SARS‐CoV‐2 Alpha,Beta, and Delta variants display enhanced Spike‐mediated syncytia formation

Severe COVID‐19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS‐CoV‐2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco‐2, Calu‐3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon‐induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS‐CoV‐2 emerging variants display enhanced syncytia formation.     

Regulation of postsurgical fibrosis by the programmed death-1 inhibitory pathway

Surgical adhesions are a common and often severe complication of abdominal or pelvic injury that cause pelvic pain, bowel obstruction, and infertility in women. Current treatments are of limited effectiveness because little is known about the cellular and subcellular processes underlying adhesiogenesis. Recently, we showed that Th1 alpha beta CD4(+) T cells mediate the pathogenesis of adhesion formation in a rodent model of this disease process. In this study, we demonstrate that in mice these T cells home directly to the site of surgically induced adhesions and control local chemokine production in a manner dependent on the CD28 T cell costimulatory pathway. Conversely, the inhibitory programmed death-1 pathway plays a central role in limiting adhesiogenesis, as programmed death-1 blockade was associated with increased T cell infiltration, chemokine production, and a concomitant exacerbation of disease. Our results reveal for the first time that the development of postsurgical fibrosis is under the tight control of positive and negative T cell costimulation, and suggest that targeting these pathways may provide promising therapies for the prevention of adhesion formation.     

IGFBP-3 mediates TGF-beta1-induced cell growth in human airway smooth muscle cells

Both insulin-like growth factor binding protein-3 (IGFBP-3) and transforming growth factor-beta (TGF-beta) have been separately shown to have cell-specific growth-inhibiting or growth-potentiating effects. TGF-beta stimulates IGFBP-3 mRNA and peptide expression in several cell types, and TGF-beta-induced growth inhibition and apoptosis have been shown to be mediated through the induction of IGFBP-3. However, a link between the growth stimulatory effects of TGF-beta and IGFBP-3-induction has not been shown. In this study, we investigated the role of IGFBP-3 in mediating TGF-beta1-induced cell growth using human airway smooth muscle (ASM) cells as our model. TGF-beta1 (1 ng/ml) treatment induced a 10- to 20-fold increase in the levels of expression of IGFBP-3 mRNA and protein. Addition of either IGFBP-3 or TGF-beta1 to the growth medium resulted in an approximately twofold increase in cell proliferation. Coincubation of ASM cells with IGFBP-3 antisense (but not sense) oligomers as well as with an IGFBP-3 neutralizing antibody (but not with control IgG) blocked the growth induced by TGF-beta1 (P < 0.001). Several IGFBP-3-associated proteins were observed in ASM cell lysates, which may have a role in the cellular responses to IGFBP-3. These findings demonstrate that IGFBP-3 is capable of mediating the growth stimulatory effect of TGF-beta in ASM cells.     

Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies

Polymeric chains of a small protein ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyubiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical–biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per ubiquitin is conjugated to another ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyubiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one ubiquitin and a specific ε-amine on another ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a ¹⁵N-enriched proximal ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-ubiquitins as well as control di-ubiquitins by the yeast proteasome-associated deubiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyubiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyubiquitin, these developments now provide tools for making fully natural polyubiquitin chains of essentially any type of linkage and length.     

Critical role of fine needle aspiration cytology and immunocytochemistry in preoperative diagnosis of pediatric renal tumors

OBJECTIVE: To evaluate accuracy and role of immunocytochemistry (ICC) in cytologic diagnosis of pediatric renal tumors. STUDY DESIGN: Fine needle aspirates from 75 cases of pediatric renal tumors were studied. Radiologic-guided aspirations were performed, with 6-7 smears stained with Papanicolaou and Giemsa stains. Smears were screened without the knowledge of final histologic diagnosis. Subsequently, clinical details, final histology and diagnosis rendered by the original cytologist were noted to judge accuracy of diagnosis by a sensitized cytologist. Five neuroblastomas that entered close differentials for Wilms tumor were also evaluated. ICC studies were also performed after staining. RESULTS: Of 58 Wilms tumors, 5 were misdiagnosed; 3 renal rhabdoid tumors and 1 clear cell sarcoma were missed on cytology. Non-Hodgkin's lymphomas presenting as renal masses were accurately diagnosed on cytology, but primitive neuroectodermal tumors (n = 3) and renal cell carcinomas (n = 2) were not accurately diagnosed. Accuracy rate improved from 65% to 92% on review by a cytologist aware of cytologic features of pediatric renal tumors. CONCLUSION: A good accuracy rate of diagnosis of pediatric renal tumors can be achieved by priming pathologists to typical features of tumors. Immunocytochemistry plays a supportive role in cases with atypical morphology or unusual presentations.     

