Organotypic human lung bud microarrays identify BMP-dependent SARS-CoV-2 infection in lung cells.

Although lung disease is the primary clinical outcome in COVID-19 patients, how SARS-CoV-2 induces lung pathology remains elusive. Here we describe a high-throughput platform to generate self-organizing and commensurate human lung buds derived from hESCs cultured on micropatterned substrates. Lung buds resemble human fetal lungs and display proximodistal patterning of alveolar and airway tissue directed by KGF. These lung buds are susceptible to infection by SARS-CoV-2 and endemic coronaviruses and can be used to track cell type-specific cytopathic effects in hundreds of lung buds in parallel. Transcriptomic comparisons of infected lung buds and postmortem tissue of COVID-19 patients identified an induction of BMP signaling pathway. BMP activity renders lung cells more susceptible to SARS-CoV-2 infection and its pharmacological inhibition impairs infection by this virus. These data highlight the rapid and scalable access to disease-relevant tissue using lung buds that recapitulate key features of human lung morphogenesis and viral infection biology.

Constitutive p53 heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors of BCL-xL.

Proapoptotic molecules directly targeting the BCL-2 family network are promising anticancer therapeutics, but an understanding of the cellular stress signals that render them effective is still elusive. We show here that the tumor suppressor p53, at least in part by transcription independent mechanisms, contributes to cell death induction and full activation of BAX by BH3 mimetic inhibitors of BCL-xL. In addition to mildly facilitating the ability of compounds to derepress BAX from BCL-xL, p53 also provides a death signal downstream of anti-apoptotic proteins inhibition. This death signal cooperates with BH3-induced activation of BAX and it is independent from PUMA, as enhanced p53 can substitute for PUMA to promote BAX activation in response to BH3 mimetics. The acute sensitivity of mitochondrial priming to p53 revealed here is likely to be critical for the clinical use of BH3 mimetics.

Phosphorylated STAT3 physically interacts with NPM and transcriptionally enhances its expression in cancer.

The signal transducer and activator of transcription 3 (STAT3) can be activated by the tyrosine kinase domain of the chimeric protein nucleophosmin/anaplastic lymphoma kinase (NPM/ALK), and has a pivotal role in mediating NPM/ALK-related malignant cell transformation. Although the role of STAT3 and wild-type NPM in oncogenesis has been extensively investigated, the relationship between both molecules in cancer remains poorly understood. In the present study, we first demonstrate that STAT3 phosphorylation at tyrosine 705 is accompanied by a concomitant increase in the expression level of NPM. Nuclear co-translocation of phosphorylated STAT3 with NPM can be triggered by interferon-alpha (IFN-α) stimulation of Jurkat cells and phosphorylated STAT3 co-localizes with NPM in cancer cells showing constitutive STAT3 activation. We further demonstrate that STAT3 phosphorylation can transcriptionally mediate NPM upregulation in IFN-α-stimulated Jurkat cells and is responsible for maintaining its expression in cancer cells showing constitutive STAT3 activation. Inhibition of STAT3 phosphorylation or knockdown of NPM expression abrogates their simultaneous transnuclear movements. Finally, we found evidence for a physical interaction between NPM and STAT3 in conditions of STAT3 activation. In conclusion, NPM is a downstream effector of the STAT3 signaling, and can facilitate the nuclear entry of phosphorylated STAT3. These observations might open novel opportunities for targeting the STAT3 pathway in cancer.

Interference with PD-L1/PD-1 co-stimulation during antigen presentation enhances the multifunctionality of antigen-specific T cells.

