Benchmarking computational methods for B-cell receptor reconstruction from single-cell RNA-seq data.

Multiple methods have recently been developed to reconstruct full-length B-cell receptors (BCRs) from single-cell RNA sequencing (scRNA-seq) data. This need emerged from the expansion of scRNA-seq techniques, the increasing interest in antibody-based drug development and the importance of BCR repertoire changes in cancer and autoimmune disease progression. However, a comprehensive assessment of performance-influencing factors such as the sequencing depth, read length or number of somatic hypermutations (SHMs) as well as guidance regarding the choice of methodology is still lacking. In this work, we evaluated the ability of six available methods to reconstruct full-length BCRs using one simulated and three experimental SMART-seq datasets. In addition, we validated that the BCRs assembled in silico recognize their intended targets when expressed as monoclonal antibodies. We observed that methods such as BALDR, BASIC and BRACER showed the best overall performance across the tested datasets and conditions, whereas only BASIC demonstrated acceptable results on very short read libraries. Furthermore, the de novo assembly-based methods BRACER and BALDR were the most accurate in reconstructing BCRs harboring different degrees of SHMs in the variable domain, while TRUST4, MiXCR and BASIC were the fastest. Finally, we propose guidelines to select the best method based on the given data characteristics.

Apoptosis induced by proteasome inhibition in cancer cells: predominant role of the p53/PUMA pathway.

The proteasome has emerged as a novel target for antineoplastic treatment of hematological malignancies and solid tumors, including those of the central nervous system. To identify cell death pathways activated in response to inhibition of the proteasome system in cancer cells, we treated human SH-SY5Y neuroblastoma cells with the selective proteasome inhibitor (PI) epoxomicin (Epoxo). Prolonged exposure to Epoxo was associated with increased levels of poly-ubiquitinylated proteins and p53, release of cytochrome c from the mitochondria, and activation of caspases. Analysis of global gene expression using high-density oligonucleotide microarrays revealed that Epoxo triggered transcriptional activation of the two Bcl-2-homology domain-3-only (BH3-only) genes p53 upregulated modulator of apoptosis (PUMA) and Bim. Subsequent studies in PUMA- and Bim-deficient cells indicated that Epoxo-induced caspase activation and apoptosis was predominantly PUMA-dependent. Further characterization of the transcriptional response to Epoxo in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; with deficiency in either p53 or PUMA significantly protected HCT116 cells against Epoxo-induced apoptosis. Our data suggest that p53 activation and the transcriptional induction of its target gene PUMA play an important role in the sensitivity of cancer cells to apoptosis induced by proteasome inhibition, and imply that antineoplastic therapies with PIs might be especially useful in cancers with functional p53.

The amyloid precursor protein protects PC12 cells against endoplasmic reticulum stress-induced apoptosis.

Endoplasmic reticulum (ER) stress is believed to play an important role in neurodegenerative disorders such as Alzheimer's disease. In the present study, we investigated the effect of the human amyloid precursor protein (APP) on the ER stress response in PC12 cells. Tunicamycin, an inhibitor of N-glycosylation, rapidly induced the expression of the ER-resident chaperone Bip/grp78, a known target gene of the unfolded protein response. Prolonged treatment with tunicamycin (>/= 12 h) resulted in the activation of executioner caspases 3 and 7. Interestingly, PC12 cells overexpressing human wild-type APP (APPwt) showed increased resistance to tunicamycin-induced apoptosis compared with empty vector-transfected controls. This neuroprotective effect was significantly diminished in cells expressing the Swedish mutation of APP (KM670/671NL). Similar effects were observed when ER stress was induced with brefeldin A, an inhibitor of ER-to-Golgi protein translocation. Of note, APP-mediated neuroprotection was not associated with altered expression of Bip/grp78 or transcription factor C/EBP homologous protein-10 (CHOP/GADD153), suggesting that APP acted either downstream or independently of ER-to-nucleus signaling. Our data indicate that APP plays an important physiological role in protecting neurons from the consequences of prolonged ER stress, and that APP mutations associated with familial Alzheimer's disease may impair this protective activity.

Gene expression during ER stress-induced apoptosis in neurons: induction of the BH3-only protein Bbc3/PUMA and activation of the mitochondrial apoptosis pathway.

