol Interactions at the Receptor-Lipid Microenvironment.

The G protein-coupled receptor (GPCR) superfamily of cell surface receptors has been shown to be functionally modulated by post-translational modifications. The glucagon-like peptide receptor-1 (GLP-1R), which is a drug target in diabetes and obesity, undergoes agonist-dependent palmitoyl tail conjugation. The palmitoylation in the C-terminal domain of GLP-1R has been suggested to modulate the receptor-lipid microenvironment. In this work, we have performed coarse-grain molecular dynamics simulations of palmitoylated and nonpalmitoylated GLP-1R to analyze the differential receptor-lipid interactions. Interestingly, the placement and dynamics of the C-terminal domain of GLP-1R are found to be directly dependent on the palmitoyl tail. We observe that both cholesterol and phospholipids interact with the receptor but display differential interactions in the presence and absence of the palmitoyl tail. We characterize important cholesterol-binding sites and validate sites that have been previously reported in experimentally resolved structures of the receptor. We show that the receptor acts like a conduit for cholesterol flip-flop by stabilizing cholesterol in the membrane core. Taken together, our work represents an important step in understanding the molecular effects of lipid modifications in GPCRs.

Interplay of Cholesterol and Actin in Neurotransmitter GPCR Signaling: Insights from Chronic Cholesterol Depletion Using Statin.

Serotonin1A receptors are important neurotransmitter receptors in the G protein-coupled receptor (GPCR) family and modulate a variety of neurological, behavioral, and cognitive functions. We recently showed that chronic cholesterol depletion by statins, potent inhibitors of HMG-CoA reductase (the rate-limiting enzyme in cholesterol biosynthesis), leads to polymerization of the actin cytoskeleton that alters lateral diffusion of serotonin1A receptors. However, cellular signaling by the serotonin1A receptor under chronic cholesterol depletion remains unexplored. In this work, we explored signaling by the serotonin1A receptor under statin-treated condition. We show that cAMP signaling by the receptor is reduced upon lovastatin treatment due to reduction in cholesterol as well as polymerization of the actin cytoskeleton. To the best of our knowledge, these results constitute the first report describing the effect of chronic cholesterol depletion on the signaling of a G protein-coupled neuronal receptor. An important message arising from these results is that it is prudent to include the contribution of actin polymerization while analyzing changes in membrane protein function due to chronic cholesterol depletion by statins. Notably, our results show that whereas actin polymerization acts as a negative regulator of cAMP signaling, cholesterol could act as a positive modulator. These results assume significance in view of reports highlighting symptoms of anxiety and depression in humans upon statin administration and the role of serotonin1A receptors in anxiety and depression. Overall, these results reveal a novel role of actin polymerization induced by chronic cholesterol depletion in modulating GPCR signaling, which could act as a potential therapeutic target.

Role of Cholesterol and its Biosynthetic Precursors on Membrane Organization and Dynamics: A Fluorescence Approach.

Cholesterol is the most representative sterol present in membranes of higher eukaryotes, and is the end product of a long and multistep biosynthetic pathway. Lathosterol and zymosterol are biosynthetic precursors of cholesterol in Kandutsch-Russell and Bloch pathways, respectively. Lathosterol differs with cholesterol merely in the position of the double bond in the sterol ring, whereas zymosterol differs with cholesterol in position and number of double bonds. In this work, we have monitored the effect of cholesterol and its biosynthetic precursors (lathosterol and zymosterol) on membrane organization and dynamics in fluid and gel phase membranes. Toward this goal, we have utilized two fluorescent membrane probes, DPH and its cationic derivative TMA-DPH. Our results using these probes show that cholesterol and its biosynthetic precursors (lathosterol and zymosterol) exhibit similar trend in maintaining membrane organization and dynamics (as reported by fluorescence anisotropy and apparent rotational correlation time), in fluid phase POPC membranes. Notably, although lathosterol and zymosterol show similar trend in maintaining membrane organization and dynamics, the corresponding change for cholesterol is different in gel phase DPPC membranes. These results demonstrate that the position and number of double bonds in sterols is an important determinant in maintaining membrane physical properties. Our results assume significance since accumulation of precursors of cholesterol have been reported to be associated with severe pathological conditions.

Smith-Lemli-Opitz syndrome: A pathophysiological manifestation of the Bloch hypothesis.