The release of cytokines by T cells strongly defines their functional activity in vivo. The ability to produce multiple cytokines has been associated with beneficial immune responses in cancer and infectious diseases, while their progressive loss is associated with T-cell exhaustion, senescence and anergy. Consequently, strategies that enhance the multifunctional status of T cells are a key for immunotherapy. Dendritic cells (DCs) are professional antigen presenting cells that regulate T-cell functions by providing positive and negative co-stimulatory signals. A key negative regulator of T-cell activity is provided by binding of programmed death-1 (PD-1) receptor on activated T cells, to its ligand PD-L1, expressed on DCs. We investigated the impact of interfering with PD-L1/PD-1 co-stimulation on the multifunctionality of T cells, by expression of the soluble extracellular part of PD-1 (sPD-1) or PD-L1 (sPD-L1) in human monocyte-derived DCs during antigen presentation. Expression, secretion and binding of these soluble molecules after mRNA electroporation were demonstrated. Modification of DCs with sPD-1 or sPD-L1 mRNA resulted in increased levels of the co-stimulatory molecule CD80 and a distinct cytokine profile, characterized by the secretion of IL-10 and TNF-α, respectively. Co-expression in DCs of sPD-1 and sPD-L1 with influenza virus nuclear protein 1 (Flu NP1) stimulated Flu NP1 memory T cells, with a significantly higher number of multifunctional T cells and increased cytokine secretion, while it did not induce regulatory T cells. These data provide a rationale for the inclusion of interfering sPD-1 or sPD-L1 in DC-based immunotherapeutic strategies.

pRb/E2F-1-mediated caspase-dependent induction of Noxa amplifies the apoptotic effects of the Bcl-2/Bcl-xL inhibitor ABT-737.

Although Bcl-2 family members control caspase activity by regulating mitochondrial permeability, caspases can, in turn, amplify the apoptotic process upstream of mitochondria by ill-characterized mechanisms. We herein show that treatment with a potent inhibitor of Bcl-2 and Bcl-xL, ABT-737, triggers caspase-dependent induction of the BH3-only protein, Mcl-1 inhibitor, Noxa. RNA interference experiments reveal that induction of Noxa, and subsequent cell death, rely not only on the transcription factor E2F-1 but also on its regulator pRb. In response to ABT-737, pRb is cleaved by caspases into a p68Rb form that still interacts with E2F-1. Moreover, pRb occupies the noxa promoter together with E2F-1, in a caspase-dependent manner upon ABT-737 treatment. Thus, caspases contribute to trigger the mitochondrial apoptotic pathway by coupling Bcl-2/Bcl-xL inhibition to that of Mcl-1, via the pRb/E2F-1-dependent induction of Noxa.

Lentiviral vectors: a versatile tool to fight cancer.

Over the years, there has been an exponential increase in the number of gene therapy approaches that are under investigation for the treatment of cancer. This can be attributed to our growing understanding of the molecular mechanisms that contribute to the onset and maintenance of cancer as well as to the development of gene delivery vectors. In this review, we will focus on the use of lentiviral vectors (LVs) in immuno gene therapy of cancer, as these efficacious gene delivery vehicles have come to the fore front because of their many attractive features. LVs have been successfully applied to generate potent dendritic cell based anti-cancer vaccines and to deliver cancer-specific receptors to T-cells. Moreover, LVs are under investigation for the modulation of cancer cells. We will describe various strategies of this 'genuine' cancer gene therapy, amongst which transfer of suicide genes, modulation of pro- and anti-apoptotic molecules, strategies to optimize chemo- and radiotherapy, expression of molecules that affect angiogenesis or affect the immunogenicity of tumor cells. These will be discussed in view of our current knowledge of tumor immunology. Finally we will discuss some important issues and future directions to push the field forward.

Focal transmural necrosis of the gallbladder with bleeding resulting in a massive haemoperitoneum.

A case of massive haemoperitoneum as a complication of focal transmural necrosis of the gallbladder with bleeding during acute cholecystitis is reported. Urgent laparotomy and cholecystectomy was performed to secure an adequate haemostasis. A review of the literature confirmed that this condition is a very rare complication of acute cholecystitis. Only 44 similar cases have been reported between 1858 and 1996. In our case, bleeding was caused by the edges of a necrotic zone in the gallbladder wall.

A comparison of sleep detection by wrist actigraphy, behavioral response, and polysomnography.

