FACI is a novel clathrin adaptor protein 2-binding protein that facilitates low-density lipoprotein endocytosis.

BACKGROUND: Cholesterol plays a vital role in multiple physiological processes. Cellular uptake of cholesterol is mediated primarily through endocytosis of low-density lipoprotein (LDL) receptor. New modifiers of this process remain to be characterized. Particularly, the role of fasting- and CREB-H-induced (FACI) protein in cholesterol homeostasis merits further investigation. METHODS: Interactome profiling by proximity labeling and affinity purification - mass spectrometry was performed. Total internal reflection fluorescence microscopy and confocal immunofluorescence microscopy were used to analyze protein co-localization and interaction. Mutational analysis was carried out to define the domain and residues required for FACI localization and function. Endocytosis was traced by fluorescent cargos. LDL uptake in cultured cells and diet-induced hypercholesterolemia in mice were assessed. RESULTS: FACI interacted with proteins critically involved in clathrin-mediated endocytosis, vesicle trafficking, and membrane cytoskeleton. FACI localized to clathrin-coated pits (CCP) on plasma membranes. FACI contains a conserved DxxxLI motif, which mediates its binding with the adaptor protein 2 (AP2) complex. Disruption of this motif of FACI abolished its CCP localization but didn't affect its association with plasma membrane. Cholesterol was found to facilitate FACI transport from plasma membrane to endocytic recycling compartment in a clathrin- and cytoskeleton-dependent manner. LDL endocytosis was enhanced in FACI-overexpressed AML12 cells but impaired in FACI-depleted HeLa cells. In vivo study indicated that hepatic FACI overexpression alleviated diet-induced hypercholesterolemia in mice. CONCLUSIONS: FACI facilitates LDL endocytosis through its interaction with the AP2 complex.

Aberrant splicing events caused by insertion of genes of interest into expression vectors.

Background: Expression of genes of interest from plasmids or lentiviral vectors is one of the most common tools in molecular and gene therapy. Aberrant splicing between the inserted gene of interest and downstream vector sequence has not been systematically analyzed. Methods: Formation of aberrant fusion transcripts and proteins was detected by RT-PCR, sequencing, Western blotting and mass spectrometry. Bioinformatic analysis was performed to identify all human and mouse genes prone to vector-dependent aberrant splicing. Selected genes were experimentally validated. Results: When we expressed human FACI in cultured cells, an aberrant splicing event was found to occur between FACI transcript and downstream plasmid sequence through one exon-exon junction in FACI that accidentally contributes a splice donor site. To explore whether this could be a general phenomenon, we searched the whole human and mouse genomes for protein-coding genes that harbor an exon-exon junction resembling a splice donor site. Almost all genes prone to this type of aberrant splicing were identified. A total of 17 genes among the hits were randomly selected for experimental validation. RT-PCR and sequencing results verified that 13 genes were aberrantly spliced on the identified exon-exon junctions. In addition, all 17 genes were aberrantly spliced on their V5 tag sequence. Aberrant fusion protein expression from all 17 genes was validated by immunoblotting. Aberrant splicing was prevented by recoding the V5 tag or the splice sites. Conclusions: Our study revealed an unexpectedly high frequency of vector-dependent aberrant splicing events. Aberrant formation of the resulting fusion proteins could undermine the accuracy of gain-of-function studies and might cause potential side effects when the therapeutic gene is expressed in vivo. Our work has implications in improving vector construction and epitope tagging for gene expression and therapy.

Production of single-cycle infectious SARS-CoV-2 through a trans-complemented replicon.

Single-cycle infectious virus can elicit close-to-natural immune response and memory. One approach to generate single-cycle severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is through deletion of structural genes such as spike (S) and nucleocapsid (N). Transcomplementation of the resulting ΔS or ΔN virus through enforced expression of S or N protein in the cells gives rise to a live but unproductive virus. In this study, ΔS and ΔN BAC clones were constructed and their live virions were rescued by transient expression of S and N proteins from the ancestral and the Omicron strains. ΔS and ΔN virions were visualized by transmission electron microscopy. Virion production of ΔS was more efficient than that of ΔN. The coated S protein from ΔS was delivered to infected cells in which the expression of N protein was also robust. In contrast, expression of neither S nor N was detected in ΔN-infected cells. ΔS underwent viral RNA replication, induced type I interferon (IFN) response, but did not form plaques. Despite RNA replication in cells, ΔS infection did not produce viral progeny in culture supernatant. Interestingly, viral RNA replication was not further enhanced upon overexpression of S protein. Taken together, our work provides a versatile platform for development of single-cycle vaccines for SARS-CoV-2.