The biosynthesis of cholesterol, an essential component of higher eukaryotic membranes, was worked out by Konrad Bloch (and Feodor Lynen) in the 1960s and they received the Nobel Prize around that time in recognition of their pioneering contributions. An elegant consequence of this was a hypothesis proposed by Konrad Bloch (the Bloch hypothesis) which suggests that each subsequent intermediate in the cholesterol biosynthesis pathway is superior in supporting membrane function in higher eukaryotes relative to its precursor. In this review, we discuss an autosomal recessive metabolic disorder, known as Smith-Lemli-Opitz syndrome (SLOS), associated with a defect in the Kandutsch-Russell pathway of cholesterol biosynthesis that results in accumulation of the immediate precursor of cholesterol in its biosynthetic pathway (7-dehydrocholesterol) and an altered cholesterol to total sterol ratio. Patients suffering from SLOS have several developmental, behavioral and cognitive abnormalities for which no drug is available yet. We characterize SLOS as a manifestation of the Bloch hypothesis and review its molecular etiology and current treatment. We further discuss defective Hedgehog signaling in SLOS and focus on the role of the serotonin1A receptor, a representative neurotransmitter receptor belonging to the GPCR family, in SLOS. Notably, ligand binding activity and cellular signaling of serotonin1A receptors are impaired in SLOS-like condition. Importantly, cellular localization and intracellular trafficking of the serotonin1A receptor (which constitute an important determinant of a GPCR cellular function) are compromised in SLOS. We highlight some of the recent developments and emerging concepts in SLOS pathobiology and suggest that novel therapies based on trafficking defects of target receptors could provide new insight into treatment of SLOS.

Metabolic depletion of sphingolipids inhibits agonist-induced endocytosis of the serotonin1A receptor.

G protein-coupled receptors (GPCRs) are vital cellular signaling machinery and currently represent ~40% drug targets. Endocytosis of GPCRs is an important process that allows stringent spatiotemporal control over receptor population on the cell surface. Although the role of proteins in GPCR endocytosis is well addressed, the contribution of membrane lipids in this process is rather unexplored. Sphingolipids are essential functional lipids in higher eukaryotes and are implicated in several neurological functions. To understand the role of sphingolipids in GPCR endocytosis, we subjected cells expressing human serotonin1A receptors (an important neurotransmitter GPCR involved in cognitive and behavioral functions) to metabolic sphingolipid depletion using fumonisin B1 , an inhibitor of sphingolipid biosynthetic pathway. Our results, using flow cytometric analysis and confocal microscopic imaging, show that sphingolipid depletion inhibits agonist-induced endocytosis of the serotonin1A receptor in a concentration-dependent manner, which was restored when sphingolipid levels were replenished. We further show that there was no change in the internalization of transferrin, a marker for clathrin-mediated endocytosis, under sphingolipid-depleted condition, highlighting the specific requirement of sphingolipids for endocytosis of serotonin1A receptors. Our results reveal the regulatory role of sphingolipids in GPCR endocytosis and highlight the importance of neurotransmitter receptor trafficking in health and disease.

Statin-induced increase in actin polymerization modulates GPCR dynamics and compartmentalization.

The function of the actin cytoskeleton in cellular motility and trafficking has been widely studied. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol and its consequences on membrane dynamics are addressed only rarely. In a recent work, we reported that chronic cholesterol depletion using statins leads to significant polymerization of the actin cytoskeleton. In this work, we explore the effect of reorganization of the actin cytoskeleton on membrane dynamics under cholesterol-depleted condition. Specifically, we explore the role of actin cytoskeleton in regulating the dynamics of the serotonin1A receptor, a crucial neurotransmitter G protein-coupled receptor (GPCR) that plays a major role in the generation and modulation of cognitive and behavioral functions. For this, we analyzed the lateral dynamics of the serotonin1A receptor in cholesterol-depleted cells (using statins) by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) measurements. Our results indicate that lateral diffusion parameters of serotonin1A receptors in normal cells are consistent with models describing the diffusion of molecules in a homogeneous membrane. Interestingly, these parameters are altered in cholesterol-depleted cells and the receptor exhibits dynamic confinement. Notably, our results show that statin-induced dynamic confinement could be reversed by destabilization of the actin cytoskeleton. On a broader perspective, these results assume significance in understanding the modulatory role of the membrane environment on the organization and dynamics of GPCRs in diseases caused by altered cholesterol biosynthesis.

Double left atrial appendage: A diagnostic dilemma.

We report a unique intraoperative finding of an additional double left atrial appendage (LAA) during an arterial switch operation with ventricular septal defect closure in a 4-month-old girl. Immediately after the procedure, a prolapsing mass within the left atrium (LA) on the transesophageal echocardiogram raised concerns of a possible thrombus. The LAA was clearly visible with a pressure monitoring line which was put intraoperatively. To investigate further, cardiopulmonary bypass was resumed, and the heart was arrested and explored. There was an appendage-like structure, separate from the one that had the pressure monitoring line, which was inverted inside. It was pulled out from outside clearly establishing a double LAA. This report illustrates an example of a diagnostic dilemma caused by a double atrial appendage which was invaginated into LA masquerading as a mass or thrombus.