Two alternative methods for detecting sleep, wrist actigraphy (ACT) and behavioral response monitoring (BRM), were compared to polysomnography (PSG). In the BRM paradigm, a threshold intensity visual or auditory stimulus generated by a palm-top computer was presented about once per minute, and subjects pressed a microswitch if the stimulus was detected. A response within 5 seconds of the stimulus was scored as "wake" and a failure to respond as "sleep." Four males and four females underwent two nights of simultaneous in-home PSG, BRM, and ACT. Each night, subjects underwent a protocol designed to generate five sleep latency trials. Subjects were awakened by alarm clocks at approximately 1-hour intervals and remained awake for 10 minutes before returning to bed for another sleep onset latency (SOL) trial. Minute-by-minute comparisons were made for PSG versus ACT and BRM. All measures were fairly sensitive in detecting sleep, but BRM was more accurate in determining SOL and subsequent wakefulness. Behavioral response monitoring using a tone resulted in more responses and arousals prior to and during light stages of sleep than BRM using a light. It is concluded that BRM has some important advantages as a simple, minimally invasive method for monitoring sleep.

Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants.

As a first step toward the exploitation of the disaccharide trehalose as a stress-protective and preservative agent in plants, we engineered trehalose biosynthesis in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) by introducing the otsA and otsB genes from Escherichia coli, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. In leaves of transgenic tobacco plants, very low levels of trehalose accumulation were obtained (0.11 mg g-1 fresh weight), whereas in transgenic potato tubers, no trehalose accumulated at all. Plant trehalase activity was shown to affect the accumulation of trehalose in these plants. An increase in trehalose accumulation, up to 0.41 and 4.04 mg g-1 fresh weight in tobacco leaves and potato micro-tubers, respectively, was noted when the potent trehalase inhibitor validamycin A was added to in vitro plants and to hydroponically grown greenhouse plants. Stunted growth and the formation of lancet-shaped leaves by trehalose-accumulating tobacco plants suggest a negative effect of trehalose biosynthesis on N. tabacum development. It is surprising that experiments with wild-type plants cultured in the presence of validamycin A indicate that, despite current belief, the capacity to synthesize trehalose may not be restricted to primitive phyla of vascular plants and certain "resurrection plants," but may exist throughout the angiosperms.

Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves.

Phytase from Aspergillus niger increases the availability of phosphorus from feed for monogastric animals by releasing phosphate from the substrate phytic acid. A phytase cDNA was constitutively expressed in transgenic tobacco (Nicotiana tabacum) plants. Secretion of the protein to the extracellular fluid was established by use of the signal sequence from the tobacco pathogen-related protein S. The specific phytase activity in isolated extracellular fluid was found to be approximately 90-fold higher than in total leaf extract, showing that the enzyme was secreted. This was confirmed by use of immunolocalization. Despite differences in glycosylation, specific activities of tobacco and Aspergillus phytase were identical. Phytase was found to be biologically active and to accumulate in leaves up to 14.4% of total soluble protein during plant maturation. Comparison of phytase accumulation and relative mRNA levels showed that phytase stably accumulated in transgenic leaves during plant growth.

The 30-kilodalton protein present in purified fusicoccin receptor preparations is a 14-3-3-like protein.

We have recently reported on the purification of the fusicoccin (FC) receptor from corn (Zea mays L.) and its identification by photoaffinity labeling (P. Aducci, A. Ballio, V. Fogliano, M.R. Fullone, M. Marra, N. Proietti [1993] Eur J Biochem 214: 339-345). Pure preparations of FC receptors, obtained under nondenaturing conditions, showed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis two doublets of proteins with apparent molecular masses of 30 and 90 kD. In the present paper we describe the isolation and identification of the primary structure of the 30-kD doublet proteins. Sequencing studies of peptides resulting from the digestion of the 30-kD protein showed a full identity with a 14-3-3-like protein from corn, named GF14. The 14-3-3 family is a class of proteins that is widely distributed in eukaryotes and is known to play various regulatory roles. The 30-kD protein has been immunologically identified by specific antibodies prepared against a synthetic peptide based on the determined amino acid sequence. A similar protein is recognized in partially purified FC receptor preparations from bean and spinach leaves.