Lemierre Syndrome in a Patient With Splenectomy Secondary to Pyruvate Kinase Deficiency, Complicated by Heparin Resistance.

Lemierre syndrome was first documented in the literature in 1936, and is defined as septic thrombophlebitis of the internal jugular vein. It is typically a result of oropharyngeal infection causing local soft tissue inflammation, which spreads to vasculature, and promotes formation of septic thrombi within the lumen, persistent bacteremia, and septic emboli. We present the case of a 24-year-old incarcerated man, who presented with leukocytosis and a right-sided tender, swollen neck after undergoing left mandibular molar extraction for an infected tooth. Computed tomography revealed a persistent thrombus in the transverse and sigmoid sinuses bilaterally, extending downwards, into the upper jugular veins. He was started on empiric intravenous vancomycin, zosyn, and heparin, but subsequently demonstrated heparin resistance, and was thus anticoagulated with a lovenox bridge to warfarin. Throughout his hospital course, hemocultures demonstrated no growth, so antibiotic treatment was deescalated to oral metronidazole and ceftriaxone. On discharge, the patient was transitioned to oral amoxicillin and metronidazole for an additional 4 weeks with continuation of anticoagulation with warfarin for a total of 3 to 6 months. This case report details a unique presentation of Lemierre syndrome with bilateral transverse sinus, sigmoid sinus, and internal jugular vein thrombosis that was presumably secondary to an odontogenic infectious focus.

Preponderance of CTLA4 Variation Associated With Autosomal Dominant Immune Dysregulation in the MYPPPY Motif.

One of the primary targets of immune checkpoint inhibition is the negative immune regulatory molecule CTLA-4. Immune-related adverse events are commonly observed following CTLA-4 inhibition in melanoma treatment, and a spectrum of these conditions are also observed in individuals with germline haploinsufficiency of CTLA4. Here we describe a heterozygous de novo missense variant of CTLA4 in a young girl with childhood-onset autoimmune hepatitis and polyarthritis, the latter responding to treatment with CTLA-4-Ig fusion protein. This variant lay within the highly conserved MYPPPY motif of CTLA-4: a critical structural determinant of ligand binding, which is also bound by the anti-CTLA-4 monoclonal antibody ipilimumab. Within the spectrum of CTLA4 variants reported, missense variants in the MYPPPY motif were overrepresented when compared to variants within a control population, highlighting the physiological importance of this motif in both the genetic and pharmacological regulation of autoimmunity and anti-tumor immunity.

A pilot study of change in fracture risk in patients with acute respiratory distress syndrome.

INTRODUCTION: Acute skeletal muscle wasting is a major contributor to post critical illness physical impairment. However, the bone response remains uncharacterized. We prospectively investigated the early changes in bone mineral density (BMD) and fracture risk in critical illness. METHODS: Patients were prospectively recruited ≤24 hours following intensive care unit (ICU) admission to a university teaching or a community hospital (August 2009 to April 2011). All were aged >18 years and expected to be intubated for >48 hours, spend >7 days in critical care and survive ICU admission. Forty-six patients were studied (55.3% male), with a mean age of 54.4 years (95% confidence interval (CI): 49.1 to 59.6) and an APACHE II score of 23.9 (95% CI: 22.4 to 25.5). Calcaneal dual X-ray absorptiometry (DXA) assessment of BMD was performed on day 1 and 10. Increase in fracture risk was calculated from the change in T-score. RESULTS: BMD did not change between day 1 and 10 in the cohort overall (0.434 (95% CI: 0.405 to 0.463) versus 0.425 g/cm(2) (95% CI: 0.399 to 0.450), P = 0.58). Multivariable logistical regression revealed admission corrected calcium (odds ratio (OR): 1.980 (95% CI: 1.089 to 3.609), P = 0.026) and admission PaO2-to-FiO2 ratio (OR: 0.916 (95% CI: 0.833 to 0.998), P = 0.044) to be associated with >2% loss of BMD. Patients with acute respiratory distress syndrome had a greater loss in BMD than those without (-2.81% (95% CI: -5.73 to 0.118%), n = 34 versus 2.40% (95% CI: 0.204 to 4.586%), n = 12, P = 0.029). In the 34 patients with acute respiratory distress syndrome, fracture risk increased by 19.4% (95% CI: 13.9 to 25.0%). CONCLUSIONS: Patients with acute respiratory distress syndrome demonstrated early and rapid bone demineralisation with associated increase in fracture risk.