Synergistic and Competitive Lipid Interactions in the Serotonin1A Receptor Microenvironment.

The interaction of lipids with G-protein-coupled receptors (GPCRs) has been shown to modulate and dictate several aspects of GPCR organization and function. Diverse lipid interaction sites have been identified from structural biology, bioinformatics, and molecular dynamics studies. For example, multiple cholesterol interaction sites have been identified in the serotonin1A receptor, along with distinct and overlapping sphingolipid interaction sites. How these lipids interact with each other and what is the resultant effect on the receptor is still not clear. In this work, we have analyzed lipid-lipid crosstalk at the receptor of the serotonin1A receptor embedded in a membrane bilayer that mimics the neuronal membrane composition by long coarse-grain simulations. Using a set of similarity coefficients, we classified lipids that bind at the receptor together as synergistic cobinding, and those that bind individually as competitive. Our results show that certain lipids interact with the serotonin1A receptor in synergy with each other. Not surprisingly, the ganglioside GM1 and cholesterol show a synergistic cobinding, along with the relatively uncommon GM1-phosphatidylethanolamine (PE) and cholesterol-PE synergy. In contrast, certain lipid pairs such as cholesterol and sphingomyelin appear to be in competition at several sites, despite their coexistence in lipid nanodomains. In addition, we observed intralipid competition between two lipid tails, with the receptor exhibiting increased interactions with the unsaturated lipid tails. We believe our work represents an important step in understanding the diversity of GPCR-lipid interactions and exploring synergistic cobinding and competition in natural membranes.

Transcatheter management of combined patent ductus arteriosus and left pulmonary artery stenosis in congenital rubella syndrome: A series of three patients and an insight into case selection for intervention.

Transcatheter closure of patent ductus arteriosus (PDA) is now the standard of care with some exceptions. Best treatment for peripheral pulmonary artery (PA) stenosis in small children is still debatable. Surgical augmentation, balloon dilatation, and stenting are the available options with each having its own risks and limitations. Here, we are reporting complete transcatheter management of three cases of congenital rubella syndrome who had PDA and left branch PA stenosis by device closure and stent placement, with successful outcome in two cases and complication, leading to surgery in one. We also share our understanding of case selection for a better outcome.

Cholesterol-Dependent Dynamics of the Serotonin1A Receptor Utilizing Single Particle Tracking: Analysis of Diffusion Modes.

G protein-coupled receptors (GPCRs) are signaling hubs in cell membranes that regulate a wide range of physiological processes and are popular drug targets. Serotonin1A receptors are important members of the GPCR family and are implicated in neuropsychiatric disorders. Cholesterol is a key constituent of higher eukaryotic membranes and is believed to contribute to the segregated distribution of membrane constituents into domains. To explore the role of cholesterol in lateral dynamics of GPCRs, we utilized single particle tracking (SPT) to monitor diffusion of serotonin1A receptors under acute and chronic cholesterol-depleted conditions. Our results show that the short-term diffusion coefficient of the receptor decreases upon cholesterol depletion, irrespective of the method of cholesterol depletion. Analysis of SPT trajectories revealed that relative populations of receptors undergoing various modes of diffusion change upon cholesterol depletion. Notably, in cholesterol-depleted cells, we observed an increase in the confined population of the receptor accompanied by a reduction in diffusion coefficient for chronic cholesterol depletion. These results are supported by our recent work and present observations that show polymerization of G-actin in response to chronic cholesterol depletion. Taken together, our results bring out the interdependence of cholesterol and actin cytoskeleton in regulating diffusion of GPCRs in membranes.

Lysine 101 in the CRAC Motif in Transmembrane Helix 2 Confers Cholesterol-Induced Thermal Stability to the Serotonin1A Receptor.

G protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins that transduce signals across the plasma membrane and orchestrate a multitude of physiological processes within cells. The serotonin1A receptor is a crucial neurotransmitter receptor in the GPCR family involved in a multitude of neurological, behavioral and cognitive functions. We have previously shown, using a combination of experimental and simulation approaches, that membrane cholesterol acts as a key regulator of organization, dynamics, signaling and endocytosis of the serotonin1A receptor. In addition, we showed that membrane cholesterol stabilizes the serotonin1A receptor against thermal deactivation. In the present work, we explored the molecular basis of cholesterol-induced thermal stability of the serotonin1A receptor. For this, we explored the possible role of the K101 residue in a cholesterol recognition/interaction amino acid consensus (CRAC) motif in transmembrane helix 2 in conferring the thermal stability of the serotonin1A receptor. Our results show that a mutation in the K101 residue leads to loss in thermal stability of the serotonin1A receptor imparted by cholesterol, independent of membrane cholesterol content. We envision that our results could have potential implications in structural biological advancements of GPCRs and design of thermally stabilized receptors for drug development.