Cytologic findings in infiltrating micropapillary carcinoma and mucinous carcinomas with micropapillary pattern

OBJECTIVE: To examine the observation that some mucinous carcinomas have a micropapillary pattern and are mucinous variants of the highly angioinvasive infiltrating micropapillary carcinomas (IMPC). STUDY DESIGN: We evaluated cytologic findings of 13 IMPC and 55 mucinous carcinomas for comparative features. RESULTS: In mucinous carcinomas, 37 of 50 (74%) had a micropapillary pattern. This group included 27 cases with pure mucinous micropapillary morphology (MUMPC), 8 MUMPC associated with a ductal carcinoma of the IMPC type (MUIDC) and 2 cases of mixed mucinous carcinomas with an MUMPC and a solid variant ofpapillary carcinoma (SVPC) component. On cytology both IMPC and mucinous carcinomas with micropapillary pattern demonstrated the micropapillary pattern, that is, angulated clusters or abortive papillae and ball-like clusters. However, the IMPC smears revealed numerous singly scattered tumor cells and larger fragments with shrub-like branching and the MUMPC had psammoma bodies. The mixed MUMPC and SVPC showed the classic cytologic features of MUMPC admixed with abundant singly dispersed tumor cells in the background representing the SVPC component. CONCLUSION: Although IMPC and the MUMPC share the micropapillary pattern on histologic examination, mucin alters the appearances in aspirates. Recognition of this morphologic spectrum will help in understanding the behavior of these tumors.     

Increases in alpha4* but not alpha3*/alpha6* nicotinic receptor sites and function in the primate striatum following chronic oral nicotine treatment

Knowledge of the effects of chronic nicotine is critical considering its widespread use in tobacco products and smoking cessation therapies. Although nicotine is well known to up-regulate alpha4* nAChR sites and function in the cortex, its actions in the striatum are uncertain because of the presence of multiple subtypes with potentially opposing effects. We therefore investigated the effect of long-term nicotine treatment on nAChR sites and function in the primate striatum, which offers the advantage of similar proportions of alpha3*/alpha6* and alpha4* nAChRs. Nicotine was given in drinking water, which resembles smoking in its intermittent but chronic delivery. Plasma nicotine and cotinine levels were similar to smokers. Chronic nicotine treatment (> 6 months) enhanced alpha4* nAChR-evoked [(3)H]dopamine release in striatal subregions, with an overall pattern of increase throughout the striatum when normalized to uptake. This increase correlated with elevated striatal alpha4* nAChRs. Under the same conditions, striatal alpha3*/alpha6* nAChR sites and function were decreased or unchanged. These divergent actions of chronic nicotine treatment on alpha4* versus alpha6* nAChRs, as well as effects on dopamine uptake, allow for a complex control of striatal activity to maintain dopaminergic function. Such knowledge is important for understanding nicotine dependence and the consequences of nicotine administration for the treatment of neurological disorders.     

Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage

A myopathy characterized by mitochondrial pathology and oxidative stress is present in patients with peripheral arterial disease (PAD). Patients with PAD differ in disease severity, mode of presentation, and presence of comorbid conditions. In this study, we used a mouse model of hindlimb ischemia to isolate and directly investigate the effects of chronic inflow arterial occlusion on skeletal muscle microanatomy, mitochondrial function and expression, and oxidative stress. Hindlimb ischemia was induced by staged ligation/division of the common femoral and iliac arteries in C57BL/6 mice, and muscles were harvested 12 wk later. Muscle microanatomy was examined by bright-field microscopy, and mitochondrial content was determined as citrate synthase activity in muscle homogenates and ATP synthase expression by fluorescence microscopy. Electron transport chain (ETC) complexes I through IV were analyzed individually by respirometry. Oxidative stress was assessed as total protein carbonyls and 4-hydroxy-2-nonenal (HNE) adducts and altered expression and activity of manganese superoxide dismutase (MnSOD). Ischemic muscle exhibited histological features of myopathy and increased mitochondrial content compared with control muscle. Complex-dependent respiration was significantly reduced for ETC complexes I, III, and IV in ischemic muscle. Protein carbonyls, HNE adducts, and MnSOD expression were significantly increased in ischemic muscle. MnSOD activity was not significantly changed, suggesting MnSOD inactivation. Using a mouse model, we have demonstrated for the first time that inflow arterial occlusion alone, i.e., in the absence of other comorbid conditions, causes myopathy with mitochondrial dysfunction and increased oxidative stress, recapitulating the muscle pathology of PAD patients.