Acute skeletal muscle wasting in critical illness.

IMPORTANCE: Survivors of critical illness demonstrate skeletal muscle wasting with associated functional impairment. OBJECTIVE: To perform a comprehensive prospective characterization of skeletal muscle wasting, defining the pathogenic roles of altered protein synthesis and breakdown. DESIGN, SETTING, AND PARTICIPANTS: Sixty-three critically ill patients (59% male; mean age: 54.7 years [95% CI, 50.0-59.6 years]) with an Acute Physiology and Chronic Health Evaluation II score of 23.5 (95% CI, 21.9-25.2) were prospectively recruited within 24 hours following intensive care unit (ICU) admission from August 2009 to April 2011 at a university teaching and a community hospital in England. Patients were recruited if older than 18 years and were anticipated to be intubated for longer than 48 hours, to spend more than 7 days in critical care, and to survive ICU stay. MAIN OUTCOMES AND MEASURES: Muscle loss was determined through serial ultrasound measurement of the rectus femoris cross-sectional area (CSA) on days 1, 3, 7, and 10. In a subset of patients, the fiber CSA area was quantified along with the ratio of protein to DNA on days 1 and 7. Histopathological analysis was performed. In addition, muscle protein synthesis, breakdown rates, and respective signaling pathways were characterized. RESULTS: There were significant reductions in the rectus femoris CSA observed at day 10 (−17.7% [95% CI, −25.9% to 8.1%]; P < .001). In the 28 patients assessed by all 3 measurement methods on days 1 and 7, the rectus femoris CSA decreased by 10.3% (95% CI, 6.1% to 14.5%), the fiber CSA by 17.5% (95% CI, 5.8% to 29.3%), and the ratio of protein to DNA by 29.5% (95% CI, 13.4% to 45.6%). Decrease in the rectus femoris CSA was greater in patients who experienced multiorgan failure by day 7 (−15.7%; 95% CI, −27.7% to 11.4%) compared with single organ failure (−3.0%; 95% CI, −5.3% to 2.1%) (P < .001), even by day 3 (−8.7% [95% CI, −59.3% to 50.6%] vs −1.8% [95% CI, −12.3% to 10.5%], respectively; P = .03). Myofiber necrosis occurred in 20 of 37 patients (54.1%). Protein synthesis measured by the muscle protein fractional synthetic rate was depressed in patients on day 1 (0.035%/hour; 95% CI, 0.023% to 0.047%/hour) compared with rates observed in fasted healthy controls (0.039%/hour; 95% CI, 0.029% to 0.048%/hour) (P = .57) and increased by day 7 (0.076% [95% CI, 0.032%-0.120%/hour]; P = .03) to rates associated with fed controls (0.065%/hour [95% CI, 0.049% to 0.080%/hour]; P = .30), independent of nutritional load. Leg protein breakdown remained elevated throughout the study (8.5 [95% CI, 4.7 to 12.3] to 10.6 [95% CI, 6.8 to 14.4] µmol of phenylalanine/min/ideal body weight × 100; P = .40). The pattern of intracellular signaling supported increased breakdown (n = 9, r = −0.83, P = .005) and decreased synthesis (n = 9, r = −0.69, P = .04). CONCLUSIONS AND RELEVANCE: Among these critically ill patients, muscle wasting occurred early and rapidly during the first week of critical illness and was more severe among those with multiorgan failure compared with single organ failure. These findings may provide insights into skeletal muscle wasting in critical illness.