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of ischemic and neurodegenerative disorders. Treatment of human SH-SY5Y neuroblastoma cells with tunicamycin, an inhibitor of protein glycosylation, rapidly induced the expression of target genes of the unfolded protein response. However, prolonged treatment also triggered a delayed, caspase-dependent cell death. Microarray analysis of gene expression changes during tunicamycin-induced apoptosis revealed that the Bcl-2 homology domain 3-only family member, Bcl-2 binding component 3/p53 upregulated modulator of apoptosis (Bbc3/PUMA), was the most strongly induced pro-apoptotic gene. Expression of Bbc3/PUMA correlated with a Bcl-xL-sensitive release of cytochrome c and the activation of caspase-9 and -3. Increased expression of Bbc3/PUMA was also observed in p53-deficient human cells, in response to the ER stressor thapsigargin, and in rat hippocampal neurons after transient forebrain ischemia. Overexpression of Bbc3/PUMA was sufficient to trigger apoptosis in SH-SY5Y neuroblastoma cells, and human cells deficient in Bbc3/PUMA showed dramatically reduced apoptosis in response to ER stress. Our data suggest that the transcriptional induction of Bbc3/PUMA may be sufficient and necessary for ER stress-induced apoptosis.

Ceramide-induced apoptosis of D283 medulloblastoma cells requires mitochondrial respiratory chain activity but occurs independently of caspases and is not sensitive to Bcl-xL overexpression.

Ceramides are potent lipid second messengers that are involved in apoptotic and hypoxic/ischaemic neurone death. We investigated the role of mitochondria and the mitochondrial apoptosis pathway in ceramide-induced cell death using human D283 medulloblastoma cells with a reduced mitochondrial DNA copy number (rho- cells) and a corresponding defect in mitochondrial respiration. Treatment with the complex I inhibitor rotenone, C2- or C8-ceramide induced cell death in D283 control cells, while rho- cells were significantly protected. In contrast, activation of the mitochondrial apoptosis pathway by transient overexpression of the pro-apoptotic Bax protein or exposure to the kinase inhibitor staurosporine induced apoptosis to a similar extent in control and rho- cells. Overexpression of the antiapoptotic protein Bcl-xL failed to inhibit the toxic effect of C2-ceramide in D283 control cells, and no significant increase in caspase-3-like protease activity could be detected during the death process. Despite this, C2-ceramide induced significant chromatin condensation and cell shrinkage in D283 control cells, reminiscent of apoptosis. These morphological alterations were associated with the activation of calpains. Both apoptotic morphology and calpain activation were attenuated in rho- cells. Our data indicate that the apoptosis-inducing effect of C2-ceramide may require mitochondrial respiratory chain activity and can occur independently of the mitochondrial apoptosis pathway, but involves the activation of calpains.

Dominant-negative suppression of HNF-1 alpha results in mitochondrial dysfunction, INS-1 cell apoptosis, and increased sensitivity to ceramide-, but not to high glucose-induced cell death.

Maturity onset diabetes of the young (MODY) 3 is a monogenic form of diabetes caused by mutations in the transcription factor hepatocyte nuclear factor (HNF)-1 alpha. We investigated the involvement of apoptotic events in INS-1 insulinoma cells overexpressing wild-type HNF-1 alpha (WT-HNF-1 alpha) or a dominant-negative mutant (DN-HNF-1 alpha) under control of a doxycycline-dependent transcriptional activator. Forty-eight h after induction of DN-HNF-1 alpha, INS-1 cells activated caspase-3 and underwent apoptotic cell death, while cells overexpressing WT-HNF-1 alpha remained viable. Mitochondrial cytochrome c release and activation of caspase-9 accompanied DN-HNF-1 alpha-induced apoptosis, suggesting the involvement of the mitochondrial apoptosis pathway. Activation of caspases was preceded by mitochondrial hyperpolarization and decreased expression of the anti-apoptotic protein Bcl-xL. Transient overexpression of Bcl-xL was sufficient to rescue INS-1 cells from DN-HNF-1 alpha-induced apoptosis. Both WT- and DN-HNF-1 alpha-expressing cells demonstrated similar increases in apoptosis when cultured at high glucose (25 mm). In contrast, induction of DN-HNF-1 alpha highly sensitized cells to ceramide toxicity. In cells cultured at low glucose, DN-HNF-1 alpha induction also caused up-regulation of the cell cycle inhibitor p27(KIP1). Therefore, our data indicate that increased sensitivity to the mitochondrial apoptosis pathway and decreased cell proliferation may account for the progressive loss of beta-cell function seen in MODY 3 subjects.