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and beta-1,3-glucanase in transgenic plants.

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and beta-1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.

[An investigation on hepatitis B infection in the women with sexual abuse].

A serological investigation on hepatitis B virus (HBV) infection in women with sexual abuse was made in mainland China. In such women, the HBV infection rate and the positivity rate of HBsAg were higher than those in normal healthy conditions. It was suggested that sexual abuse was a important factor for HBV infection and the women with sexual abuse were one of the most dangerous sources of the HBV infection. The results of investigation also showed that in women with sexual abuse, HBV infection was not related to whether they were suffering from Venereal Diseases (VD) or not; but the existence of VD might probably exacerbate the process of hepatitis B.

Direct screening for high-level expression of an introduced alpha-amylase gene in plants.

A method is described for obtaining transgenic plants with a high level of expression of the introduced gene. Tobacco protoplasts were transformed with an expression construct containing a translational fusion between mature alpha-amylase from Bacillus licheniformis and the signal peptide of the tobacco PR-S protein. A total number of 5200 transformed protoplasts was cultured to microcalli and screened for alpha-amylase expression by incubation on media containing starch followed by staining with iodine. The calli were divided into four classes, based on the resulting halo sizes on the plates. The halo sizes were found to correlate with the expression levels in transgenic plants regenerated from the calli. The expression levels varied between 0 and 0.5% of soluble leaf protein in the regenerated transgenic plants. Wider implications of this method are discussed.

Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction.

As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis alpha-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum expression levels of about 0.3% of total soluble protein were obtained. No apparent effect of the presence of the enzyme on plant phenotype was observed. The molecular weight of the enzyme produced in tobacco was around 64 kD. The difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein. Application studies on the liquefaction of starch were done with transgenic seeds containing the recombinant alpha-amylase. The resulting hydrolysis products were virtually identical with those obtained from degradation with alpha-amylase from Bacillus licheniformis.

Herpes simplex virus type 1 colitis: an unusual cause of diarrhea.

Herpetic infections of the gastrointestinal tract are a well-recognized entity. Involvement of the colon seems to be very rare. A 78-yr-old woman developed bloody diarrhea and abdominal discomfort 2 months after surgical treatment for adenocarcinoma of the transverse colon. Colonoscopy revealed diffuse hemorrhagic, erosive, aphtoid, and ulcerative lesions. Histology showed nonspecific inflammatory changes. Herpes simplex virus type 1 (HSV-1) was isolated from endoscopic biopsy and stool specimens. The patient responded rapidly to symptomatic treatment with loperamide. This case demonstrates the potential for HSV-1 to induce infectious colitis; failure to obtain microbiologic evaluations and the rapid response to empiric, symptomatic treatment may be responsible for the rarity of diagnosis of this infection. The implications of this diagnosis are probably more relevant in immunosuppressed individuals, and may be important in the elderly population.

Serine esterases: structural conservation during animal evolution and variability in enzymatic properties in the genus Drosophila.

Both general esterases and acetylcholinesterases have been shown to be members of a homologous superfamily of serine esterases. A comparison of N-terminal sequences demonstrates that esterase-4 and -5 from Drosophila mojavensis belong to this family as well, with esterase-6 and esterase-P from D. melanogaster being the closest relatives. In order to investigate the presence of immunologically related esterases in other Drosophila species, crude larval extracts from five species were applied to two immunoaffinity columns with antibodies directed against esterase-4 and esterase-5 from D. mojavensis. The substrate preference for either 1- or 2-naphthyl acetate was determined. Both esterase-4 and esterase-5 from D. mojavensis are 'normally' specific for 2-naphthyl esters, but at least three of the cross-reacting esterases from the other species have a preference for 1-naphthyl esters. This difference in substrate preference is another example of the variability observed with Drosophila esterases.