Inositol Phosphoryl Transferase, Ipt1, Is a Critical Determinant of Azole Resistance and Virulence Phenotypes in Candida glabrata.

In this study, we have specifically blocked a key step of sphingolipid (SL) biosynthesis in Candida glabrata by disruption of the orthologs of ScIpt1 and ScSkn1. Based on their close homology with S. cerevisiae counterparts, the proteins are predicted to catalyze the addition of a phosphorylinositol group onto mannosyl inositolphosphoryl ceramide (MIPC) to form mannosyl diinositolphosphoryl ceramide (M(IP)2C), which accounts for the majority of complex SL structures in S. cerevisiae membranes. High throughput lipidome analysis confirmed the accumulation of MIPC structures in ΔCgipt1 and ΔCgskn1 cells, albeit to lesser extent in the latter. Noticeably, ΔCgipt1 cells showed an increased susceptibility to azoles; however, ΔCgskn1 cells showed no significant changes in the drug susceptibility profiles. Interestingly, the azole susceptible phenotype of ΔCgipt1 cells seems to be independent of the ergosterol content. ΔCgipt1 cells displayed altered lipid homeostasis, increased membrane fluidity as well as high diffusion of radiolabeled fluconazole (3H-FLC), which could together influence the azole susceptibility of C. glabrata. Furthermore, in vivo experiments also confirmed compromised virulence of the ΔCgipt1 strain. Contrarily, specific functions of CgSkn1 remain unclear.

Late presentation of aortopulmonary window: a contemporary series.

Late presentation of aortopulmonary window (APW) beyond infancy is uncommon and many of these cases are inoperable due to development of progressive pulmonary hypertension and Eisenmenger syndrome. Outcome data in this cohort is thus sparse and the aim of this study was to analyze the outcomes in patients with APW operable beyond 1 year of age. Between September 2016 and March 2020, in a single center, 12 consecutive patients older than 1 year, undergoing surgery for APW, were included in the study. The median age and weight at presentation were 7.5 years (interquartile range (IQR) 4-9.5) and 15 kg (IQR 11.7-19.5). Ten (83.3%) patients had type 1 APW (proximal type) and 2 (16.6%) had a type 2 APW (distal type). Eight (66.6%) patients had associated lesions. Transaortic patch closure of APW was done in all cases. Seven (58.3%) patients were extubated within 3.5 h of admission in intensive care. There were no early deaths or during follow-up. The median follow-up duration was 20.5 months (IQR 7.5-24), and all patients were in New York Heart Association (NYHA) class I at last follow-up. Follow-up echocardiography did not reveal any significant residual shunts necessitating any additional procedure and a consistent decrease in pulmonary artery pressures. Surgery in patients with APW beyond 1 year of age is possible in selected patients. The early and intermediate surgical outcomes in patients who remain operable are excellent.

Membrane Dipole Potential: An Emerging Approach to Explore Membrane Organization and Function.

Biological membranes are complex organized molecular assemblies of lipids and proteins that provide cells and membrane-bound intracellular organelles their individual identities by morphological compartmentalization. Membrane dipole potential originates from the electrostatic potential difference within the membrane due to the nonrandom arrangement (orientation) of amphiphile and solvent (water) dipoles at the membrane interface. In this Feature Article, we will focus on the measurement of dipole potential using electrochromic fluorescent probes and highlight interesting applications. In addition, we will focus on ratiometric fluorescence microscopic imaging technique to measure dipole potential in cellular membranes, a technique that can be used to address novel problems in cell biology which are otherwise difficult to address using available approaches. We envision that membrane dipole potential could turn out to be a convenient tool in exploring the complex interplay between membrane lipids and proteins and could provide novel insights in membrane organization and function.

Effect of Local Anesthetics on Dipole Potential of Different Phase Membranes: A Fluorescence Study.

The molecular mechanism behind the action of local anesthetics is not well understood. Phenylethanol (PEtOH) is an ingredient of essential oils with a rose-like odor, and it has previously been used as a local anesthetic. In this work, we explored the effect of PEtOH on dipole potential in membranes representing biologically relevant phases, employing the dual-wavelength ratiometric method utilizing the potential-sensitive probe di-8-ANEPPS. Our results show that PEtOH reduces membrane dipole potential in membranes of all biologically relevant phases (gel, liquid-ordered, and fluid) in a concentration-dependent manner. To the best of our knowledge, these results constitute one of the early reports describing reduction of membrane dipole potential induced by local anesthetics, irrespective of membrane phase.